ABSTRACT Rationale Hereditary angioedema (HAE) is a potentially life-threatening illness most commonly due to deficiency or dysfunction of C1-esterase inhibitor (C1-INH). While specific treatments are available to thwart acute exacerbations, they are extremely costly and some can be associated with rare but serious side effects. The heparins are long known to augment C1-INH activity and case reports / series have documented their efficacy in treating HAE. Objective to determine if unfractionated heparin and two low-molecular weight heparins (enoxaparin and nadroparin) can augment C1-INH activity ex vivo in the sera of patients with HAE and in an in vitro biochemical assay. Methods C1-INH activity in the absence or presence of the heparin formulations were analyzed by two different methods. To measure C1-INH activity ex vivo , a commercially available assay was utilized with patient sera, excess amounts of C1s, and a substrate of C1s which, upon cleavage by C1s, produces a chromogenic product. To determine biochemically the C1-INH activity in vitro , a pharmacologic grade C1-INH, recombinant C1s (C1s-CCP12SP), and a peptide substrate of C1s were employed. Microscale thermophoresis was used to determine whether C1-INH binds to heparin. Main results in patient sera, nadroparin was superior to enoxaparin and unfractionated heparin in augmenting C1-INH activity, followed by enoxaparin and then unfractionated heparin. In the in vitro biochemical assay, all three heparins augmented C1-INH-C1s binding linearly in a dose-dependent fashion. Microscale thermophoresis assay demonstrated that nadroparin binds to C1-INH, providing a mechanism by which heparin facilitates the interaction between C1-INH and the proteases known to produce bradykinin, the mediator of HAE. Conclusion low-molecular weight heparin augments C1-INH activity and should be studied as a potential treatment for acute HAE.

Abstract Background and Aims Complement mediated kidney diseases including C3 glomerulopathy (C3G), atypical uremic syndrome (aHUS), lupus nephritis (LN), and immunoglobulin A nephropathy (IgAN) are chronic inflammatory conditions characterized by dysregulated complement activation. Traditionally, complement activation is measured in plasma. However, patients with kidney disease often have an abundance of complement activation fragments in their urine—including soluble C5b-9 (sC5b-9), a marker of terminal pathway activation—highlighting the potential value of sC5b-9 measurement in urine for diagnosis and risk management in patients with C3G, aHUS, LN, IgAN, and other kidney diseases. Herein we evaluated the analytical performance of the Quidel MicroVue sC5b-9 Plus EIA for measurement of sC5b-9 in urine. Method The Quidel MicroVue sC5b-9 Plus EIA was evaluated using randomly collected healthy urine samples or healthy urine samples spiked with a known concentration of sC5b-9, as well as chronic kidney disease (CKD) urine samples. Results The analytical performance of the Quidel MicroVue sC5b-9 Plus EIA for measurement of sC5b-9 in urine met acceptability criteria for accuracy and precision. Assay accuracy was (100 ± 20%) and intra-assay precision as well as inter-assay precision was ≤ 11% coefficient of variability (CV). sC5b-9 was not detected in healthy urine samples (N = 40), but sC5b-9 was detected in a subset of urine samples from patients with CKD (N = 10). Positive linearity was observed over a wide range of concentrations. sC5b-9 measured in urine was not affected by hemoglobin or triglyceride, and was stable following three freeze-thaw cycles. Lastly, sC5b-9 measured in urine was stable when stored at −20°C and –80°C for 4 weeks. Long term stability testing at –80°C is underway. Conclusion Quidel MicroVue sC5b-9 Plus EIA is an accurate, reliable assay for detection of sC5b-9 in urine samples.

Introduction A strong epidemiologic link exists between cigarette smoke (CS) exposure and susceptibility to tuberculosis (TB). Macrophage and murine studies showed that CS and nicotine impair host-protective immune cells against Mycobacterium tuberculosis (MTB) infection. While CS and nicotine may activate T regulatory cells (Tregs), little is known about how CS may affect these immunosuppressive cells with MTB infection. Methods We investigated whether CS-exposed Tregs could exacerbate MTB infection in co-culture with human macrophages and in recipient mice that underwent adoptive transfer of Tregs from donor CS-exposed mice. Results We found that exposure of primary human Tregs to CS extract impaired the ability of unexposed human macrophages to control an MTB infection by inhibiting phagosome-lysosome fusion and autophagosome formation. Neutralizing CTLA-4 on the CS extract-exposed Tregs abrogated the impaired control of MTB infection in the macrophage and Treg co-cultures. In Foxp3 + GFP + DTR + (Thy1.2) mice depleted of endogenous Tregs, adoptive transfer of Tregs from donor CS-exposed B6.PL(Thy1.1) mice with subsequent MTB infection of the Thy1.2 mice resulted in a greater burden of MTB in the lungs and spleens than those that received Tregs from air-exposed mice. Mice that received Tregs from donor CS-exposed mice and infected with MTB had modest but significantly reduced numbers of interleukin-12-positive dendritic cells and interferon-gamma-positive CD4 + T cells in the lungs, and an increased number of total programmed cell death protein-1 (PD-1) positive CD4 + T cells in both the lungs and spleens. Discussion Previous studies demonstrated that CS impairs macrophages and host-protective T effector cells in controlling MTB infection. We now show that CS-exposed Tregs can also impair control of MTB in co-culture with macrophages and in a murine model.

While the mechanisms underlying the functions of the complement system in the central nervous system (CNS) and systemically, namely opsonization, chemotaxis, membrane lysis, and regulation of inflammation are the same, the plethora of functions that complement orchestrates in the central nervous system (CNS) is complex. Strictly controlled expression of complement effector molecules, regulators and receptors across the gamut of life stages (embryogenesis, development and maturation, aging and disease) dictate fascinating contributions for this ancient system. Furthermore, it is becoming apparent that complement functions differ widely across distinct brain regions. This review provides a comprehensive overview of the newly identified roles for complement in the brain, including its roles in CNS development and function, during aging and in the processes of neurodegeneration. The diversity and selectively of beneficial and detrimental activities of complement, while challenging, should lead to precision targeting of specific components to provide disease modifying treatments for devastating psychiatric and neurodegenerative disorders that are still without effective treatment.

P< 0.01); conversely, IRF recurrence rate did not impact long-term kidney outcome (p¼ 0.24).Seven patients received a diagnosis of cancer during long-term follow up (median 3.5 years, range 3-9).Cancer risk did not seem to be attributable to a specific regimen of immunosuppressive therapy.At the last observation, 29/30 patients were alive as only 1 patient died of cancer after 3-y of follow-up.CONCLUSIONS: In most patients, different therapeutic approaches reduced the fibrotic mass and maintained stable kidney function in the long-term follow-up.There was a strong association between IRF and cancer within 10 years of IRF diagnosis, suggesting that cancer screening should be performed regularly.Because of the cancer risk, other suitable therapeutic strategies should be investigated.

Eculizumab is a monoclonal anti-C5 antibody used in the treatment of atypical hemolytic uremic syndrome (aHUS).We monitored complement inhibition in 16 eculizumab-treated patients suffering from HUS or transplant rejection (not aHUS patients). Blood samples were obtained one to four weeks after the last eculizumab injection.We observed that eculizumab efficiently blocked the terminal pathway (TP) through classical pathway (CP) activation measured by kinetic hemolytic assay (HA) (< 10%) but incompletely blocked the TP through alternative pathway (AP) activation measured by rabbit (APH50 > 23%) or chicken erythrocytes HA (AP100 > 15%). Conversely, functional ELISA revealed a complete blockade of TP through AP activation in all patients (< 10%). C5a and sC5b9 levels were not correlated with residual APH50 or AP100.Similar results were obtained after in vitro addition of increasing amounts of eculizumab to a control serum (in vitro APH50 > 60% and AP100 > 20%). We also showed that ELISA was less sensitive than HA.

To establish a simple, stable method for measuring total complement activity in plasma.Total complement activity (TCA) of plasma was measured using a classical method (CH50 method) and a self-hemolysis colorimetric method (new method). Human red blood cells (RBCs) were used as a hemolysis indicator system and rabbit–anti-human RBC antibody instead of the traditional hemolysin (rabbit–anti-sheep RBC antibody) in the new method. TCA in intensive care unit (ICU) patients and healthy individuals was measured using the new method.TCA using the new method and the CH50 method were significantly correlated. TCA in ICU patients was significantly lower than that in healthy individuals.The self-hemolysis colorimetric method is a simple and stable method, and has potential value in clinical applications.

Atlantic Mackerel (Scombrus scombrus) is a novel species in Iceland, caught during the heavy feeding period. The feeding has an impact on quality and stability of the products, which requires optimization of storage and processing conditions. Physicochemical changes of brined and un-brined mackerel were analysed during frozen storage (6, 9, 12 months) at −18 °C vs. −25 °C with the aim of investigating the suitability of using well-fed frozen mackerel as raw material for canned and hot-smoked products. Heat treatments to a core temperature of 90 °C (representing canning) and 75 °C (representing hot-smoking) were applied. Prolonged frozen storage showed negative effects on the raw material prior to heat processing due to an increased level of lipid oxidation, where fish stored at −18 °C was of significantly poorer quality than fish stored at −25 °C. Moreover, the results indicated that heat treatment resulting in a core temperature of 75 °C showed higher water content, liquid holding capacity, heating yield as well as lower maximum shear force of texture compared to mackerel heated to a core temperature of 90 °C. Overall, analyses indicated that the fatty summer mackerel was well suitable for production of canned and hot-smoked products.

Eculizumab is a monoclonal anti-C5 antibody used in the treatment of atypical hemolytic uremic syndrome (aHUS).We monitored complement inhibition in 16 eculizumab-treated patients suffering from HUS or transplant rejection (not aHUS patients). Blood samples were obtained one to four weeks after the last eculizumab injection.We observed that eculizumab efficiently blocked the terminal pathway (TP) through classical pathway (CP) activation measured by kinetic hemolytic assay (HA) (< 10%) but incompletely blocked the TP through alternative pathway (AP) activation measured by rabbit (APH50 > 23%) or chicken erythrocytes HA (AP100 > 15%). Conversely, functional ELISA revealed a complete blockade of TP through AP activation in all patients (< 10%). C5a and sC5b9 levels were not correlated with residual APH50 or AP100.Similar results were obtained after in vitro addition of increasing amounts of eculizumab to a control serum (in vitro APH50 > 60% and AP100 > 20%). We also showed that ELISA was less sensitive than HA.

The failure to clear apoptotic cells is linked to defects in development and autoimmunity. Complement component C1q is required for efficient engulfment of apoptotic cells (efferocytosis), and C1q deficiency leads to the development of lupus. We recently identified a novel molecular mechanism for C1q-dependent efferocytosis in murine macrophages. C1q elicited the expression of Mer tyrosine kinase (Mer), a receptor that regulates efficient efferocytosis and prevention of autoimmunity. To characterize the C1q-dependent signal transduction mechanism, pathway analysis of the transcriptome from C1q-activated macrophages was performed, and it identified the adiponectin signaling pathway as significantly upregulated with C1q. Adiponectin is structurally homologous to C1q and regulates cellular metabolism via downstream activation of 5′adenosine monophosphate-activated protein kinase (AMPK). Macrophage stimulation with C1q resulted in the activation of AMPK, and silencing of AMPK expression using siRNA-inhibited C1q-dependent efferocytosis. Adiponectin signaling also stimulates activation of nuclear receptors, and inhibition of the nuclear receptor retinoid X receptor abrogated C1q-dependent Mer expression and efferocytosis. Furthermore, adiponectin elicited Mer expression and Mer-dependent efferocytosis in macrophages similar to cells stimulated with C1q. Collectively, our results suggest that C1q and adiponectin share a common signal transduction cascade to promote clearance of apoptotic cells, and identify a novel molecular pathway required for efficient efferocytosis.

Abstract Failure to efficiently clear apoptotic cells (efferocytosis) is associated with autoimmunity. Complement component C1q is required for efferocytosis, and deficiency in C1q leads to development of autoimmunity. We recently identified a novel molecular mechanism for C1q-dependent efferocytosis in mouse macrophages. We found that C1q elicited the expression and function of Mer tyrosine kinase and the Mer ligand, Ga6: a receptor-ligand pair that mediates efferocytosis. To define the signal transduction pathway downstream of C1q, pathway analysis was performed using the transcriptome from C1q-treated macrophages. This analysis revealed that the adiponectin signaling pathway was significantly upregulated with C1q. Adiponectin is a well characterized adipokine with critical roles in glucose and fatty acid metabolism, and it is structurally homologous to C1q. Similar to C1q, adiponectin triggered expression of Mer that correlated with enhanced engulfment of apoptotic cells, and a soluble Mer-Fc fusion protein inhibited adiponectin-dependent efferocytosis. Furthermore, C1q triggered activation of AMP kinase, a central regulator of adiponectin signaling and cellular metabolism. Inhibition of AMPK signaling resulted in inability to upregulate C1q-dependent Mer expression in macrophages. Our results suggest that C1q and adiponectin activate macrophages via a common signaling pathway, and this pathway may contribute to the molecular crosstalk between inflammation and metabolism.

Despite evidence that hydrogen sulfide (H2S) is an important signaling molecule involved in oxygen sensing, it has been technically difficult to measure H2S production, metabolism or distribution at the cellular and sub‐cellular level in living tissue. Here, we examine the potential for cellular H2S measurement using 150–200 nm diameter silicate nanoparticles (Photonic Explorers for Bioanalysis with Biologically Localized Embedding; PEBBLEs) and demonstrate its applicability. PEBBLES were synthesized with a hydrophilic core containing fluorescein mercuric acetate (FMA), an analyte‐sensitive dye that becomes irreversibly quenched when bound to sulfur moieties, and a hydrophobic shell that is only permeable toH2S. PEBBLES can be delivered into cells via a gene gun, or as in the present experiments, ingested by mouse bone marrow‐derived macrophages (BMDM+P). BMDM+P were exposed to a range of H2S concentrations (10 nM‐10 mM H2S as Na2S) or exposed to hypoxia for 1 h and examined by flow cytometry and confocal microscopy. Increasing H2S concentration dose‐dependently quenched FMA and exposure to 1 nM H2S was readily resolved. Furthermore, hypoxia increased intracellular H2S production. This is the first demonstration of intracellular H2S production in any tissue and clearly shows that during hypoxia intracellular H2S concentration increases. This supports our hypothesis that oxygen sensing is the balance between intrinsic intracellular H2S production and its O2‐dependent metabolism and that this is the key couple in oxygen sensing. It also affirms the validity of this method in H2S research. Support: NSF IOS 1051627

Failure to efficiently clear apoptotic cells is linked to defects in development and autoimmunity. Complement component C1q is required for efficient clearance of apoptotic cells, and C1q deficiency is the strongest known susceptibility factor for autoimmunity associated with lupus. Despite the importance of C1q in apoptotic cell clearance, the mechanism by which C1q enhances apoptotic cell clearance is poorly understood. Here we describe a novel C1q-dependent engulfment mechanism for apoptotic cells. We found that macrophage activation with C1q resulted in cycloheximide-sensitive enhanced engulfment, indicating a requirement for de novo protein synthesis. To investigate the cycloheximide-sensitive pathway, C1q-elicited macrophage transcripts were identified by microarray. C1q triggered the expression of Mer tyrosine kinase and Gas 6: a receptor-ligand pair that mediates clearance of apoptotic cells. This novel pathway is specific to C1q since MBL, a related collectin, failed to upregulate Mer expression and function. Soluble Mer-Fc fusion protein inhibited C1q-dependent engulfment of apoptotic cells. In addition, Mer-/- macrophages failed to respond to C1q with enhanced engulfment of apoptotic cells, indicating a requirement for Mer. Our results suggest that C1q elicits a macrophage phenotype specifically tailored for apoptotic cell clearance, and these data are consistent with the established requirement for C1q in prevention of autoimmunity.

While it has been known for some time that CD93 regulates several processes involved in innate immunity and inflammation including phagocytosis and adhesion, the function of CD93 in disease progression is only now being elucidated. Recent in vivo studies in mice, and genome wide studies in mice and humans, have provided clues about its molecular function. Following a comprehensive review of CD93 expression patterns, this review will focus on recent findings over the last three years that address the putative function of CD93 in inflammation and innate immunity.

Complement component C1q is a member of a family of soluble proteins called defense collagens, which are important in host defense and apoptotic cell clearance. Failure to efficiently clear apoptotic cells in the absence of C1q is associated with autoimmunity. Here, we review the literature describing a central role for C1q in the enhancement of phagocyte function and focus specifically on C1q in apoptotic cell clearance. In addition, we highlight our recent findings demonstrating that C1q elicits a macrophage phenotype that is tailored specifically for clearance of apoptotic cells.