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A 14-year-old female patient with sickle cell disease developed a severe delayed hemolytic transfusion reaction (DHTR) leading to multiple transfusions and intensive care management. To better understand the extent to which the classical complement pathway was contributing to her DHTR, we utilized the complement hemolysis using human erythrocytes (CHUHE) assay and the classical complement pathway inhibitor, PIC1. Residual discarded de-identified plasma and erythrocytes from the patient obtained from routine phlebotomy was acquired. These reagents were used in the CHUHE assay in the presence of increasing concentrations of PIC1. Complement-mediated hemolysis of the patient’s erythrocytes occurred in her plasma and complement permissive buffer. Increasing concentrations of PIC1 dose-dependently inhibited hemolysis to levels found for the negative control - complement inhibitor buffer. Complement-mediated hemolysis was demonstrated by the CHUHE assay for this patient with sickle cell disease and severe DHTR. PIC1 inhibition of hemolysis suggested that the classical complement pathway was contributing to her DHTR. J Hematol. 2021;10(1):18-21 doi: https://doi.org/10.14740/jh553

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.

The EPICC peptides are a family of peptides that have been developed from the sequence of the capsid protein of human astrovirus type 1 and previously shown to inhibit the classical and lectin pathways of complement. The EPICC peptides have been further optimized to increase aqueous solubility and identify additional mechanisms of action. Our laboratory has developed the lead EPICC molecule, PA-dPEG24 (also known as RLS-0071), which is composed of a 15 amino acid peptide with a C-terminal monodisperse 24-mer PEGylated moiety. RLS-0071 has been demonstrated to possess other mechanisms of action in addition to complement blockade that include the inhibition of neutrophil-driven myeloperoxidase (MPO) activity, inhibition of neutrophil extracellular trap (NET) formation as well as intrinsic antioxidant activity mediated by vicinal cysteine residues contained within the peptide sequence. RLS-0071 has been tested in various ex vivo and in vivo systems and has shown promise for the treatment of both immune-mediated hematological diseases where alterations in the classical complement pathway plays an important pathogenic role as well as in models of tissue-based diseases such as acute lung injury and hypoxic ischemic encephalopathy driven by both complement and neutrophil-mediated pathways (i.e., MPO activity and NET formation). Next generation EPICC peptides containing a sarcosine residue substitution in various positions within the peptide sequence possess aqueous solubility in the absence of PEGylation and demonstrate enhanced complement and neutrophil inhibitory activity compared to RLS-0071. This review details the development of the EPICC peptides, elucidation of their dual-acting complement and neutrophil inhibitory activities and efficacy in ex vivo systems using human clinical specimens and in vivo efficacy in animal disease models.

The EPICC peptides are a family of peptides that have been developed from the sequence of the capsid protein of human astrovirus type 1 and previously shown to inhibit the classical and lectin pathways of complement. The EPICC peptides have been further optimized to increase aqueous solubility and identify additional mechanisms of action. Our laboratory has developed the lead EPICC molecule, PA-dPEG24 (also known as RLS-0071), which is composed of a 15 amino acid peptide with a C-terminal monodisperse 24-mer PEGylated moiety. RLS-0071 has been demonstrated to possess other mechanisms of action in addition to complement blockade that include the inhibition of neutrophil-driven myeloperoxidase (MPO) activity, inhibition of neutrophil extracellular trap (NET) formation as well as intrinsic antioxidant activity mediated by vicinal cysteine residues contained within the peptide sequence. RLS-0071 has been tested in various

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.

Perinatal hypoxic ischemic encephalopathy (HIE) remains a major contributor of infant death and long-term disability worldwide. The role played by the complement system in this ischemia-reperfusion injury remains poorly understood. In order to better understand the role of complement activation and other modifiable mechanisms of injury in HIE, we tested the dual-targeting anti-inflammatory peptide, RLS-0071 in an animal model of HIE. Using the well-established HIE rat pup model we measured the effects of RLS-0071 during the acute stages of the brain injury and on long-term neurocognitive outcomes. Rat pups subject to hypoxia-ischemia insult received one of 4 interventions including normothermia, hypothermia and RLS-0071 with and without hypothermia. We measured histopathological effects, brain C1q levels and neuroimaging at day 1 and 21 after the injury. A subset of animals was followed into adolescence and evaluated for neurocognitive function. On histological evaluation, RLS-0071 showed neuronal protection in combination with hypothermia (P = 0.048) in addition to reducing C1q levels in the brain at 1hr (P = 0.01) and at 8 hr in combination with hypothermia (P = 0.005). MRI neuroimaging demonstrated that RLS-0071 in combination with hypothermia reduced lesion volume at 24 hours (P

Introduction Autoimmune hemolytic anemia (AIHA) is a serious manifestation of systemic lupus erythematosus (SLE) associated with significant morbidity and mortality. In order to more fully understand the causative pathways, we utilized sera from subjects with SLE and active AIHA, or a history of AIHA, to evaluate the classical complement pathway, anti-erythrocyte antibodies, and immune complexes. Methods To evaluate antibody-mediated complement activation on the surface of erythrocytes, as occurs in AIHA, blood type O erythrocytes were incubated with sera from 19 subjects with SLE and a history of AIHA. Circulating anti-erythrocyte antibodies and immune complexes were measured with ELISA-based assays. Results In total, 90% of subjects with SLE and a history of AIHA, but not active clinical hemolysis, had measurable anti-erythrocyte antibodies. Of those with anti-erythrocyte antibody, 53% demonstrated complement opsonization on the erythrocyte surface >twofold above negative control and 29% generated the anaphylatoxin C5a. Conclusions For subjects with SLE and a history of AIHA, the persistence of circulating anti-erythrocyte antibodies and resultant erythrocyte complement opsonization and anaphylatoxin generation suggests the possibility that these complement effectors contribute to chronic morbidity and risk of AIHA relapse.

Peptide Inhibitor of Complement C1 (PIC1) is a family of 15 amino acid peptides that inhibit complement activation via the classical and lectin pathways and inhibit myeloperoxidase. PIC1 peptides were originally derived from a region of limited homology with defensin human neutrophil peptide 1 (HNP-1). Despite having undergone extensive rearrangements of amino acid sequence subsequently, PIC1 peptides retain the defensin-like characteristics of being cysteine rich and amphiphilic. To date, defensin-like antimicrobial activity for PIC1 has not been explored. Here we report the antimicrobial activity of PIC1 for multiple pathogenic bacteria tested in minimum inhibitory concentration (MIC)-type assays. PIC1variant PA-dPEG24 was found to have antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, Neisseria meningitidis, Neisseria gonorrhoeae, Gardnerella vaginalis, and Prevotella bivia. Confocal microscopy demonstrated PIC1 localized to the surface of P. aeruginosa and S. aureus consistent with the defensins. Testing PIC1 variants with amino acid substitutions revealed differences in complement inhibition and antimicrobial effects suggesting these occur via independent mechanisms. PIC1 inhibited P. aeruginosa growth in normal human serum suggesting the antimicrobial effect was dominant versus the survival benefit resulting from complement inhibition. In summary, these experiments demonstrate that PIC1 peptides have broad antimicrobial activity against pathogenic bacteria similar to defensins.

Hemoglobin is the natural carrier of oxygen in red blood cells (RBCs). While intracellular hemoglobin provides life-sustaining oxygen transport, extracellular free hemoglobin displays toxicity due to inherent peroxidase activity generating reactive oxygen species that subsequently react with the hemoglobin molecule to produce toxic heme degradation products resulting in free radicals, oxidative stress damage, and lipid peroxidation. We have recently demonstrated that Peptide Inhibitor of Complement C1 (PIC1) inhibits peroxidase activity of the heme-based enzyme myeloperoxidase. To elucidate whether PIC1 could inhibit peroxidase activity of hemoglobin, we evaluated the consequence of PIC1 on RBC lysates, methemoglobin, and myoglobin using tetramethylbenzidine (TMB) as an oxidation target. PIC1 reversibly and dose-dependently prevented TMB oxidation to tetramethylbenzidine diimine by RBC lysates, methemoglobin, and myoglobin, having comparable activity to the inhibitor 4-aminobenzoic acid hydrazide. PIC1 inhibited TMB oxidation of RBC lysates similar to L-cysteine suggesting that the two cysteine residues contained in PIC1 may mediate peroxidase activity. PIC1 also inhibited heme destruction by NaOCl for RBC lysates, hemoglobin, and myoglobin as assayed by preservation of the Soret absorbance peak in the presence of NaOCl and reduction in free iron release. In conclusion, PIC1 inhibits peroxidase activity of hemoglobin and myoglobin likely via an antioxidant mechanism.

BackgroundA 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.MethodsSixteen 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.ResultsSix 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.ConclusionsSeveral 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.

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.

Infection with Staphylococcus aureus does not induce long-lived protective immunity for reasons that are not completely understood. Human and murine vaccine studies support a role for Abs in protecting against recurring infections, but S. aureus modulates the B cell response through expression of staphylococcus protein A (SpA), a surface protein that drives polyclonal B cell expansion and induces cell death in the absence of costimulation. In this murine study, we show that SpA altered the fate of plasmablasts and plasma cells (PCs) by enhancing the short-lived extrafollicular response and reducing the pool of bone marrow (BM)-resident long-lived PCs. The absence of long-lived PCs was associated with a rapid decline in Ag-specific class-switched Ab. In contrast, when previously inoculated mice were challenged with an isogenic SpA-deficient S. aureus mutant, cells proliferated in the BM survival niches and sustained long-term Ab titers. The effects of SpA on PC fate were limited to the secondary response, because Ab levels and the formation of B cell memory occurred normally during the primary response in mice inoculated with wild-type or SpA-deficient S. aureus mutant. Thus, failure to establish long-term protective Ab titers against S. aureus was not a consequence of diminished formation of B cell memory; instead, SpA reduced the proliferative capacity of PCs that entered the BM, diminishing the number of cells in the long-lived pool.

BACKGROUND The agglutination-based cross-matching method is sensitive for antibody binding to red blood cells but is only partially predictive of complement-mediated hemolysis, which is important in many acute hemolytic transfusion reactions. Here, we describe complement hemolysis using human erythrocytes (CHUHE) assays that directly evaluate complement-mediated hemolysis between individual serum-plasma and red blood cell combinations. The CHUHE assay is used to evaluate correlations between agglutination titers and complement-mediated hemolysis as well as the hemolytic potential of plasma from type A blood donors. STUDY DESIGN AND METHODS Plasma or serum from each type A blood donor was incubated with AB or B red blood cells in the CHUHE assay and measured for free hemoglobin release. RESULTS CHUHE assays for serum or plasma demonstrate a wide, dynamic range and high sensitivity for complement-mediated hemolysis for individual serum/plasma and red blood cell combinations. CHUHE results suggest that agglutination assays alone are only moderately predictive of complement-mediated hemolysis. CHUHE results also suggest that plasma from particular type A blood donors produce minimal complement-mediated hemolysis, whereas plasma from other type A blood donors produce moderate to high-level complement-mediated hemolysis, depending on the red blood cell donor. CONCLUSION The current results indicate that the CHUHE assay can be used to assess complement-mediated hemolysis for plasma or serum from a type A blood donor, providing additional risk discrimination over agglutination titers alone.

BACKGROUND Acute hemolytic transfusion reactions have a broad clinical presentation from mild and transitory signs and symptoms to shock, disseminated intravascular coagulation, renal failure, and death. We have recently developed a rat model of acute intravascular hemolysis showing that the classical complement pathway mediates antibody-dependent hemolysis. The objective of this study was to evaluate the role of the classical pathway inhibitor peptide inhibitor of complement C1 (PIC1) in this animal model. STUDY DESIGN AND METHODS Male Wistar rats received a 15% transfusion of human red blood cells (RBCs) and blood was isolated from the animals up to 120 minutes. Animals received PIC1 either 2 minutes before or 0.5 minutes after transfusion. Sham-, vehicle-, and cobra venom factor (CVF)-treated animals were used as control groups with a subset of rats also receiving an equivalent dose of intravenous immunoglobulin (IVIG) before transfusion. Blood was analyzed for transfused RBC survival by flow cytometry and free hemoglobin (Hb) in isolated plasma by spectrophotometry. RESULTS Vehicle-treated rats showed decreased human RBC survival and increased free Hb as expected. Rats receiving PIC1 before transfusion showed increased human RBC survival and decreased Hb similar to CVF-treated rats. Notably, rats receiving PIC1 after initiation of transfusion showed similar decreases in hemolysis as animals receiving PIC1 before transfusion. Compared to IVIG and saline controls, PIC1-treated animals demonstrated decreased hemolysis and protection from acute kidney injury. CONCLUSIONS These results demonstrate that PIC1 has efficacy in an animal model of acute intravascular hemolysis in both prevention and rescue scenarios.

Fungal infections are rare causes of acute surgical wound infections, butCandidais not an infrequent etiology in chronic wound infections.Trichophytonspecies is a common cause of tinea capitis but has not been reported as a cause of neurosurgical wound infection. We report a case ofTrichophyton tonsuranscausing a nonhealing surgical wound infection in a 14-year-old male after hemicraniectomy. His wound infection was notable for production of purulent exudate from the wound and lack of clinical improvement despite empiric treatment with multiple broad-spectrum antibiotics targeting typical bacterial causes of wound infection. Multiple wound cultures consistently grewTrichophytonfungus, and his wound infection clinically improved rapidly after starting terbinafine and discontinuing antibiotics.

BACKGROUND A patient with B+ sickle cell disease received 3 units of red blood cells (RBCs) from two O+ donors and developed fever and hypotension after the first unit, consistent with an acute transfusion reaction (ATR). Anti-B titers in plasma from each O+ donor were markedly elevated and nondiscriminatory. In order to evaluate the potential for the transfused units to produce complement-mediated hemolysis of B+ RBCs, hemolytic complement testing was performed. STUDY DESIGN AND METHODS Plasma from each donor was diluted in veronal buffer and incubated with B+ RBCs, and free hemoglobin was measured by spectrophotometer in the complement hemolysis using human erythrocytes (CHUHE) assay. Peptide inhibitor of complement C1 (PIC1) was used to confirm antibody-initiated complement pathway activation. RESULTS A 96-fold difference (p = 0.014) in hemolysis was measured between plasma samples from the two O+ donors using the CHUHE assay. The extremely high degree of hemolysis produced by the one plasma was inhibited by PIC1 in a dose-dependent manner. CONCLUSION These results indicate that hemolytic complement testing with the CHUHE assay can be used to assess the risk of antibody-initiated, complement-mediated hemolysis from a transfusion beyond what can be achieved with antibody titers alone.

Staphylococcus aureus is a crucial human pathogen expressing various immune-evasion proteins that interact with the host-cell molecules. Clumping factor A (ClfA) is a microbial surface protein that promotes S. aureus binding to fibrinogen, and is associated with septic arthritis and infective endocarditis. In order to identify the major human serum proteins that bind the ClfA, we utilized recombinant ClfA region A in a plate-based assay. SDS-PAGE analysis of the bound proteins yielded five prominent bands, which were analysed by MS yielding apolipoprotein E (ApoE) as the predominant protein. ClfA-sufficient S. aureus bound purified ApoE by more than one log greater than an isogenic ClfA-deficient mutant. An immunodot-blot assay yielded a linearity model for ClfA binding to human ApoE with a stoichiometric-binding ratio of 1.702 at maximal Pearson's correlation coefficient (0.927). These data suggest that ApoE could be a major and novel binding target for the S. aureus virulence factor ClfA. Thus, ClfA recruitment of serum ApoE to the S. aureus surface may sequester ApoE and blunt its host defence function against S. aureus-invasive infections to humans. In this context, compounds that can block or suppress ClfA binding to ApoE might be utilized as prophylactic or therapeutic agents.

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.

Reactive oxygen species (ROS) are natural byproducts of oxidative respiration that are toxic to organs and tissues. To mitigate ROS damage, organisms have evolved a variety of antioxidant systems to counteract these harmful molecules, however in certain pathological conditions these protective mechanisms can be overwhelmed. We have recently demonstrated that Peptide Inhibitor of Complement C1 (PIC1) mitigates peroxidase activity of the heme bearing proteins myeloperoxidase, hemoglobin, and myoglobin through a reversible process. To determine if this property of PIC1 was antioxidant in nature, we tested PIC1 in a number of well-established antioxidant assays. PIC1 showed dose-dependent antioxidant activity in a total antioxidant (TAC) assay, hydroxyl radical antioxidant capacity (HORAC) assay, oxygen radical antioxidant capacity (ORAC) assay as well as the thiobarbituric acid reactive substances (TBARS) assay to screen for PIC1 antioxidant activity in human plasma. The antioxidant activity of PIC1 in the TAC assay, as well as the HORAC/ORAC assay demonstrated that this peptide acts via the single electron transport (SET) and hydrogen atom transfer (HAT) mechanisms, respectively. Consistent with this mechanism of action, PIC1 did not show activity in a metal chelating activity (MCA) assay. PIC1 contains two vicinal cysteine residues and displayed similar antioxidant activity to the well characterized cysteine-containing tripeptide antioxidant molecule glutathione (GSH). Consistent with the role of the cysteine residues in the antioxidant activity of PIC1, oxidation of these residues significantly abrogated antioxidant activity. These results demonstrate that in addition to its described complement inhibiting activity, PIC1 displays in vitro antioxidant activity.

In this study, we report the first case of Mycobacterium tuberculosis endocarditis in an immunocompetent child born in the United States. Mass spectrometry of the vegetation identified coagulation, humoral immune proteins, neutrophil granule proteins, and histones. Few neutrophils on histopathology suggest that neutrophil extracellular traps may contribute to tuberculous endocardiac mass formation.

Capnocytophaga is an opportunistic gram-negative anaerobic bacillus found in the oropharyngeal cavity of mammals and is associated with periodontal disease in humans. Sepsis, osteomyelitis, lung abscess, endocarditis, and meningitis have been reported in humans following animal bites. Perinatal infection with Capnocytophaga is infrequent and is generally considered to have a low risk of morbidity to the mother and fetus. We report a case of neonatal Capnocytophaga sepsis associated with the development of severe cystic periventricular leukomalacia.

Background Prevention of acute hemolytic transfusion reactions is a worldwide concern. The objective of this study was to develop a simple rat model of complement-mediated acute intravascular hemolysis. Study Design and Methods Human AB red blood cells (RBCs) were incubated with complement-sufficient or complement-deficient Wistar rat serum (WRS) in the presence and absence of human RBC antibody in vitro to elucidate the mechanism of hemolysis. To study the role of complement in acute intravascular hemolysis in vivo, Wistar rats were treated either with or without cobra venom factor (CVF) to deplete complement activity. Human AB RBCs were then injected into both groups of rats, followed by serial blood draws up to 2 hours. Venous blood clearance and lysis of transfused RBCs at each time point were measured by flow cytometry and spectrophotometry. RBC sequestration was determined in the liver, spleen, and kidney by immunohistochemistry. Results In vitro incubation of human RBCs with WRS demonstrated that RBC lysis was mediated via the classical complement pathway and that hemolysis was antibody dependent. Transfusion of human RBCs into rats showed significantly less hemolysis in the CVF group versus untreated group. RBC sequestration in the spleen and liver 2 hours posttransfusion were not quantitatively different between the two groups. Conclusions Given the much higher degree of similarity for rat and human complement compared to mice, this simple rat model is ideal for testing novel inhibitors of classical pathway activation for the prevention and treatment of acute intravascular hemolysis.

In cystic fibrosis (CF), lung damage is mediated by a cycle of obstruction, infection, inflammation and tissue destruction. The complement system is a major mediator of inflammation for many diseases with the effectors C5a and C3a often playing important roles. We have previously shown in a small pilot study that CF sputum soluble fraction concentrations of C5a and C3a were associated with clinical measures of CF disease. Here we report a much larger study of 34 CF subjects providing 169 testable sputum samples allowing longitudinal evaluation comparing C5a and C3a with clinical markers. Levels of the strongly pro-inflammatory C5a correlated negatively with FEV1% predicted (P < 0.001), whereas the often anti-inflammatory C3a correlated positively with FEV1% predicted (P = 0.01). C5a concentrations correlated negatively with BMI percentile (P = 0.017), positively with worsening of an acute pulmonary exacerbation score (P = 0.007) and positively with P. aeruginosa growth in sputum (P = 0.002). C5a levels also correlated positively with concentrations of other sputum markers associated with worse CF lung disease including neutrophil elastase (P < 0.001), myeloperoxidase activity (P = 0.006) and DNA concentration (P < 0.001). In contrast to C5a, C3a levels correlated negatively with worse acute pulmonary exacerbation score and correlated negatively with sputum concentrations of neutrophil elastase, myeloperoxidase activity and DNA concentration. In summary, these data suggest that in CF sputum, increased C5a is associated with increased inflammation and poorer clinical measures, whereas increased C3a appears to be associated with less inflammation and improved clinical measures.

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.

INTRODUCTION Uncontrolled HIV infection is known to activate the complement system, leading to an increase in chronic inflammation. Whether or not this activation of complement persists and contributes to chronic inflammation in subjects with HIV infection that is well controlled through use of antiretroviral therapy has not been studied. METHODS We conducted an observational, cross-sectional study using sera from 305 adults with well-controlled HIV infection and 30 healthy controls. Sera was tested for markers of complement activation (C3a and C5a levels), complement function (CH50 assay), and immunoglobulin levels (IgG1-IgG4) as IgG can activate complement. We evaluated the association of well-controlled HIV infection with C3a, C5a, CH50, IgG1-IgG4, and total IgG levels using both univariate and multivariate analyses, controlling for factors such as age, sex, race, comorbidities (including hepatitis C coinfection), smoking status, and statin use. RESULTS Well-controlled HIV infection was associated with a 54% increase in complement activation as measured by C3a levels compared with healthy controls (P < 0.0001). Hepatitis C coinfection was associated with a further 52% increase in complement activation, as measured by C3a levels, over HIV alone (P = 0.003). CONCLUSION These results suggest that complement activation may contribute to a proinflammatory state even in well-controlled HIV infection. Furthermore, hepatitis C virus coinfection may be even more proinflammatory, in complement activation, compared with HIV infection alone.

In the setting of infectious disease emergencies, rapid and accurate identification of the causative agent is critical to optimizing antimicrobial therapy in a timely manner. It is clearly evident that the age of molecular diagnostics is now upon us, with real-time PCR becoming the standard of diagnosis for many infectious disease emergencies in either monoplex or multiplex format. Other molecular techniques such as whole or partial genome sequencing, microarrays, broad-range PCR, restriction fragment length polymorphisms, and molecular typing are also being used. However, for most small clinical laboratories, implementation of these advanced molecular techniques is not feasible owing to the high cost of instrumentation and reagents. If these tests are not available in-house, samples can be sent to national reference laboratories (eg, Mayo Medical Laboratories and Quest Diagnostics) for real-time PCR assays that can be completed in 1 day. It is anticipated that over time commercial real-time PCR tests and instrumentation will become more standardized and affordable, allowing individual laboratories to conduct tests locally, thus further reducing turnaround time. Although real-time PCR has been proved to expand our diagnostic capability, it must be stressed that such molecular methodology constitutes only an additional tool in the diagnosis of infectious diseases in emergency situations. Phenotypic methodologies (staining, cultures, biochemical tests, and serology) still play a critical role in identifying, confirming, and providing antibiotic susceptibility testing for many microbial pathogens. As multiplex assays become increasingly available, there will be even greater temptation for taking a “shotgun” approach to diagnostic testing. These new technologies will not substitute for a proper history and physical examination leading to a thoughtful differential diagnosis. None the less, these new molecular tests increase the capability of the diagnostician to rapidly identify the microbiological etiology of an infection. An added advantage of rapid diagnostic tests often not emphasized is the capability to rule out certain diagnoses for which unnecessary antimicrobial therapy may otherwise be instituted and/or continued.

Guidelines for the treatment of Lyme arthritis were published by the Infectious Diseases Society of America in 2006 and recommended oral doxycycline for initial therapy. We report here the case of a young girl treated with intravenous ceftriaxone who subsequently developed drug-induced autoimmune hemolytic anemia and renal failure. Her severe sequelae highlight the importance of antimicrobial stewardship. We review here the goals of antimicrobial stewardship and several strategies for achieving them. In addition, we briefly discuss the rare adverse drug event experienced by our patient.

Staphylococcus aureus is a major pathogen for immunologically intact humans and its pathogenesis is a model system for evasion of host defences. Antibodies and complement are essential elements of the humoral immune system for prevention and control of S. aureus infections. The specific hypothesis for the proposed research is that S. aureus modifies humoral host defences by cleaving IgG that has bound to the bacterial surface, thereby inhibiting opsonophagocytosis. S. aureus was coated with pooled, purified human IgG and assayed for the shedding of cleaved IgG fragments using ELISA and Western blot analysis. Surface-bound IgG was shed efficiently from S. aureus in the absence of host blood proteins. Broad-spectrum protease inhibitors prevented cleavage of IgG from the S. aureus surface, suggesting that staphylococcal proteases are responsible for IgG cleavage. Serine protease inhibitors and cysteine protease inhibitors decreased the cleavage of surface-bound IgG; however, a metalloprotease inhibitor had no effect. Using protease inhibitors to prevent the cleavage of surface-bound IgG increased the binding of complement C3 fragments on the surface of S. aureus, increased the association with human neutrophils and increased phagocytosis by human neutrophils.

Staphylococcus aureus is a significant human pathogen that causes skin-structure, invasive, and hospital-associated infections worldwide. The complement system is vital to innate defense against many bacterial infections. As shown with other pathogens, mechanisms for circumventing complement attack may include recruitment of the complement regulatory protein factor H (fH). In the present study, we show that S. aureus binds fH in a dose-dependent and time-dependent manner. Interestingly, this interaction does not require complement activation nor C3-fragment presence and occurs efficiently in the absence of other serum components suggesting a mechanism other than bridging between intermediary molecules. However, fH binding is greater when incubated with normal human serum compared to heat-inactivated serum, which suggests that complement activation may enhance fH binding. S. aureus-bound fH was found to inhibit the alternative pathway through disruption of the alternative pathway C3 convertase as shown by an increase in Bb release and a decrease in total C3-fragment deposition. Furthermore, S. aureus-bound fH retains cofactor activity for factor-I mediated cleavage of C3b. These studies show that the acquisition of fH to the S. aureus surface inhibits complement-mediated opsonization via disruption of the alternative pathway convertase; thus, we report an immune-evasion mechanism not previously described for S. aureus.

Previous work from our laboratories has demonstrated that purified, recombinant human astrovirus coat protein (HAstV CP) binds C1q and mannose-binding lectin (MBL) inhibiting activation of the classical and lectin pathways of complement, respectively. Analysis of the 787 amino acid CP molecule revealed that residues 79-139 share limited sequence homology with human neutrophil defensin-1 (HNP-1), a molecule previously demonstrated to bind C1q and MBL, inhibiting activation of the classical and lectin pathways of complement, respectively. A 30 amino acid peptide derived from this region of the CP molecule competitively inhibited the binding of wild-type CP to C1q. The parent peptide and various derivatives were subsequently assayed for C1q binding, inhibition of C1 and C4 activation as well as suppression of complement activation in hemolytic assays. The parent peptide and several derivatives inhibited complement activation in these functional assays to varying degrees. One peptide derivative in particular (E23A) displayed superior inhibition of complement activation in multiple assays of classical complement pathway activation. Further analysis revealed homology to a plant defensin allowing development of a proposed structural model for E23A. Based upon these findings, we hypothesize that further rationale optimization of E23A may result in a promising therapeutic inhibitor for the treatment of inflammatory and autoimmune diseases in which dysregulated activation of the classical and lectin pathways of complement contribute to pathogenesis.

The human complement system is important in the immunological control ofStaphylococcus aureusinfection. We showed previously thatS. aureussurface protein clumping factor A (ClfA), when expressed in recombinant form, bound complement control protein factor I and increased factor I cleavage of C3b to iC3b. In the present study, we show that, compared to the results for the wild type, when isogenic ClfA-deficientS. aureusmutants were incubated in serum, they bound less factor I, generated less iC3b on the bacterial surface, and bound fewer C3 fragments. It has been shown previously that two amino acids in ClfA (P336and Y338) are essential for fibrinogen binding. However,S. aureusexpressing ClfA(P336A Y338S) was less virulent than ClfA-deficient strains in animal models. This suggested that ClfA contributed toS. aureusvirulence by a mechanism different than fibrinogen binding. In the present study, we showed thatS. aureusexpressing ClfA(P336A Y338S) was more susceptible to complement-mediated phagocytosis than a ClfA-null mutant or the wild type. Unlike ClfA, ClfA(P336A Y338S) did not enhance factor I cleavage of C3b to iC3b and inhibited the cofactor function of factor H. Fibrinogen enhanced factor I binding to ClfA and theS. aureussurface. Twenty clinicalS. aureusstrains all expressed ClfA and bound factor I. High levels of factor I binding by clinical strains correlated with poor phagocytosis. In summary, our results suggest that the interaction of ClfA with factor I contributes toS. aureusvirulence by a complement-mediated mechanism.

Human astroviruses (HAstVs) constitute a family of non-enveloped, RNA viruses which cause infantile gastroenteritis. We have previously demonstrated that purified HAstV coat protein (CP), multiple copies of which compose the viral capsid, bind C1q resulting in inhibition of classical complement pathway activity. The objective of this study was to further analyze the mechanism by which CP inhibits C1 activation. CP inhibited C1 activation, preventing cleavage of C1s to its active form in the presence of heat-aggregated IgG, a potent classical pathway activator. CP also inhibited generation of the potent anaphylatoxin C5a. CP dose-dependently bound to C1q, the isolated globular heads and the collagen-like regions of the C1q molecule. When CP was added to C1, C1s dissociated from C1q suggesting that CP functionally displaces the protease tetramer (C1s-C1r-C1r-C1s). Given the structural and functional relatedness of C1q and MBL, we subsequently investigated the interactions between CP and MBL. CP bound to purified MBL and was able to inhibit mannan-mediated activation of the lectin pathway. Interestingly, CP did not bind to a variant of MBL that replaces a lysine residue (Lys55) critical for binding to MASP-2, a functional homolog of C1s. Finally, CP was shown to cross the species barrier to inhibit C3 activation and MAC formation in rat serum. These findings suggest CP inhibits C1 and MBL activation via a novel mechanism of interference with the normal interaction of the recognition molecule with its cognate serine proteases.