Association Between Updated Guideline-Based Palivizumab Administration and Hospitalizations for Respiratory Syncytial Virus Infections.

BACKGROUND: Since its introduction, palivizumab has been used to prevent respiratory syncytial virus (RSV) infection in high-risk populations. Recommendations for palivizumab administration changed in 2014. We examined whether adherence to 2014 palivizumab guidelines affected RSV hospitalization rates. METHODS: This was a retrospective sequential period analysis comparing the incidence of RSV hospitalization in patients younger than 2 years of age before and after implementation of 2014 palivizumab use criteria. Hospitalization data were prospectively collected through age-based surveillance for the post-2014 guideline period (November 1, 2014 to April 1, 2015 RSV season). Comparative data were collected retrospectively for hospitalizations during the pre-2014 guideline period of 2 previous RSV seasons (November 1, 2012 to April 1, 2013 and November 1, 2013 to April 1, 2014). The primary outcome was RSV hospitalization rate, and number of palivizumab doses administered was analyzed as a secondary outcome. RESULTS: During the study period, 194 RSV hospitalizations occurred. The rate of RSV hospitalization was 5.37 per 1000 children <24 months in the pre-2014 guideline period versus 5.78 per 1000 children <24 months in the post-2014 guideline period (difference of +0.4, 95% confidence interval: -1.2 to +2, P = 0.622). During the pre-2014 guideline period, 21.7 doses per 1000 children <24 months of palivizumab were administered, which decreased to 10.3 doses per 1000 children <24 months in the post-2014 guideline period, yielding a reduction of 11.4 doses per 1000 children <24 months (95% confidence interval: 14.3-8.4, P < 0.001). CONCLUSIONS: The implementation of 2014 palivizumab use criteria was not associated with an increased incidence of RSV hospitalization for children younger than 2 years of age but was associated with significantly less use of palivizumab.

A 1-Year-Old with Mycobacterium tuberculosis Endocarditis with Mass Spectrometry Analysis of Cardiac Vegetation Composition.

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.

Glucose-based dialysis fluids inhibit innate defense against Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus peritonitis is a serious complication of Chronic Peritoneal Dialysis (CPD) and associated with a higher risk for severe and recurrent infections compared with other bacteria. We have previously shown that complement-mediated effectors essential for optimal opsonophagocytosis of S. aureus are inhibited by high glucose concentrations. Since most commonly used peritoneal dialysis (PD) fluids are glucose-based, we hypothesized that glucose-based PD fluids likely inhibit complement host defenses against S. aureus. METHODS: Commercially available PD fluids were tested: glucose-based (Dianeal), Dianeal supplemented with amino acids, icodextrin-based (Extraneal) and amino acid-based (Nutrineal). Control PD fluid was generated to simulate Dianeal excluding the glucose. Three commercially available glucose concentrations were tested: Dianeal 1.5% (15 gm/1000 ml), Dianeal 2.5% (25 gm/1000 ml) and Dianeal 4.25% (42.5 gm/1000 ml). Complement effectors against S. aureus were analyzed including opsonization with C3-fragments, anaphylatoxin generation, and phagocytosis efficiency. We also evaluated clinical strains, including MRSA strains, and specific complement activation pathways. RESULTS: Glucose-based PD fluids inhibited complement opsonization of S. aureus (≥7-fold reduction) and inhibited S. aureus-induced generation of anaphylatoxins C3a and C5a (>10-fold reduction) compared to non-glucose based PD fluids. Dianeal 1.5%, 2.5% and 4.25%, all similarly inhibited C3-mediated opsonization. Glucose-based PD fluids showed a ≥4-fold reduction in opsonization of clinical strains of S.aureus, including MRSA strains. Decreased opsonization of S.aureus in the glucose-based PD fluid compared with non-glucose based fluids correlated with decreased phagocytosis by neutrophils. CONCLUSION: Complement-mediated opsonophagocytosis of S. aureus and anaphylatoxin generation were severely inhibited in glucose-based PD fluids compared with non-glucose-based PD fluids. By inhibiting complement host defenses, glucose-based PD fluids may increase the risk of and severity of S. aureus peritonitis for CPD patients using these fluids.

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.

Clinical hypothermia temperatures increase complement activation and cell destruction via the classical pathway.

BACKGROUND: Therapeutic hypothermia is a treatment modality that is increasingly used to improve clinical neurological outcomes for ischemia-reperfusion injury-mediated diseases. Antibody-initiated classical complement pathway activation has been shown to contribute to ischemia-reperfusion injury in multiple disease processes. However, how therapeutic hypothermia affects complement activation is unknown. Our goal was to measure the independent effect of temperature on complement activation, and more specifically, examine the relationship between clinical hypothermia temperatures (31-33°C), and complement activation. METHODS: Antibody-sensitized erythrocytes were used to assay complement activation at temperatures ranging from 0-41°C. Individual complement pathway components were assayed by ELISA, Western blot, and quantitative dot blot. Peptide Inhibitor of complement C1 (PIC1) was used to specifically inhibit activation of C1. RESULTS: Antibody-initiated complement activation resulting in eukaryotic cell lysis was increased by 2-fold at 31°C compared with 37°C. Antibody-initiated complement activation in human serum increased as temperature decreased from 37°C until dramatically decreasing at 13°C. Quantitation of individual complement components showed significantly increased activation of C4, C3, and C5 at clinical hypothermia temperatures. In contrast, C1s activation by heat-aggregated IgG decreased at therapeutic hypothermia temperatures consistent with decreased enzymatic activity at lower temperatures. However, C1q binding to antibody-coated erythrocytes increased at lower temperatures, suggesting that increased classical complement pathway activation is mediated by increased C1 binding at therapeutic hypothermia temperatures. PIC1 inhibited hypothermia-enhanced complement-mediated cell lysis at 31°C by up to 60% (P = 0.001) in a dose dependent manner. CONCLUSIONS: In summary, therapeutic hypothermia temperatures increased antibody-initiated complement activation and eukaryotic cell destruction suggesting that the benefits of therapeutic hypothermia may be mediated via other mechanisms. Antibody-initiated complement activation has been shown to contribute to ischemia-reperfusion injury in several animal models, suggesting that for diseases with this mechanism hypothermia-enhanced complement activation may partially attenuate the benefits of therapeutic hypothermia.

Complement inhibition significantly decreases red blood cell lysis in a rat model of acute intravascular hemolysis.

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.

Hyperglycemia inhibits complement-mediated immunological control of S. aureus in a rat model of peritonitis.

Hyperglycemia from diabetes is associated with increased risk of infection from S. aureus and increased severity of illness. Previous work in our laboratory demonstrated that elevated glucose (>6 mM) dramatically inhibited S. aureus-initiated complement-mediated immune effectors. Here we report in vivo studies evaluating the extent to which a hyperglycemic environment alters complement-mediated control of S. aureus infection in a rat peritonitis model. Rats were treated with streptozocin to induce diabetes or sham-treated and then inoculated i.p. with S. aureus. Rats were euthanized and had peritoneal lavage at 2 or 24 hours after infection to evaluate early and late complement-mediated effects. Hyperglycemia decreased the influx of IgG and complement components into the peritoneum in response to S. aureus infection and decreased anaphylatoxin generation. Hyperglycemia decreased C4-fragment and C3-fragment opsonization of S. aureus recovered in peritoneal fluids, compared with euglycemic or insulin-rescued rats. Hyperglycemic rats showed decreased phagocytosis efficiency compared with euglycemic rats, which correlated inversely with bacterial survival. These results suggest that hyperglycemia inhibited humoral effector recruitment, anaphylatoxin generation, and complement-mediated opsonization of S. aureus, suggesting that hyperglycemic inhibition of complement effectors may contribute to the increased risk and severity of S. aureus infections in diabetic patients.

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

Previous 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.

An 11-year-old male with refractory osteomyelitis.

We present a case of empirical treatment failure for chronic osteomyelitis in a previously healthy 11-year-old male involving the distal phalanx of the right first digit. After initial debridement, empiric antibiotics were started for presumed Staphylococcus aureus infection. Operative bacterial cultures yielded no growth. Despite three weeks of antistaphylococcal antibiotics the patient's symptoms worsened and the destruction of bone progressed. A repeat plain X-ray revealed a new lesion in the proximal phalanx of the right second digit. The recognition of multifocal osteomyelitis led to reexamination of bone tissue specimens using special stains which demonstrated rare broad-based budding yeast. Fungal cultures eventually grew Blastomyces dermatitidis. Treatment with amphotericin B led to rapid clinical improvement. This case illustrates that clinicians must remain vigilant for warning signs that empiric treatment may be failing for presumptive Staphylococcus aureus, provoking reconsideration of the differential diagnosis and an intensification of efforts to evaluate for alternative etiologies.