Changes in serum C3d and C5b-9 levels in elderly patients with idiopathic membranous nephropathy and their clinical significance / 中华老年医学杂志

Objective:To investigate changes in serum C3d and C5b-9 levels in elderly patients with idiopathic membranous nephropathy(IMN)and their correlations with prognosis.Methods:Two hundred thirty-one elderly patients with IMN and 96 non-elderly patients with IMN confirmed by kidney biopsy at the First Affiliated Hospital of Zhengzhou University from January 2015 to May 2017 were enrolled.During the same period, 118 healthy individuals receiving health checkups were included as controls.Patients were divided into the low C3d group( n=112)and the high C3d group( n=113)according to the median level of serum C3d.Serum C3d and C5b-9 levels were measured by enzyme-linked immunosorbent assays. Results:Serum C3d and C5b-9 levels in elderly IMN patients were 0.23(0.15, 0.45)mg/L and 0.28(0.20, 1.23)mg/L, respectively, which were higher than those in healthy controls[0.18(0.13, 0.22)mg/L, 0.22(0.16, 0.26)mg/L, respectively]( Z=-4.261 and -6.213, P<0.001). Serum C3d levels in elderly and non-elderly IMN patients were correlated negatively with the estimated glomerular filtration rate( r=-0.155 and -0.426, P=0.019 and 0.000), but positively with serum creatinine, anti-phospholipase A2 receptor(PLA2R)antibody levels and 24 h urinary protein( r=0.184, 0.326, 0.407, 0.321 and 0.145, P=0.005, 0.001, 0.000, 0.001 and 0.027). Kaplan-Meier survival analysis showed that the cumulative renal survival rate in elderly IMN patients was lower in the high C3d group than in the low C3d group(47.8% vs.70.8%, Log Rank χ2=7.399, P=0.007). Multivariate Cox regression analysis showed that high C3d levels were an independent risk factor for poor renal outcomes in elderly IMN patients( HR=2.288, 95% CI: 1.082-4.839, P=0.030). Conclusions:High serum C3d levels are associated with increases in urinary protein excretion and anti-PLA2R antibody levels, renal function decline, and poor renal outcomes in elderly IMN patients.

Gene testing for osteonecrosis of the femoral head in systemic lupus erythematosus using targeted next-generation sequencing: A pilot study.

BACKGROUND: Previous publications indicated that genetic predisposition might play important roles in the onset of osteonecrosis of the femoral head (ONFH) in systemic lupus erythematosus (SLE). Some gene loci such as complement C3d receptor 2 (CR2), nitric oxide synthase 3 (NOS3), collagen type II alpha 1 chain (COL2A1), protein tyrosine phosphatase non-receptor type 22 (PTPN22), and transient receptor potential cation channel subfamily V member 4 (TRPV4) were reported to be involved in this process. AIM: To investigate whether the risk of ONFH in SLE is associated with single nucleotide variations (SNVs) in these five genes. METHODS: SNVs in the CR2, NOS3, COL2A1, PTPN22, and TRPV4 genes were examined by using FastTarget and Illumina Miseq sequencing technologies in 49 cases of SLE with ONFH. Burrows-wheeler aligner was used to align the sequencing reads to hg19, and GATK and Varscan programs were used to perform SNV calling. PolyPhen-2, SIFT, and MutationTaster were used to assess the functional effects of non-synonymous SNVs. RESULTS: Six of the 49 patients were confirmed to have low frequency SNVs, including one patient with SNVs in NOS3 (exon 6: c.814G>A: p.E272K and exon 7: c.814G>A: p.E272K.), four in COL2A1 (rs41263847: exon 29: c.1913C>T: p.T638I, exon 28: c.1706C>T: p.T569I, and rs371445823: exon 8: c.580G>A: p.A194T, exon 7: c.373G>A: p.A125T), and one in CR2 (rs45573035: exon 2: c.200C>G: p.T67S). CONCLUSION: The onset of ONFH in SLE might be associated with the identified SNVs in NOS3, COL2A1, and CR2.

Preanalytical classical and alternative complement pathway activity loss.

INTRODUCTION: Complement functional analyses provide insight into the integrity of the entire complement reaction cascade. These tests are suitable for investigating suspected complement deficiencies. Falsely reduced test outcomes may result from preanalytical instabilities of individual complement components. To generate rationale for this or potential alternative practices, this study aimed to extend the knowledge on the preanalytical stability of widely used tests to screen the complement system. We assessed the influence of time, temperature and EDTA on classical (CH50) and alternative pathway (AP50) functional assay test results. MATERIALS AND METHODS: We used nephelometric (C3d) and immunofixation (C3c) techniques to support the investigation of the preanalytical phase of basic complement system activity tests. Quantitative determination of classical and alternative pathway function was performed with a haemolytic activity assay and a C5b-9 neo-epitope ELISA-based assay respectively. Blood of five healthy volunteers was sampled and complement components allowed to degrade under different conditions. RESULTS: CH50 and AP50 remain stable for approximately one week in serum samples incubated on ice. CH50 activity decreased almost twice as fast in EDTA plasma compared to serum at room temperature. AP50 activity contrastingly, decreased twice as slow in EDTA plasma compared to serum at room temperature. CONCLUSION: Serum on ice remains the preferred specimen for functional complement analyses. In the absence of serum transported on ice, serum kept at room temperature (not exceeding 24h) is suitable for classical and alternative pathway analyses. For alternative pathway analyses specifically, the C3-stabilising effect of EDTA allows for the extended use of EDTA plasma (not over 4 days). In these conditions, at least 85% of baseline complement activity remains.

[Expression of C3d in normal human liver tissues with non-immunologic osmostic mechanism].

Objective: To observe the deposition of complement C3d at different development stages in human normal organs and tissues, and investigate the significance of its deposition. Methods: Using immunohistochemical methods, the deposition of C3d was detected at different development stages of 60 normal human organs and tissue specimens and double staining was performed in some specimens. Ninty-five cases of other organs or tissues were collected as control group. Results: In 50 of 60 livers, it was observed the deposition of C3d in Glisson's capsule and periportal sheath, with irregular linear network-like disposition surrounding the portal sheath. In different age groups, the expression of C3d was more beyond the 20 year-old group than 3 to 20 year-old group. There wasn't any expression of C3d under 3-year-old group. Under the immuning electron micrograph, C3d depositing at the Glisson's capsule was observed, without immuning compounding. Thirty in 40 spleens, deposition of C3d in capsules, arteries of lymphatic sheath, follicles in the spleen was observed. Conclusions: The deposition of C3d in Glisson's capsule, splenic trabeculae, fibrous sheath, endarterium of liver and spleen arterioles, within normal human tissues from patients elder than 3 years, are osmosis/immunogenic deposition. The deposition of C3d is a normal physiological phenomenon, and treatment of the deposition of C3d should be avoided, as it is an immune complex or immuning reaction phenomenon.

Delivery route determines the presence of immune complexes on umbilical cord erythrocytes.

OBJECTIVE: Umbilical cord blood offers a unique opportunity to study the basal level of immunoglobulin complexes. This study aims to determine the presence of immune complexes and complement deposition on erythrocytes from umbilical cord blood from normal, full-term pregnancies. METHODS: In vitro pre-formed IgA, IgG, and IgM complexes were used as positive control for flow cytometry detection, and for C3d deposition. Blood samples (34) of umbilical cord blood taken from vaginal and cesarean deliveries were tested for the presence of immunoglobulin complexes. RESULTS: Fourteen samples from vaginal deliveries and 20 samples from cesarean deliveries were assessed. IgG and IgM complexes were detected on erythrocytes, whereas no IgA complexes or complement deposition was observed. Interestingly, the percentage of IgG complexes was higher on erythrocytes from vaginal delivery samples compared to those from cesarean deliveries. No other associations between immune complexes and other maternal or newborn variables were found. CONCLUSIONS: IgG and IgM complexes seem to be normally present on umbilical cord erythrocytes. Erythrocytes from vaginal deliveries have a higher percentage of IgG complexes present compared to that from cesarean deliveries. Since no C3d activity was detected, these complexes are non-pathological and should be part of the newborn's initial innate immune response.

Expression of C3d in normal human liver tissues with non-immunologic osmostic mechanism / 中华病理学杂志

Objective@#To observe the deposition of complement C3d at different development stages in human normal organs and tissues, and investigate the significance of its deposition.@*Methods@#Using immunohistochemical methods, the deposition of C3d was detected at different development stages of 60 normal human organs and tissue specimens and double staining was performed in some specimens. Ninty-five cases of other organs or tissues were collected as control group.@*Results@#In 50 of 60 livers, it was observed the deposition of C3d in Glisson′s capsule and periportal sheath, with irregular linear network-like disposition surrounding the portal sheath. In different age groups, the expression of C3d was more beyond the 20 year-old group than 3 to 20 year-old group. There wasn′t any expression of C3d under 3-year-old group. Under the immuning electron micrograph, C3d depositing at the Glisson′s capsule was observed, without immuning compounding. Thirty in 40 spleens, deposition of C3d in capsules, arteries of lymphatic sheath, follicles in the spleen was observed.@*Conclusions@#The deposition of C3d in Glisson′s capsule, splenic trabeculae, fibrous sheath, endarterium of liver and spleen arterioles, within normal human tissues from patients elder than 3 years, are osmosis/immunogenic deposition. The deposition of C3d is a normal physiological phenomenon, and treatment of the deposition of C3d should be avoided, as it is an immune complex or immuning reaction phenomenon.