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

A novel peptide inhibitor of classical and lectin complement activation including ABO incompatibility.

International audiencePrevious experiments from our laboratories have identified peptides derived from the human astrovirus coat protein (CP) that bind C1q and mannose binding lectin (MBL) inhibiting activation of the classical and lectin pathways of complement, respectively. The purpose of this study was to evaluate the function of these coat protein peptides (CPPs) in an in vitro model of complement-mediated disease (ABO incompatibility), preliminarily assess their in vivo complement suppression profile and develop more highly potent derivatives of these molecules. E23A, a 30 amino acid CPP derivative previously demonstrated to inhibit classical pathway activation was able to dose-dependently inhibit lysis of AB erythrocytes treated with mismatched human O serum. Additionally, when injected into rats, E23A inhibited the animals' serum from lysing antibody-sensitized erythrocytes, providing preliminary in vivo functional evidence that this CPP can cross the species barrier to inhibit serum complement activity in rodents. A rational drug design approach was implemented to identify more potent CPP derivatives, resulting in the identification and characterization of a 15 residue peptide (polar assortant (PA)), which demonstrated both superior inhibition of classical complement pathway activation and robust binding to C1q collagen-like tails. PA also inhibited ABO incompatibility in vitro and demonstrated in vivo complement suppression up to 24h post-injection. CPP's ability to inhibit ABO incompatibility in vitro, proof of concept in vivo inhibitory activity in rats and the development of the highly potent PA derivative set the stage for preclinical testing of this molecule in small animal models of complement-mediated disease

Peptide Inhibitor of Complement C1 (PIC1) Rapidly Inhibits Complement Activation after Intravascular Injection in Rats.

The complement system has been increasingly recognized to play a pivotal role in a variety of inflammatory and autoimmune diseases. Consequently, therapeutic modulators of the classical, lectin and alternative pathways of the complement system are currently in pre-clinical and clinical development. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement and is referred to as Peptide Inhibitor of Complement C1 (PIC1). In this study, we determined that the lead PIC1 variant demonstrates a salt-dependent binding to C1q, the initiator molecule of the classical pathway. Additionally, this peptide bound to the lectin pathway initiator molecule MBL as well as the ficolins H, M and L, suggesting a common mechanism of PIC1 inhibitory activity occurs via binding to the collagen-like tails of these collectin molecules. We further analyzed the effect of arginine and glutamic acid residue substitution on the complement inhibitory activity of our lead derivative in a hemolytic assay and found that the original sequence demonstrated superior inhibitory activity. To improve upon the solubility of the lead derivative, a pegylated, water soluble variant was developed, structurally characterized and demonstrated to inhibit complement activation in mouse plasma, as well as rat, non-human primate and human serum in vitro. After intravenous injection in rats, the pegylated derivative inhibited complement activation in the blood by 90% after 30 seconds, demonstrating extremely rapid function. Additionally, no adverse toxicological effects were observed in limited testing. Together these results show that PIC1 rapidly inhibits classical complement activation in vitro and in vivo and is functional for a variety of animal species, suggesting its utility in animal models of classical complement-mediated diseases

Complement Activation and STAT4 Expression Are Associated with Early Inflammation in Diabetic Wounds

<div><p>Diabetic non-healing wounds are a major clinical problem. The mechanisms leading to poor wound healing in diabetes are multifactorial but unresolved inflammation may be a major contributing factor. The complement system (CS) is the most potent inflammatory cascade in humans and contributes to poor wound healing in animal models. Signal transducer and activator of transcription 4 (STAT4) is a transcription factor expressed in immune and adipose cells and contributes to upregulation of some inflammatory chemokines and cytokines. Persistent CS and STAT4 expression in diabetic wounds may thus contribute to chronic inflammation and delayed healing. The purpose of this study was to characterize CS and STAT4 in early diabetic wounds using db/db mice as a diabetic skin wound model. The CS was found to be activated early in the diabetic wounds as demonstrated by increased anaphylatoxin C5a in wound fluid and C3-fragment deposition by immunostaining. These changes were associated with a 76% increase in nucleated cells in the wounds of db/db mice vs. controls. The novel classical CS inhibitor, Peptide Inhibitor of Complement C1 (PIC1) reduced inflammation when added directly or saturated in an acellular skin scaffold, as reflected by reduced CS components and leukocyte infiltration. A significant increase in expression of STAT4 and the downstream macrophage chemokine CCL2 and its receptor CCR2 were also found in the early wounds of db/db mice compared to non-diabetic controls. These studies provide evidence for two new promising targets to reduce unresolved inflammation and to improve healing of diabetic skin wounds.</p></div

Immunohistochemistry showing longitudinal changes in expression of STAT4 in diabetic db/db and heterozygous control mice.

<p><b>(A)</b> Representative micrographs showing STAT4 immunostaining (red) and nuclear staining using DAPI (blue) of formalin fixed paraffin embedded whole wounds of db/db-/- and control mice at the time of wounding (T0) and after 8 and 48 hours, respectively. Magnification: 100x; (B) High power (400x) images of the dermis (top) and adipose tissue (bottom) layers of the dbdb-/- and control mice at 48 hours post-wounding. Solid arrows indicate peri-nuclear localization of STAT4 and dashed arrows indicate nuclear localization. Insets represent higher magnification of the nuclear (top) or peri-nuclear (bottom) localization of the signal. (C) Grading of STAT4 staining in the dermis and dermal adipose tissue of diabetic db/db and control mice. A scale of “0” to”3” was used to quantify the abundance of staining. A number of 5–8 micrographs/section from n = 3 mice/group were graded by 4 independent observers in a blinded manner. * P ≤0.05.</p

Pegylated versions of PA inhibit complement activity in a hemolytic assay.

<p>(A) Hemolytic assays using factor B-depleted serum were performed with PA dissolved in DMSO and its pegylated derivatives dissolved in water. Factor B-depleted serum was incubated with 0.77mM of each peptide and then added to sensitized sheep erythrocytes. (B) Titration of increasing amounts of PA and PA-dPEG24 in the hemolytic assay. Water and DMSO were used as vehicle controls in the presence of factor B-depleted serum. GVBS⁺⁺ is a buffer- only control. Values are the means of three independent experiments. Error bars represent the SEM.</p

PA binds to MBL and ficolins and does not displace C1s from C1q.

<p>(A) The AcPA and CP2 peptides were adsorbed to a microtiter plate and incubated with a constant amount of MBL or K55Q. Bound MBL was detected with a polyclonal goat anti-MBL sera followed by HRP-conjugated anti-goat sera. (B) The various ficolins indicated in the figure were adsorbed to the microtiter plate and incubated with biotinylated PA followed by a neutravidin conjugate. BSA was used as a negative control for binding. (C) Partially purified C1 was mixed with increasing amounts of CP or PA and added to a microtiter plate coated with monoclonal antibody to C1q. After washing, C1s signal present in intact C1 complexes was measured by ELISA using polyclonal antibody to C1s (CP, red line; PA, blue line) or polyclonal antibody to C1q (green line) to confirm C1q was bound to the plate. Data represent the means of three independent experiments. Error bars denote SEM.</p

Complement activation and cellular inflammation for acute skin wounds in diabetic mice.

<p>(A) C5a concentration in wound beds of diabetic and control mice absorbed by filter paper and assayed by ELISA. Left and right wounds averaged for n = 3 mice in each group and time point: 10 min (P = 0.05) 2h (P = 0.002), 4h (P = 0.001), 24h (P = 0.05). * P ≤0.05 vs. hetero (control). (B) C3-fragment deposition (C3 opsonization) in the subcutaneous tissue at the edges of the wound beds of diabetic and control mice assayed by immunofluorescence (n = 2). (C) Nucleated cell infiltration into the subcutaneous tissue at the edges of the wound beds of diabetic and control mice assayed by DAPI fluorescence (n = 2). Data are means ± SEM.</p

Analysis of leukocyte inflammation of wounds for diabetic mice treated with PIC1 at 14 days.

<p>Representative wound histology (H&E) for (A) control scaffold only mice, and (B) PIC1 impregnated skin scaffold. (C) Averaged inflammatory index of leukocytes (predominantly neutrophils) for db/db mice at day 14 post wounding for wounds treated with PIC1 complement inhibitor in a skin scaffold or control skin scaffold, n = 6 in each group. Data are means ± SEM. * P ≤0.05 vs. saline control.</p