Mild Clinical Course of COVID-19 in 3 Patients Receiving Therapeutic Monoclonal Antibodies Targeting C5 Complement for Hematologic Disorders.

BACKGROUND Patients receiving immunosuppressive therapies might be more susceptible to COVID-19. Conversely, an exaggerated inflammatory response to the SARS-CoV-2 infection might be blunted by certain forms of immunosuppression, which could be protective. Indeed, there are data from animal models demonstrating that complement may be a part of the pathophysiology of coronavirus infections. There is also evidence from an autopsy series demonstrating complement deposition in the lungs of patients with COVID-19. This raises the question of whether patients on anti-complement therapy could be protected from COVID-19. CASE REPORT Case 1 is a 39-year-old woman with an approximately 20-year history of paroxysmal nocturnal hemoglobinuria (PNH), who had recently been switched from treatment with eculizumab to ravulizumab prior to SARS-CoV-2 infection. Case 2 is a 54-year-old woman with a cadaveric renal transplant for lupus nephritis, complicated by thrombotic microangiopathy, who was maintained on eculizumab, which she started several months before she developed the SARS-CoV-2 infection. Case 3 is a 60-year-old woman with a 14-year history of PNH, who had been treated with eculizumab since 2012, and was diagnosed with COVID-19 at the time of her scheduled infusion. All 3 patients had a relatively mild course of COVID-19. CONCLUSIONS We see no evidence of increased susceptibility to SARS-CoV-2 in these patients on anti-complement therapy, which might actually have accounted for the mild course of infection. The effect of anti-complement therapy on COVID-19 disease needs to be determined in clinical trials.

Absence of complement component 3 does not prevent classical pathway-mediated hemolysis.

Complement component 3 (C3) is emerging as a potential therapeutic target. We studied complement-mediated hemolysis using normal and C3-depleted human sera, wild-type (WT) and C3-deficient rat sera, and WT and C3 knockout rat models. In all of the in vitro and in vivo experiments, we found that the loss of C3 did not prevent classical pathway-mediated hemolysis, but it did almost abolish alternative pathway-mediated hemolysis. Experiments using preassembled classical pathway C3 convertases confirmed that C4b2a directly activated complement component 5 (C5), leading to membrane attack complex formation and hemolysis. Our results suggest that targeting C3 should effectively inhibit hemolysis and tissue damage mediated by the alternative pathway of complement activation, but this approach might have limited efficacy in treating classical pathway-mediated pathological conditions.

Aspergillus Niger peritonitis in a peritoneal dialysis patient treated with eculizumab.

The complement system plays a vital role in preventing life-threatening infections by ensuring optimal functioning of the host immune system. Its dysregulation has been implicated in causing glomerular, hematological, and transplant-related disorders. Eculizumab a novel monoclonal antibody against complement component C5 has emerged in the recent past as the standard of care offering an effective rescue and maintenance therapy against many of these disorders. Its use has been associated with increased risk of infections predominantly with encapsulated organisms. There is no data in the literature on its effects in end-stage kidney disease (ESKD) or dialysis patients. We describe here a very rare case of Aspergillus Niger peritonitis in an ESKD patient on peritoneal dialysis (PD) receiving maintenance eculizumab therapy for atypical hemolytic uremic syndrome. Given that murine models with the same defect as that induced by eculizumab is vulnerable to invasive Aspergillosis, it is suggested that the fungal peritonitis in this patient was the result of the eculizumab therapy.

Anticomplement monoterpenoid glucosides from the root bark of Paeonia suffruticosa.

Six new (1-6) and 19 known monoterpenoid glucosides were isolated from the root bark of Paeonia suffruticosa. The monoterpenoid glucosides 1, 2, 7, 10-19, and 22 exhibited anticomplement effects with CH50 and AP50 values ranging from 0.14 to 2.67 mM and 0.25 to 3.67 mM, respectively. In a mechanistic study, suffrupaeoniflorin A (1) interacted with C1q, C3, C5, and C9, while galloylpaeoniflorin (12) and galloyloxypaeoniflorin (19) acted on C1q, C3, and C5 components in the complement activation cascade.

Pharmacologic C5-complement suppression reduces blood loss during on-pump cardiac surgery.

BACKGROUND: Inflammation contributes to morbidity following on-pump cardiac surgery. Complement activation during cardiopulmonary bypass has been associated with the postoperative bleeding and tissue injury. This study examines the pharmacology and impact on blood loss of complement C5 suppression with pexelizumab in patients undergoing cardiac surgery with cardiopulmonary bypass. METHODS: Pexelizumab, a humanized monoclonal antibody single-chain fragment that binds to the human C5 complement component, was studied in a Phase II multicentered clinical trial. CABG (n = 800) and CABG with concomitant valve surgery (n = 114) patients were evaluated. Patients were randomized to either: pexelizumab bolus (2.0 mg/kg) + placebo infusion; pexelizumab bolus (2.0 mg/kg) + pexelizumab infusion (0.05 mg/kg/hour for 24 hours); or placebo bolus + placebo infusion. Pharmacology, chest tube drainage, and transfusion requirements were assessed. RESULTS: Mean maximum pexelizumab serum concentration was similar for bolus and bolus + infusion-treated patients. Complement-dependent serum hemolytic activity was completely suppressed within 1 hour following pexelizumab bolus, however, suppression was maintained for a longer duration in the bolus + infusion compared to the bolus-only treated patients. A reduction in chest tube drainage was observed for all pexelizumab-treated patients, although transfusion of blood products was similar across all study groups. CONCLUSION: Pexelizumab administration inhibits complement-dependent hemolytic activity and is associated with a reduction in postoperative chest tube drainage in patients undergoing cardiac surgery requiring cardiopulmonary bypass. Further, clinical studies are needed to assess the value of complement attenuation in this setting.

In vitro cleavage by asbestos fibers of the fifth component of human complement through free-radical generation and kallikrein activation.

Chrysotile and crocidolite fibers incubated in normal human plasma (NHP) generated from the C5 component of complement C5a-type fragments that stimulated polymorphonuclear leukocyte (PMN) chemotaxis. Absorption of NHP with antiserum against C5a totally abolished neutrophil chemotactic activity. Asbestos fibers also produced C5a small peptides in the presence of ethylene glycol bis(beta-aminoethyl ether) N,N,N'N'-tetraacetic acid (EGTA) but not ethylene diamine tetraacetic acid (EDTA). Activation of C5 was significantly inhibited when asbestos fibers were pretreated with iron chelators such as sodium dithionite (DTN), deferoxamine (DFX), or ascorbate (AA). Concentration-related inhibition of C5 activation was also observed when asbestos fibers were added concurrently to plasma in the presence of DFX, 1,3-dimethyl-2-thiourea (DMTU), a strong hydroxyl scavenger, or aprotinin (APR), a specific protease inhibitor. Further, chrysotile and crocidolite significantly increased plasma kallikrein activity. Data demonstrate that asbestos-induced C5 activation plays a role in inflammatory reactions characteristic of asbestosis through mechanisms involving iron ions, hydroxyl radicals, and oxidized C5-ike fragments. The ferrous ions present at the asbestos fiber surface trigger this activation and catalyze, via Fenton reaction, the production of hydroxyl radicals, which in turn convert native C5 to an oxidized C5-like form. This product is then cleaved by kallikrein, activated by the same asbestos fibers, yielding an oxidized C5a with the same functional properties as C5a.

Novel approaches in the treatment of lupus nephritis.

Lupus nephritis can be managed successfully in the majority of cases; most therapies, however, are associated with significant side-effects. Several new agents aiming at specific stages in the pathogenesis of lupus are in different phases of clinical trials. The central role of lymphocytes makes them targets of various therapeutic approaches. Lymphocyte depletion can be achieved by high-dose chemotherapy with or without bone marrow transplantation. Nucleoside analogs selectively deplete mononuclear cells; antibodies against T or B cell surface antigens target specific subsets of lymphocytes. Synchronized plasmapheresis has been used in an attempt to delete pathogenic lymphocyte clones activated by plasmapheresis. Treating patients with DNase or neutralizing pathogenic antibodies by administering specific binding peptides or inducing specific anti-idiotype antibodies may prevent immune complex formation and/or deposition. Blocking the complement cascade or some of the inflammatory mediators like thromboxane A2 may be efficacious even if immune complex deposition could not be prevented. Inducing antigen-specific tolerance or interfering with important interactions between T-lymphocytes and other cells by blocking CD40 ligand or decreasing the level of interleukin-10 are some of the other approaches currently under clinical investigation.

Complement activation by myeloperoxidase products released from stimulated human polymorphonuclear leukocytes.

Purified human myeloperoxidase (MPO) converted human C5 to an activated form, i.e. the C5 protein adopted a configuration expressing a binding site for C6; the resulting C56 complex then reacted with C7, C8 and C9 forming a hemolytic C5-9 complex. For the activation by myeloperoxidase chloride and hydrogen peroxide were essential. This indicates that the peroxidase acted through the generation of HOCl which had been shown earlier to oxidize and activate C5. Human polymorphonuclear leukocytes (PMN) were stimulated in vitro by incubation with opsonized zymosan; thereafter the supernatants were tested for C5 activating potency. Stimulated PMN release H2O2 and MPO that produces hypochlorite and secondarily various chloramines. As a trap for the labile hypochlorite generated excess taurine was added to the PMN suspensions during the incubation. Hypochlorite is then stoichiometrically converted to the relatively stable taurine chloramine. In order to rule out interfering activities of proteolytic enzymes released from the PMN and known to attack C5, the supernatants were ultracentrifuged, and the ultrafiltrates, containing only low molecular weight compounds, were used for the further studies. They contained taurine chloramine, estimated photometrically, and they activated C5 upon incubation, assayed functionally by reactive lysis. Azide, an inhibitor of myeloperoxidase, and catalase which destroys H2O2, essential for MPO-catalyzed oxidations, prevented the generation of C5 activating potency and of chloramines. Unstimulated PMN produced neither oxidants nor C5 activating potency. When taurine was omitted from the PMN suspensions during stimulations much less oxidant was found in the supernatants and less C5 activating potency. These findings indicate that the C5 activating agent was produced by stimulated PMN through MPO-generated hypochlorite, trapped as taurine chloramine. In the absence of added taurine the hypochlorite formed by MPO oxidized endogenous amines that also activated C5. Further studies suggested that among these was some monochloramine derived from endogenous ammonia. Activation of the terminal complement reaction sequence by MPO released from stimulated PMN may represent a third pathway to complement activation contributing to and reinforcing complement and PMN functions at the site of inflammation or tissue injury.

Heparin-coated circuit during cardiopulmonary bypass. A clinical study using closed circuit, centrifugal pump and reduced heparinization.

A prospective randomized study was performed to investigate the effect of surface coating with covalently endpoint-attached heparin (Carmeda Bio Active Surface) and reduced general heparinization on haematological indices and complement C5 activation. Care was taken to optimize the rheological design of the system using centrifugal pump and a closed system without venting or machine suction. Twenty patients scheduled for aortocoronary bypass grafting (EF > 0.5) participated in the study. Ten patients were randomized to be treated with heparin-coated equipment (CBAS) and reduced i.v. heparin (1.5 mg.kg-1) while 10 patients treated with identical but noncoated equipment and full heparinization (3 mg.kg-1) served in a Control group. A vacuum suction was used to collect the blood from the operating field and it was autotransfused at weaning from extracorporeal circulation (ECC). Blood samples were obtained from the venous (precircuit) and arterial (postcircuit) side. We used a new and very specific method for detection of C5a based on monoclonal antibodies. The concentration of C5a was low in both groups during the operation but a significant increase was seen on days 1 and 2. In the Control group there was an increase from 10.2 ng.ml-1 +/- 1.2 to 27.5 ng.ml-1 +/- 4.8 on day 2 and in the CBAS group from 10.7 ng.ml-1 +/- 1.2 to 35.6 ng.ml-1 +/- 11.6 on day 2 (NS between groups). The granulocytes and total leukocyte count increased at the end of ECC and was maintained at the elevated level throughout the study period. The amount of free haemoglobin was high in the autotransfused blood in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional assay of C5-activating and nonactivating cobra venom factor preparations in the mouse system.

This paper deals with a new, functional assay of cobra venom factor (CVF) preparations with or without C5-activating property. Existing methods lack sensitivity and use diluted human complement as target of inactivation. An adapted assay using diluted mouse serum as complement source was hampered by underestimation of C3 depletion by bystander lysis and an overvaluation of C5 consumption resulting from C3 inactivation in the reagent used. These disadvantages prompted us to develop the new assay which is based on the incubation of CVF preparations with undiluted mouse serum. After incubation, residual total C activity, as well as functional C3 and C5 are estimated by titration. The procedure permits the assessment of CVF activities with minimal interference from undesired processes. The conditions in the new assay approach the in vivo situation in mice by the use of undiluted serum from the same animal species.

Non-enzymic activation of the fifth component of human complement, by oxygen radicals. Some properties of the activation product, C5b-like C5.

Purified human C5 was converted non-enzymically to an activated form as defined by its ability to participate in reactive lysis. This conversion occurred following exposure to systems that generate oxygen radicals, namely addition of H2O2 in the presence of ascorbic acid and iron or the addition of xanthine oxidase, acetaldehyde and iron. The conversion of C5 to a functionally active species was iron-dependent and inhibited by hydroxyl radical scavengers such as DMSO. The findings suggest that OH. is the active oxygen species that converts C5. The conversion product of C5, termed C5(H2O2), is C5b-like due to its ability to bind C6 and cause reactive lysis. C5(H2O2) is much more stable than C5b obtained by complement convertases. Although C5(H2O2) has lost the binding site of native C5 for C3b it can be cleaved by complement-derived convertases; the cleavage is, however, less efficient than in the case of native C5. The resulting cleavage product, which is C5a-like, is chemotactic although C5(H2O2) is not chemotactic. C5(H2O2) serves as a better substrate for plasma kallikrein than native C5, resulting in the generation of a C5a-like chemotactic product. These data indicate that oxygen radicals can bring about a conformational change in C5, causing it to behave as a functionally activated molecule of the complement system. This may have implications for the role of complement and its activation in the inflammatory response.