Anti-C2 Antibody ARGX-117 Inhibits Complement in a Disease Model for Multifocal Motor Neuropathy

Background and ObjectivesTo determine the role of complement in the disease pathology of multifocal motor neuropathy (MMN), we investigated complement activation, and inhibition, on binding of MMN patient-derived immunoglobulin M (IgM) antibodies in an induced pluripotent stem cell (iPSC)-derived motor neuron (MN) model for MMN.MethodsiPSC-derived MNs were characterized for the expression of complement receptors and membrane-bound regulators, for the binding of circulating IgM anti-GM1 from patients with MMN, and for subsequent fixation of C4 and C3 on incubation with fresh serum. The potency of ARGX-117, a novel inhibitory monoclonal antibody targeting C2, to inhibit fixation of complement was assessed.ResultsiPSC-derived MNs moderately express the complement regulatory proteins CD46 and CD55 and strongly expressed CD59. Furthermore, MNs express C3aR, C5aR, and complement receptor 1. IgM anti-GM1 antibodies in serum from patients with MMN bind to MNs and induce C3 and C4 fixation on incubation with fresh serum. ARGX-117 inhibits complement activation downstream of C4 induced by patient-derived anti-GM1 antibodies bound to MNs.DiscussionBinding of IgM antibodies from patients with MMN to iPSC-derived MNs induces complement activation. By expressing complement regulatory proteins, particularly CD59, MNs are protected against complement-mediated lysis. Yet, because of expressing C3aR, the function of these cells may be affected by complement activation upstream of membrane attack complex formation. ARGX-117 inhibits complement activation upstream of C3 in this disease model for MMN and therefore represents an intervention strategy to prevent harmful effects of complement in MMN.

Case Report: Drug-Induced Immune Haemolytic Anaemia Caused by Cefoperazone-Tazobactam/ Sulbactam Combination Therapy

There has previously been a report of a patient developing haemolytic anaemia following exposure to cefoperazone. Another case has been reported involving the detection of cefoperazone-dependent antibodies in the absence of immune haemolytic anaemia. To date, no serological evidence has been reported to suggest that cefoperazone can lead to drug-induced immune haemolytic anaemia (DIIHA). This report aims to fill these gaps in knowledge by describing a case of DIIHA caused by cefoperazone-dependent antibodies. A 59-year-old man developed fatal haemolytic anaemia while receiving cefoperazone-tazobactam or cefoperazone-sulbactam for the treatment of a lung infection that occurred after craniocerebral surgery. This eventually led to renal function impairment. Prior to the discontinuation of cefoperazone treatment, the patient showed strong positive (4+) results for both anti-IgG and anti-C3d direct antiglobulin test (DAT), while cefoperazone-dependent IgM and IgG antibodies were detected. The patient's plasma and O-type RBCs were incubated with tazobactam or sulbactam solution at 37°C for 3 h, the results of DAT for anti-IgG and anti-C3d were both positive. Forty-three days after the discontinuation of cefoperazone, the results of DAT for anti-IgG and anti-C3d were negative. Meanwhile incubation of the patient's fresh serum and his own RBCs with cefoperazone at 37°C, gave rise to mild haemolysis, and the results of DAT for both anti-IgG and anti-C3d were positive. It is suggested that cefoperazone-dependent antibodies can activate complement, and the non-immunologic protein adsorption effect of tazobactam or sulbactam can enhance IgG and complement binding to RBCs. This may promote the formation of immunocomplexes and complement activation, thereby aggravating haemolysis.

Ficolin-2 inhibitors are present in sera after prolonged storage at −80 °C

Ficolins can activate the lectin pathway of the complement system that provides innate immune protection against pathogens, marks host cellular debris for clearance, and promotes inflammation. Baseline inflammation increases with aging in a phenomenon known as “inflammaging.” Although IL-6 and C-reactive protein are known to increase with age, contributions of many complement factors, including ficolins, to inflammaging have been little studied.Ficolin-2 is abundant in human serum and can recognize many target structures; therefore, ficolin-2 has potential to contribute to inflammaging. We hypothesized that inflammaging would alter ficolin-2 levels among older adults and examined 360 archived sera collected from older individuals. We found that these sera had apparently reduced ficolin-2 levels and that 84.2% of archived sera exhibited ficolin-2 inhibitors, which suppressed apparent amounts of ficolin-2 detected by enzyme-linked immunosorbent assay. Fresh serum samples were obtained from donors whose archived sera showed inhibitors, but the fresh sera did not have ficolin-2 inhibitors. Ficolin-2 inhibitors were present in other long-stored sera from younger persons. Furthermore, noninhibiting samples and fresh sera from older adults had apparently normal amounts of ficolin-2. Thus, ficolin-2 inhibitors may arise as an artifact of long-term storage of serum at −80 °C.

USE OF MUELLER-HINTON BROTH AND AGAR IN THE GERM TUBE TEST

Candida albicans is often isolated from clinical samples, thus its presumptive differentiation from other species of the same genus can be based on its ability to form the germ tube in human serum. Nevertheless, there are two other species that share this characteristic: C. dubliniensis and C. africana. The aim of this study was to compare four different substrates to perform the germ tube (GT) test. The Candida spp. isolates were identified using a manual system (135 C. albicans, 24 C. tropicalis and one C. dubliniensis). The germ tube test was performed with fresh, previously frozen serum and Mueller-Hinton (MH) broth and agar. GT was observed in 96% (130/136) of the isolates through the fresh serum technique, 94% (128/136) through previously frozen serum, 92% (125/136) in MH agar, and 90% (122/136) in MH broth. The sensitivity of each test was higher than 90%, with 100% specificity. Both the MH agar and broth were able to identify the true positives, and false positives were not found. However, some C. albicans isolates were not identified. MH agar and broth may be used in laboratory for the rapid presumptive identification of C. albicans, as an alternative method for germ tube test.