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•Patient with myasthenia gravis presented severe bulbar symptoms.•Eculizumab (ECZ) decreased serum CH50 level and improved the symptoms.•When ECZ was postponed, serum CH50 increased and symptoms worsened.•Symptoms severity correlated temporally with serum CH50 during ECZ therapy.•Serum CH50 may be a marker of ECZ-induced complement blockade in our case. The correlation between serum 50 % hemolytic complement (CH50) level and myasthenic symptom severity has not been known in patients with anti-acetylcholine receptor (anti-AChR)-positive myasthenia gravis (MG) during eculizumab treatment. A patient with anti-AChR-positive MG showed severe bulbar symptoms. Eculizumab administration decreased CH50 level and improved the symptoms. However, shortly after the second administration of eculizumab was postponed due to the development of pneumonia, his serum CH50 level returned almost to the level it was at before the initiation of eculizumab therapy and myasthenic symptoms worsened. Even after his pneumonia was completely cleared in response to an antibiotic, the severe myasthenic symptoms persisted. After eculizumab was resumed, serum CH50 level was reduced to below the limit of detection within 24 h, and the symptom steadily improved. His symptom severity was correlated temporally with serum CH50 level during eculizumab therapy. Our case suggests that serum CH50 level may be a marker of eculizumab-induced complement blockade and an indicator of a potential worsening of myasthenic symptoms during eculizumab treatment.

A study was performed to investigate the reproducibility of haemagglutinin-inhibition (HI) and virus neutralising (VN) assays for detection of anti-influenza antibody. Participants in 11 laboratories from eight countries measured antibody to egg-grown A/Japan/434/2003, cell-grown A/Japan/434/2003 and A/Panama/2007/99 (H3N2) viruses in 18 human and two post-infection ferret sera. There was significant intra-laboratory assay variability for VN compared to HI. For replicate assays within laboratories, 14/410 (3%) and 130/631 (21%) titres differed by >2-fold ( p < 0.0001), and 0/410 (0%) and 35/631 (6%) titres differed by >5-fold ( p < 0.0001) by HI and VN, respectively. Although both assays showed inter-laboratory variation, VN assays were significantly more variable than HI. Median geometric coefficients of variation (GCV) for VN assays with each virus were 256%, 323% and 359% compared to 138%, 155% and 261% with HI. A serum standard improved inter-laboratory agreement and reduced median GCVs. This study raises concern about comparability of serology results from H5N1 vaccine trials and it is proposed that an International Standard for influenza H5N1 antibody is developed.

Background The aim of the present study was to develop a haemolytic assay for the study of the complement system in dairy goats ( Capra aegagrus hircus ) and to characterize the major goat complement system proteins. Results The commonly used sheep erythrocyte sensitized with rabbit antibodies were not sensitive to lysis by goat serum, but the combination of human red blood cells (RBC) plus rabbit antibodies was the best option found for goat complement assay. A buffer based on HEPES instead of the classical veronal (barbitone) was developed. Three proteins were isolated: factor H, C1q and C3 and these were compared with the corresponding human proteins. A novel affinity chromatography technique was developed for isolation of factor H. Conclusions Human RBC plus rabbit antibodies were a suitable option for haemolytic assays. The isolated proteins are similar to the human counterparts.

For thrombotic microangiopathies (TMAs), the diagnosis of atypical hemolytic uremic syndrome (aHUS) is made by ruling out Shiga toxin-producing Escherichia coli (STEC)-associated HUS and ADAMTS13 activity-deficient thrombotic thrombocytopenic purpura (TTP), often using the exclusion criteria for secondary TMAs. Nowadays, assays for ADAMTS13 activity and evaluation for STEC infection can be performed within a few hours. However, a confident diagnosis of aHUS often requires comprehensive gene analysis of the alternative complement activation pathway, which usually takes at least several weeks. However, predisposing genetic abnormalities are only identified in approximately 70% of aHUS. To facilitate the diagnosis of complement-mediated aHUS, we describe a quantitative hemolytic assay using sheep red blood cells (RBCs) and human citrated plasma, spiked with or without a novel inhibitory anti-complement factor H (CFH) monoclonal antibody. Among 45 aHUS patients in Japan, 24% (11/45) had moderate-to-severe (≥50%) hemolysis, whereas the remaining 76% (34/45) patients had mild or no hemolysis (<50%). The former group is largely attributed to CFH-related abnormalities, and the latter group has C3-p.I1157T mutations (16/34), which were identified by restriction fragment length polymorphism (RFLP) analysis. Thus, a quantitative hemolytic assay coupled with RFLP analysis enabled the early diagnosis of complement-mediated aHUS in 60% (27/45) of patients in Japan within a week of presentation. We hypothesize that this novel quantitative hemolytic assay would be more useful in a Caucasian population, who may have a higher proportion of CFH mutations than Japanese patients.

C3 nephritic factors are autoantibodies that prolong the half-life or prevent regulation of the alternative pathway C3 convertase, resulting in uncontrolled complement activation. They are strongly associated with renal disease but their role in pathogenesis remains controversial. Here we optimized and compared a panel of assays to identify and interrogate nephritic factor activities. Of 101 patients with histologic or clinically evident disease, 48 were positive in some or all assays. In the presence of properdin, binding of autoantibody was detected in 39 samples and convertase stabilization was detected in 36. Forty-two of 48 nephritic factors tested prevented convertase decay by factor H, and most of these by decay accelerating factor (28) and complement receptor 1 (34). Representative properdin-independent nephritic factors had no effect on C5 cleavage and terminal pathway activity, while properdin-dependent nephritic factors enhanced activity. Biacore analysis of four purified IgG samples confirmed resistance to decay and showed that properdin-independent nephritic factors increased convertase half-life over 50-fold, whereas properdin-dependent nephritic factors increased the half-life 10- to 20-fold and also increased activity of the C3 convertase up to 10-fold. Thus, our study provides a rational approach to detect and characterize nephritic factors in patients.

In this study, blood cell counts and serum C3, C4, and CH50 values at baseline and after more than 6-month drug use were measured to elucidate changes in blood cells and complements during relapse prevention therapies for aquaporin-4 antibody-positive neuromyelitis optica spectrum disorder (AQP4+ NMOSD). A total of 70 patients with AQP4+ NMOSD (87% female, median age 56 years) were enrolled. They were divided into the following treatment groups: glucocorticoids and/or immunosuppressants (GC/IS, n = 22), inebilizumab/rituximab (anti-CD19/20, n = 13), satralizumab (anti-IL-6R, n = 22), and eculizumab/ravulizumab (anti-C5, n = 13). At baseline, the blood counts and complement levels did not differ among the groups. At follow-up, the neutrophil and platelet counts in the anti-IL-6R group decreased from those at baseline (p < 0.0001 and p < 0.001, respectively). Compared with the GC/IS, anti-CD19/20, and anti-C5 groups, the anti-IL-6R group had lower levels of C3 (p < 0.0001, p < 0.01, and p < 0.05, respectively) and C4 (p < 0.0001, p < 0.01, p < 0.001, respectively). Furthermore, the anti-C5 group had significantly lower CH50 levels than the GC/IS, anti-CD19/20, and anti-IL-6R groups (p < 0.0001, p < 0.0001, p < 0.05, respectively). In addition, the anti-IL-6R group had lower CH50 levels than the GC/IS and anti-CD19/20 groups (p < 0.001 and p < 0.05, respectively). The present study demonstrated that anti-IL-6R therapy broadly and mildly suppressed the complement system and decreased the neutrophil and platelet counts. It also showed that anti-C5 therapy strongly suppressed total complement activity but did not affect the C3 and C4 levels or blood counts. These findings may have implications for the mode of action of the drugs and the risk of adverse drug reactions, including infections.

Immunodeficient mice, like the NOD-SCID-Gamma (NSG) strain, are important for the study of xenogeneic cells because of their lack of lymphocytes, dysfunctional hemolytic complement factor 5 (C5), and macrophage defects making them permissive hosts. Nonetheless, cellular barriers remain that limit engraftment of foreign cells such as monocytic phagocytes. Accordingly, we created a line of mice that allows for depletion of monocytic cells by breeding NSG mice with macrophage Fas-induced apoptosis (MaFIA) mice resulting in a stable line of NSG-MaFIA mice. NSG-MaFIA mice were generated by crossing NSG and MaFIA mice, with the hybrids backcrossed for nine generations to NSG mice. Flow cytometry was used to detect the expression of the MaFIA gene construct among blood leukocytes. Functional and confirmatory studies evaluated the successful transfer of the MaFIA transgene into the NSG genetic background. Apoptosis of monocytic cells was achieved through administration of a homodimerizer drug. The phenotypic characteristics of NSG mice were confirmed in NSG-MaFIA mice by flow cytometry, CBC analysis, testing of radiation sensitivity, and sequencing of the C5 gene. The permissiveness of NSG-MaFIA mice for xenogeneic engraftment was tested by transfusion of human red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs). The MaFIA transgene was hybridized into NSG mice as exhibited by expression of a fluorescent marker. Functional expression of the MaFIA transgene was evidenced by weight loss and decreased fluorescence after homodimerizer treatment. NSG-MaFIA mice are lymphopenic, are sensitive to X-ray irradiation, and carry a mutated C5 gene. Transfusion of human RBCs resulted in similar clearance in NSG and NSG-MaFIA mice, without homodimerizer treatment, indicating a similar innate immune response. Moreover, transfusion of human RBCs or PBMCs after depletion of monocytic cells led to prolonged circulation of RBCs and rapid engraftment of leukocytes. A novel NSG-MaFIA mouse line was developed that has use in the study of monocytic cells and in the development of better humanized mouse models. Transfusion of human blood cells into cell-depleted NSG-MaFIA mice increased the persistence of the human cells in the circulation, indicating a role for monocytic cells in the removal of xenogeneic cells from immunodeficient mice.

Complement may contribute to platelet destruction in immune thrombocytopenia (ITP), but serum complement levels of ITP patients are not well defined. This study characterized C3, C4, and CH50 levels from 108 ITP patients in comparison with 120 healthy subjects. Results of complement testing performed using commercially available turbidimetric immunoassays were retrospectively analyzed. Mean complement levels in patients with ITP were compared with levels from a sample of 120 healthy subjects, and subgroups of ITP patients were compared. Regression analyses evaluated for relations between low complement levels and disease severity and response to ITP treatments. One hundred eight patients with ITP were included. Mean C3, C4, and CH50 were significantly lower in patients with ITP compared with healthy subjects, largely driven by the 32% of patients with ITP with substantial reductions in one or more assays. Patients requiring treatment had lower mean C4 (18.1 vs 23.1 mg/dL; P = .042) and CH50 (50.4 vs 63.0 mg/dL; P = .004). Mean C3 was higher in splenectomized versus nonsplenectomized patients (120.6 vs 101.0 mg/dL; P = .035). In multivariable analyses, reduced complement did not predict treatment response to corticosteroids, intravenous immunoglobulin, or thrombopoietin receptor agonists but low C4 levels did predict more severe ITP (relative to nonsevere disease, odds ratio for severe/refractory disease: 6.28; 95% confidence interval, 0.75‐52.54; P = .090). Complement levels in patients with ITP were generally consistent over repeat measurements. Complement levels are reduced in one‐third of patients with ITP and are associated with more severe disease. Additional study is needed to evaluate if hypocomplementemia is predictive of response to emerging complement‐directed therapies.

Pig-to-human organ transplantation has drawn attention in recent years due to the potential use of pigs as an alternative source of human donor organs. While GGTA1 knockout (GTKO) can protect xenografts from hyperacute rejection, complement-dependent cytotoxicity might still contribute to this type of rejection. To prolong the xenograft survival, we utilized a T2A-mediated pCMV-hCD55-T2A-hCD59-Neo vector and transfected the plasmid into GTKO Diannan miniature pig fetal fibroblasts. After G418 selection combined with single-cell cloning culture, four colonies were obtained, and three of these were successfully transfected with the hCD55 and hCD59. One of the three colonies was selected as donor cells for somatic cell nuclear transfer (SCNT). Then, the reconstructed embryos were transferred into eight recipient gilts, resulting in four pregnancies. Three of the pregnant gilts delivered, yielding six piglets. Only one piglet carried hCD55 and hCD59 genetic modification. The expression levels of the GGTA1, hCD55, and hCD59 in the tissues and fibroblasts of the piglet were determined by q-PCR, fluorescence microscopy, immunohistochemical staining, and western blotting analyses. The results showed the absence of GGTA1 and the coexpression of the hCD55 and hCD59. However, the mRNA expression levels of hCD55 and hCD59 in the GTKO/hCD55/hCD59 pig fibroblasts were lower than that in human 293T cells, which may be caused by low copy number and/or CMV promoter methylation. Furthermore, we performed human complement-mediated cytolysis assays using human serum solutions from 0 to 60%. The result showed that the fibroblasts of this triple-gene modified piglet had greater survival rates than that of wild-type and GTKO controls. Taken together, these results indicate that T2A-mediated polycistronic vector system combined with SCNT can effectively generate multiplex genetically modified pigs, additional hCD55 and hCD59 expression on top of a GTKO genetic background markedly enhance the protective effect towards human serum-mediated cytolysis than those of GTKO alone. Thus, we suggest that GTKO/hCD55/hCD59 triple-gene-modified Diannan miniature pig will be a more eligible donor for xenotransplantation.

Complement-mediated hemolytic uremic syndrome (CM-HUS) following chemotherapy for pediatric acute lymphoid neoplasms has rarely been reported. We report the case of an 8-year-old boy with T-lymphoblastic lymphoma (T-LBL) who developed CM-HUS with complement factor H (CFH) mutations (S1191L, V1197A) during induction therapy. Safe administration of chemotherapy after CM-HUS recovery was challenging. By closely monitoring hemolytic and renal parameters during the 2-year treatment period, we observed four episodes of microangiopathic hemolytic anemia (MAHA) with hypocomplementemia and low haptoglobin but no renal dysfunction or thrombocytopenia. Here, we describe the MAHA and CM-HUS episodes in the hopes of elucidating the complex pathophysiology of disorders associated with CFH mutation.