补体5a受体在阿霉素致急性心力衰竭心肌损伤的早期研究

目的:探讨补体系统补体5a受体(complement 5a receptor,C5aR)在急性心力衰竭(心衰)早期,对心脏功能影响以及对心肌损伤的作用。方法:选择12 w龄C57BL/6J野生型及C5aR敲除小鼠各12只,分别随机分为野生小鼠对照组、野生小鼠心衰组、C5aR敲除小鼠对照组和C5aR敲除小鼠心衰组等共4组,每组6只。采用单次腹腔注射阿霉素20 mg/kg建立小鼠急性心衰模型,对照组采用同计量0.9%氯化钠液注射;阿霉素注射3d后显微超声检测小鼠心室射血分数和短轴缩短率,处死后测量体质量以及心脏质量;运用半定量PCR、蛋白印迹技术Western Blotting、免疫荧光等实验方法观察C5aR在野生小鼠心脏中的表达。C5aR敲除小鼠心脏组织采用HE染色观察心肌形态、Masson染色观察心肌纤维化程度,WGA染色观察心肌横截面大小,免疫组化染色观察转化生长因子-β(TGF-β)及α-平滑肌肌动蛋白(α-SMA)在心脏中表达。结果:与野生小鼠对照组相比,野生小鼠心衰组中C5aR的mRNA及蛋白表达均显著上调(P<0.05);与野生小鼠心衰组相比,C5aR敲除小鼠心衰组体质量及血压均显著升高(均为P<0.05),心室射血分数和短轴缩短率均显著升高(均为P<0.05),C5aR敲除小鼠心衰组胶原沉积及α-SMA表达均显著降低(均为P<0.05),TGF-β表达显著降低(P<0.01)。结论:C5aR在阿霉素诱导心衰模型中表达上调、加重了心肌损伤且促进了心脏纤维化,而C5aR敲除保护小鼠心功能并抑制纤维化,提示C5aR在小鼠急性心衰早期具有促进心脏纤维化作用。     

Eculizumab Improves Posttransplant Thrombotic Microangiopathy Due to Antiphospholipid Syndrome Recurrence but Fails to Prevent Chronic Vascular Changes

Thrombotic microangiopathy (TMA) is one of the hallmark vascular lesions of antiphospholipid syndrome nephropathy (APSN). These lesions are at high risk of recurrence after kidney transplantation. The complement pathway is thought to be active in this process. We used eculizumab to treat three consecutive kidney transplant recipients with posttransplant TMA due to APSN recurrence that was resistant to plasmapheresis and explored the complement deposition and apoptotic and vascular cell markers on the sequential transplant biopsies. Treatment with eculizumab resulted in a rapid and dramatic improvement of the graft function in all three patients and in improvement of the TMA lesions within the graft. None of these patients had TMA flares after eculizumab was withdrawn. At the time of TMA diagnosis, immunofluorescence studies revealed intense C5b‐9 and C4d depositions at the endothelial cell surface of the injured vessels. Moreover, C5b‐9 colocalized with vessels exhibiting a high rate of apoptotic cells. Examination of sequential biopsies during eculizumab therapy showed that TMA lesions, C4d and apoptotic markers were rapidly cleared but the C5b‐9 deposits persisted for several months as a footprint of the TMA. Finally, we noticed that complement inhibition did not prevent the development of the chronic vascular changes associated with APSN. Eculizumab seems to be an efficient method for treating severe forms of posttransplant TMA due to APSN recurrence. Terminal complement inhibition does not prevent the development of chronic APSN.     

补体在抗体介导排斥反应中作用的研究进展

随着免疫抑制剂的发展及诊断手段的提高,器官移植后细胞性排斥反应得到很好的预防和治疗,但器官移植致敏患者数量不断增加,抗体介导排斥反应越米越受到关注.补体在抗体介导排斥反应中有重要作用,本文主要对补体在抗体介导排斥反应中的作用机制,补体在抗体介导排斥反应诊断中的应用,以及针对补体的抗体介导排斥反应治疗方案等方面的新进展作一综述.     

Role of anti-polyethylene glycol (PEG) antibodies in the allergic reactions to PEG-containing Covid-19 vaccines: Evidence for immunogenicity of PEG

A small fraction of recipients who receive polyethylene-glycol (PEG)-containing COVID-19 mRNA-LNP vaccines (Comirnaty and Spikevax) develop hypersensitivity reactions (HSRs) or anaphylaxis. A causal role of anti-PEG antibodies (Abs) has been proposed, but not yet been proven in humans. We used ELISA for serial measurements of SARS-CoV-2 neutralizing Ab (anti-S) and anti-PEG IgG/IgM Ab levels before and after the first and subsequent booster vaccinations with mRNA-LNP vaccines in a total of 291 blood donors. The HSRs in 15 subjects were graded and correlated with anti-PEG IgG/IgM, just as the anti-S and anti-PEG Ab levels with each other. The impacts of gender, allergy, mastocytosis and use of cosmetics were also analyzed. Serial testing of two or more plasma samples showed substantial individual variation of anti-S Ab levels after repeated vaccinations, just as the levels of anti-PEG IgG and IgM, which were over baseline in 98–99 % of unvaccinated individuals. About 3-4 % of subjects in the strongly left-skewed distribution had 15–45-fold higher values than the median, referred to as anti-PEG Ab supercarriers. Both vaccines caused significant rises of anti-PEG IgG/IgM with >10-fold rises in about ∼10 % of Comirnaty, and all Spikevax recipients. The anti-PEG IgG and/or IgM levels in the 15 vaccine reactors (3 anaphylaxis) were significantly higher compared to nonreactors. Serial testing of plasma showed significant correlation between the booster injection-induced rises of anti-S and anti-PEG IgGs, suggesting coupled anti-S and anti-PEG immunogenicity. Conclusions: The small percentage of people who have extreme levels of anti-PEG Ab in their blood may be at increased risk for HSRs/anaphylaxis to PEGylated vaccines and other PEGylated injectables. This risk might be further increased by the anti-PEG immunogenicity of these vaccines. Screening for anti-PEG Ab “supercarriers” may help predicting reactors and thus preventing these adverse phenomena.     

hs-CRP、补体C3、PA及皮质醇水平在脑卒中患者疾病进展中的变化

目的 观察超敏C反应蛋白(hs-CRP)、补体C3、前白蛋白(PA)及皮质醇水平在脑卒中患者疾病进展中的变化情况.方法 收集2014年3月至2015年3月期间我院收治的脑卒中患者90例为研究对象,在患者入院第1天、第7天、第14天和第30天采用免疫比浊法检测患者血清中的hs-CRP、补体C3水平,采用化学方法检测血清中PA水平,采用化学发光法检测血清皮质醇水平,比较上述四种指标在脑卒中患者疾病进展中的变化情况.结果 (1)患者入院后第7天[(25.03±5.68)mg/L]和第14天[(14.26±4.355)mg/L]的血清hs-CRP水平较第1天[(6.08±1.17)mg/L]显著升高,差异有统计学意义(P<0.05),入院第14天的血清Hs-CRP水平较第7天下降,差异有统计学意义(P<0.05);入院后第7天的血清C3水平较第1天显著下降,差异有统计学意义(P<0.05),入院第14天[(0.82±0.10)g/L]、第30天[(0.91±0.18)g/L]的血清C3水平与第1天比较差异无统计学意义(P>0.05);患者入院第7天[(197.68±20.08)g/L]的血清PA水平较第1天[(287.98±23.56)g/L]显著下降,差异有统计学意义(P<0.05),入院第14天[(258.93±23.15)g/L]的血清PA水平较第7天显著上升,差异有统计学意义(P<0.05);患者入院第7天[(496.78±38.99)nmol/L]和第14天[(467.06±32.58)nmol/L]的血清皮质醇水平较第1天[(397.83±42.06)nmol/L]显著上升(P<0.05),第14天的血清皮质醇水平较第7天有所下降(P<0.05).(2)脑卒中严重程度与营养代谢严重程度呈正相关关系.结论 hs-CRP、补体C3、PA和皮质醇水平在脑卒中患者疾病进展中呈现明显的时间相关性,处于急性期的患者变化最为明显.     

Deciphering IgM interference in IgG anti-HLA antibody detection with flow beads assays

In flow beads assays, the interference of IgM for IgG anti-HLA antibodies detection is not precisely understood. Using the screening flow beads assay for class I HLA antibodies, we analyzed the binding of two IgG mAbs, the anti-class I HLA W6/32 and an anti-beta-2-microglobulin, in the presence of an anti-beta-2-microglobulin IgM mAb. In neat serum, the IgM mAb impaired the detection of both IgG. In EDTA-treated serum, the interference was stronger for the anti-beta-2-microglobulin IgG than for W6/32, in agreement with the finding in surface plasmon resonance that this IgM competed with the anti-beta-2-microglobulin IgG but not with W6/32. The IgM interference was higher in neat than in EDTA-treated serum for both IgG mAbs. The IgM interference was also analyzed with class II single antigen flow beads and sera from two kidney recipients containing IgG and IgM donor specific antibodies. Anti-HLA IgG detection was partially corrected by EDTA, and restored by IgM inactivation with DTT, confirming the results observed with the mAbs. Therefore, three mechanisms can explain the IgM interference for IgG anti-HLA antibodies in flow beads assays: direct competition for antigen, steric hindrance and complement activation.     

Interactions between mannose-binding lectin and MASPs during complement activation by the lectin pathway

The lectin pathway of complement performs a key role within the immune system by recognising pathogens through patterns of sugar moieties displayed on their cell surfaces and neutralising them via an antibody-independent reaction cascade. While particularly important during early childhood before the adaptive immune system is established, or when adaptive immunity is compromised, it has a protective function throughout life, neutralising invading pathogens directly and helping to stimulate and direct an effective immune response. Complement activation is initiated when complexes comprising mannose-binding lectin (MBL) or serum ficolins and MBL-associated serine protease-2 (MASP-2) bind to pathogens. Binding induces conformational changes in these complexes, leading to autoactivation of the MASPs, which in turn activate the downstream reaction cascade. A major goal in complement research is to understand the molecular events that trigger complement activation. Over the last few years, structure-function studies have improved our knowledge of the way in which MBL binds to MASPs by defining the portions of these proteins that interact and by solving the structures of key protein fragments. In this review, I will summarise the main findings of these studies and describe current theories to explain how the components combine to initiate the reaction cascade.     

Serine proteases of the classical and lectin pathways: similarities and differences

C1r, C1s, MBL-associated serine protease (MASP)-1, MASP-2 and MASP-3 are mosaic serine proteases of the classical and lectin pathways of complement. They form a family of enzymes with identical domain organization and similar overall structure, but with different enzymatic properties. MASP-2 of the lectin pathway can autoactivate and cleave C4 and C2 components. In the classical pathway two enzymes mediate these functions: C1r autoactivates and activates C1s, while C1s cleaves C4 and C2. The substrate specificity and the biological function of MASP-1 and MASP-3 have not yet been completely resolved. MASP-1 can autoactivate and the activated MASP-1 has more relaxed substrate specificity than the other members of the family. It was demonstrated that MASP-1 can specifically cleave C2, C3 and fibrinogen, but the physiological relevance of these findings has to be proved. We do not know how MASP-3 becomes activated and its biological function is also not clear. In this review, we will summarize current knowledge about the structure and function of these proteases. Special emphasis will be laid on the specificity, autoactivation and evolution of these enzymes.     

Absence of functional alternative complement pathway alleviates lupus cerebritis

The complement inhibitor, Crry, which blocks both the classical and alternative pathways, alleviates CNS disease in the lupus model, MRL/MpJ-Tnfrsf6lpr (MRL/lpr) mice. To understand the role of the alternative pathway, we studied mice deficient in a key alternative pathway protein, complement factor B (fB). Immune deposits (IgG and C3) were reduced in the brains of MRL/lpr fB-deficient (fB-/-MRL/lpr) compared to fB-sufficient (MRL/lpr) mice, indicating reduced complement activation. Reduced neutrophil infiltration (22% of MRL/lpr mice) and apoptosis (caspase-3 activity was reduced to 33% of MRL/lpr mice) in these mice indicates that the absence of the alternative pathway was neuroprotective. Furthermore, expression of phospho (p)-Akt (0.16+/-0.02 vs. 0.35+/-0.13, p<0.03) was increased, while expression of p-PTEN (0.40+/-0.06 vs. 0.11+/-0.07, p<0.05) was decreased in fB-/-MRL/lpr mice compared to their MRL/lpr counterparts. The expression of fibronectin, laminin and collagen IV was significantly decreased in fB-/-MRL/lpr mice compared to MRL/lpr mice, indicating that in the lupus setting, tissue integrity was maintained in the absence of the alternative pathway. Absence of fB reduced behavioral alterations in MRL/lpr mice. Our results suggest that in lupus, the alternative pathway may be the key mechanism through which complement activation occurs in brain, and therefore it might serve as a therapeutic target for lupus cerebritis.