Anti-neutrophil Cytoplasmic Antibodies in New-onset Systemic Lupus Erythematosus and Lupus Nephritis

This study aims to investigate the role of Antineutrophil cytoplasmic antibodies (ANCA) in patients with new-onset systemic lupus erythematosus (SLE). Sixty SLE patients, 28 of whom had lupus nephritis (LN), and 60 normal controls were enrolled; Serum ANCA was measured by enzyme linked immunosorbent assay (ELISA). The clinical and laboratory parameters of the patients were also recorded. Results show that twenty SLE patients were seropositive for ANCA, which was significantly higher than in normal controls. LN patients had significantly higher positive rate of ANCA than patients without nephritis. Compared with ANCA-negative patients, the ANCA-positive patients had significantly higher incidence of nerves system disorder, myocarditis, renal involvement and serositis. The positive rate of gamma-globulin, anti-dsDNA and anti-Sm antibodies were significantly higher in ANCA-positive patients. Elevated IgG and ESR, decreased serum C3/C4 appeared more often in ANCA-positive patients. In addition, serum ANCA level correlated positively with disease activity. Taken together, ANCA might be used as a potential complementary parameter to differentiate LN from SLE without nephritis. In addition, ANCA may serve as a useful marker of the disease activity of SLE.     

Characterization of Anti-Mesangial Cell Antibodies and Their Target Antigens in Patients with Lupus Nephritis

The pathogenetic mechanisms of lupus nephritis (LN) remain to be elucidated. In our previous study, autoantibodies against human glomerular mesangial cells (HMC) were identified in sera of most patients with lupus nephritis. The current study is to investigate the binding characteristics of anti-mesangial cell antibodies to human mesangial cell membrane. Serum samples were collected from 54 patients with renal biopsy proven lupus nephritis, 12 patients with systemic lupus erythematosus without clinical renal involvement, and 15 healthy subjects. Membrane proteins were obtained from in vitro cultured HMC by sonication and sequential centrifugation. DNase I were employed to remove DNA fragments in sera and membrane protein preperation and IgG F(ab′)₂ was obtained by pepsin digestion. Western Blot analysis was used to characterize the antibody and antigen interaction. In results, 25 of 54 (46.3%) sera from patients with lupus nephritis had anti-mesangial cell antibodies targeted at 74 kDa, 63 kDa, 52 kDa and 42 kDa protein bands of HMC membrane. Only four of 12 (33.3%) sera from patients without renal involovement recognized the protein bands at 74 kDa and 63 kDa, but not 52 kDa and 42 kDa. DNase treatment of the HMC membrane and the sera did not affect the binding. IgG F(ab′)₂ from sera of 10 patients with positive anti-mesangial cell antibodies could still bind the 63 kDa protein. In conclusion, anti-mesangial cell antibodies from sera of patients with lupus nephritis could bind membrane proteins of HMC directly without a DNA bridge and the binding was through antigen–antibody interation. Anti-mesangial cell antibodies might play some role in the pathogenesis of lupus nephritis(LN).     

Increased Level of Soluble HLA Class I Antigens in Systemic Lupus Erythematosus: Correlation with Anti-DNA Antibodies and Leukopenia

The concentration of soluble HLA class I (sHLA-I) was measured by ELISA in serum samples from 30 well-characterised SLE patients at high and low disease activity states and from 100 healthy controls. HLA-A allotypes in the patients were analysed by a PCR-based typing technique. A higher level of sHLA-I was found in SLE patient sera both at high and low disease activity than in controls (P< 0.001). The sHLA-I level was further increased during active disease (P< 0.01). Concentrations of sHLA-I correlated with anti-dsDNA antibodies at high disease activity, but not with disease activity as analysed by a modified SLEDAI. Numbers of leukocytes and lymphocytes, as well as levels of C1q and C3 correlated inversely with sHLA-I concentration. In five serial samples from ten patients the sHLA-I level co-varied with disease activity. Presence of HLA allotype A9 was associated with higher sHLA-I levels in both patients (P< 0.001) and controls (P< 0.001). We conclude that the increased sHLA-I concentration in SLE patients was related to several laboratory parameters reflecting disease activity suggesting that sHLA-I molecules are connected with the disease process. Increased sHLA-I level due to HLA-A allotype was not a disease susceptibility factor for SLE.     

年龄、职业和免疫因素与系统性红斑狼疮及狼疮性肾炎的关系

目的评价年龄、职业和免疫冈素与系统性红斑狼疮及狼疮性。肾炎的关系。方法收集2009年本院风湿免疫科住院的172例系统性红斑狼疮患者,将病人分为狼疮性肾炎和非狼疮性肾炎组,分析两组病人的年龄分布情况,并对这两组病人的抗核抗体（ANA）、抗ENA抗体和补体C3、C4进行检测。结果本研究的系统性红斑狼疮病人中,农民患者人数最多,占38．3％;狼疮性肾炎和非狼疮性肾炎组患者的年龄分布一致,均主要集中存15～44年龄段;两组患者的核型和15种抗体的阳性率没有统计学差异;狼疮性肾炎和非狼疮性肾炎组的C3与C4值均具有正相关的趋势（相关系数为0．85）,并且狼疮性肾炎组病人的C3值明显低于非狼疮性肾炎组（P=0．03）。结论性别、年龄、工作生活环境及ANA、C3、C4对系统性红斑狼疮的诊断具有很大的价值,其中补体c3对评价系统性红斑狼疮和狼疮性肾炎有较大的意义。     

Clonotypic Dominance and Variable Gene Elements of Pathogenic Anti-DNA Autoantibodies from a Single Patient with Lupus

This study explores the usage and diversity of the variable gene elements expressed by human lupus antibodies to DNA bearing the 0–81 idiotype, a marker of pathogenic anti-DNA autoantibodies. Rather than studying DNA-specific clonotypes from different patients, a panel of idiotype positive anti-DNA autoantibody-secreting clones from a single individual were analysed. By cloning and nucleotide-sequeneing the heavy-chain variable gene segments, evidence was found for dominance of clonotypic patterns. Also noted was a high rate of diversification among the variable (V_H), diversity (D_h) and junctional (J_H) gene segments utilized, with a pattern of mutations indicative of antigenic selection. These features suggest that the clones secreting the lupus pathogenic autoantibodies have been selected over multiple generations through an affinity-maturation process that is reminiscent of antigen-driven immune responses.     

血清补体C1q与狼疮肾炎及糖尿病肾病的相关性分析

目的 探讨血清补体C1q在狼疮肾炎与糖尿病肾病中的变化,为临床狼疮肾炎与糖尿病肾病的诊断与鉴别诊断提供理论依据.方法 选取2015年10月至2016年10月我院住院治疗狼疮肾炎患者33例,糖尿病肾病患者57例,同期体检健康人104例作为对照组,分别测定其血清补体C1q水平.结果 狼疮肾炎组血清补体C1q水平明显低于糖尿病肾病组(P=0.00)和对照组(P=0.001);糖尿病肾病组与对照组比较差异无统计学意义.结论 血清补体C1q水平在各组受试者中不同,检测血清补体C1q水平可用于狼疮肾炎与糖尿病肾病的诊断与鉴别诊断.     

Reduced Paraoxonase 1 Activity as a Marker for Severe Coronary Artery Disease

Paraoxonase-1 (PON1), a high-density-lipoprotein- (HDL-) associated enzyme, has the potential to protect against atherogenesis. We examine the relationships between plasma PON1 activity and the progression of atherosclerosis as well as coronary artery disease (CAD). Fasting blood samples were collected from female apolipoprotein E-deficient (apoE^−/−) mice and 149 patients undergoing coronary angiography for the biochemical parameters measurement. The severity of CAD was defined using angiographic Gensini score (GSS). Compared to 3-month-old apoE^−/− mice, aged mice had significantly lower PON1 activity, which is negatively correlated with the size of atherosclerotic lesion and plasma interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) levels. In study patients, PON1 activity was correlated with age, sex, and HDL-cholesterol, apolipoprotein AI, and high-sensitivity C-reactive protein (hs-CRP) levels and was significantly lower in CAD group than that in non-CAD control group. Interestingly, PON1 activity in severe CAD group (GSS > 40) was further significantly reduced compared to those in mild and moderate subgroups (GSS  ≤ 40) (P < 0.01). There is a significant correlation between PON1 activity and the severity of CAD as assessed by GSS (r = −0.393, P < 0.001). PON1 activity may be a potential biomarker for the severity of CAD.     

Antibodies to Citrullinated Vimentin are a Specific and Sensitive Marker for the Diagnosis of Rheumatoid Arthritis

Background

The last 5 years have seen the emergence and establishment of antibodies to citrullinated antigens as the diagnostic marker for rheumatoid arthritis (RA). Initially, these were detected using a synthetic peptide, which has undergone a number of modifications to give a diagnostic test with a sensitivity of 65–80% and a specificity of >95%. Antibodies to citrullinated vimentin were first described in 1994 as a highly specific marker for RA (anti-Sa). However, no easily performed assay for these antibodies has been available.

Methods

We have examined the use of a ELISA-based assay with a mutated citrullinated vimentin (MCV) antigen (Orgentec, Mainz, Germany) to assess the diagnostic and prognostic utility of this antibody in RA.

Results

Antibodies to MCV were detected in the sera of 74% RA patients (specificity 96%), 2% systemic lupus erythematosus, 14% Sjögren’s syndrome, and 2% scleroderma. Anti-MCV was not detected in sera from healthy blood donors. There was no difference in the frequency of antibodies detected in RA patients with early (<2 years) or chronic (>2 years) disease. There was no significant variation in anti-MCV antibody concentrations in early RA patients over a 52-week period. No significant change was observed with time between the two treatment groups of methotrexate alone or methotrexate plus infliximab.

Conclusions

Antibodies to MCV are a specific and sensitive marker for the diagnosis of RA.

     

Development of a Bead-Based Multiplex Immunoassay for Simultaneous Quantitative Detection of IgG Serum Antibodies against Measles,Mumps, Rubella,and Varicella-Zoster Virus

Enzyme-linked immunosorbent assay (ELISA) is normally used to quantify the amount of serum IgG antibodies against measles, mumps, rubella, and varicella-zoster virus (MMRV). However, this method is time- and material-consuming. Therefore, a multiplex immunoassay for the simultaneous quantitative detection of antibodies against MMRV was developed. In-house as well as commercially available antigens can be used, making the assay available for all laboratories. The multiplex assay is much more sensitive than the separate ELISAs and has a high specificity, and only 5 μl of serum is needed. Heterologous inhibition did not exceed 11.5%, while homologous inhibition varied between 91.3 and 97.9%. Good correlations with the in-house ELISAs for measles (R² = 0.98), mumps (R² = 0.97), and rubella (R² = 0.97) virus as well as with the ELISA kit for varicella-zoster virus (R² = 0.95) were obtained. In conclusion, the MMRV multiplex assay is a good alternative to the conventional ELISAs and suitable for use in serosurveillance and vaccine studies.     

结直肠肿瘤的新标记——CDX2

CDX2是一种新发现的特异性的核转录因子，对正常和肿瘤性的肠上皮均有相对特异性和敏感性，用它作免疫组织化学染色可以用来诊断胃肠道起源的肿瘤以及辅助明确转移性肿瘤是否来源于肠道。目前与它相对应的常用的抗体是CDX2－88，它是一种单克隆抗体（小鼠IgG1、k抗体）。本文就CDX2与结直肠肿瘤关系的研究及应用作一综述。     

New Lineal Immunoenzymatic Assay for Simultaneous Detection of p24 Antigen and HIV Antibodies

 The aim of this study was to evaluate a new lineal immunoenzymatic assay for the simultaneous detection of HIV-1/HIV-2 antibodies and p24 antigen. A total of 320 serum samples were obtained from individuals infected by HIV (HIV 1, n=183; HIV 2, n=2), individuals with risk factors for HIV infection (n=49), recipients of multiple transfusions (n=40), and blood donors (n=46). The Western blot for detection of HIV antibodies and an enzyme immunoassay for detection of p24 antigen, both established techniques, were used for direct comparison. In cases of recent infection, p24 antigen was generally detected at the same time by the lineal test and the established assay. The p24 antigen sensitivity was about 200 pg of HIV antigen per milliliter. The results seem to indicate that the new test could be used with sufficient reliability for screening biological samples (sensitivity, 99.5%; specificity, 94.8%). Use of the lineal immunoenzymatic assay may shorten the amount of time needed to diagnose acute infection with HIV to approximately 2 weeks.     

Development of a Western Blot Assay for Detection of Antibodies against Coronavirus Causing Severe Acute Respiratory Syndrome

To identify a major antigenic determinant for use in the development of a rapid serological diagnostic test for severe acute respiratory syndrome (SARS) coronavirus infection and to study the immune response during SARS coronavirus infection in humans, we cloned the full length and six truncated fragments of the nucleocapsid gene, expressed them, and purified them as glutathione S-transferase-tagged recombinant proteins. The reactivities of the recombinant proteins to a panel of antibodies containing 33 SARS coronavirus-positive sera and 66 negative sera and to antibodies against other animal coronaviruses were screened. A truncated 195-amino-acid fragment from the C terminus of the nucleocapsid protein (N195) was identified that had a strong ability to detect antibodies against SARS coronavirus. No cross-reaction was found between the N195 protein and antibodies against chicken, pig, and canine coronaviruses. The N195 protein was used to develop a Western blot assay to detect antibodies against SARS coronavirus in 274 clinically blinded samples. The specificity and sensitivity of this test were 98.3 and 90.9%, respectively. The correlation between our Western blotting assay and an immunofluorescence assay (IFA) was also analyzed. The results of our Western blot assay and IFA for the detection of SARS coronavirus-positive sera were the same. Thus, the N195 protein was identified as a suitable protein to be used as an antigen in Western blot and other possible assays for the detection of SARS coronavirus infection.     

Simultaneous Quantitation of Antibodies to Neutralizing Epitopes on Virus-Like Particles for Human Papillomavirus Types 6, 11, 16, and 18 by a Multiplexed Luminex Assay

Several different methods have been developed to quantitate neutralizing antibody responses to human papillomaviruses (HPVs), including in vivo neutralization assays, in vitro pseudoneutralization assays, competitive radioimmunoassays (cRIAs), and enzyme-linked immunosorbent assays. However, each of these techniques possesses one or more limitations that preclude testing large numbers of patient sera for use in natural history studies and large vaccine clinical trials. We describe here a new multiplexed assay, by using the Luminex Laboratory MultiAnalyte Profiling (LabMAP3) assay system, that can simultaneously quantitate neutralizing antibodies to human papillomavirus types 6, 11, 16, and 18 in 50 μl of serum. The HPV-Luminex competitive immunoassay measures titers of polyclonal antibodies in serum capable of displacing phycoerythrin-labeled detection monoclonal antibodies binding to conformationally sensitive, neutralizing epitopes on the respective virus-like particles. This competitive Luminex immunoassay was found to be as sensitive, accurate, and precise as the currently used cRIAs. An effective HPV vaccine will most likely require several distinct genotypes to protect against multiple cancer causing papillomaviruses. The HPV-Luminex immunoassay should prove to be a useful tool in simultaneously quantitating antibody immune responses to multiple HPV genotypes for natural history infection studies and for monitoring the efficacy of prospective vaccines.     

Mammaglobin, a Valuable Diagnostic Marker for Metastatic Breast Carcinoma

Identification of metastasis and occult micrometastases of breast cancer demands sensitive and specific diagnostic markers. In this study, we assessed the utility of a mouse monoclonal antibody to human mammaglobin for one such purpose. Immunohistochemical stains were performed on paraffin-embedded sections from a total of 284 cases, which consisted of primary breast invasive carcinomas (41 cases) with matched metastases to ipsilateral axillary lymph nodes, metastatic breast carcinoma to liver (1 case) and kidney (1 case), non-breast neoplasms (161 cases), and normal human tissues (39 cases). The results showed 31 of the 41 cases of primary breast cancer with axillary lymph node metastases were positive for mammaglobin (76%). In the meantime, we documented expression of mammaglobin in occasional cases of endometrial carcinoma (17%). Our data further validated that mammaglobin is a valuable diagnostic marker for metastatic carcinoma of breast origin, although endometrial carcinoma should be considered as a major differential diagnosis.     

Severe OHSS: yes, there is a strategy to prevent it!

Effective measures to prevent ovarian hyperstimulation syndrome (OHSS) remain controversial. It became almost 'common knowledge' that there is no strategy that may completely prevent OHSS. Extensive clinical experience (albeit not derived from prospective randomized studies) clearly documents the ability of a single administration of gonadotrophin-releasing hormone (GnRH) agonist to effectively trigger ovulation, while completely eliminating any threat of clinically significant OHSS. This strategy cannot be used if the pituitary is down-regulated (as is the case in most assisted reproductive cycles today), however, the newly-introduced GnRH antagonists open new opportunities for implementing this strategy, since the pituitary preserves its responsiveness to GnRH agonists. Combining GnRH antagonist-based ovarian stimulation (particularly in 'high responders'), with GnRH agonist-driven ovulation triggering will make severe OHSS a disease of the past in assisted reproduction.     

Antibodies to Collagen: Comparative Epitope Mapping in Women with Silicon Breast Implants, Systemic Lupus Erythematosus and Rheumatoid Arthritis

Women with silicone breast implants have a significantly increased frequency of antibodies to collagen types I and II. To characterize the specificity of these antibodies; 70 women without a specific autoimmune disease, according to the criteria of the American College of Rheumatology, but who had silicone breast implants were studied for the presence of serum antibodies to native and denatured human types I and II collagen by ELISA. Positive sera were further studied by immunoblotting using peptides derived by cyanogen bromide digestion of the collagens. Samples of 82 women with systemic lupus erythematosus (SLE), 94 women with rheumatoid arthritis (RA), and 133 healthy controls were studied concurrently. There was a high frequency of autoantibodies to collagen in each of the study groups when compared to the healthy controls. However, and of particular interest, the epitope specificity of the autoantibodies differed markedly. Sera from women with silicone implants reacted strongly in an individual-specific manner with multiple peptides of type I collagen, whereas sera from women with SLE and RA reacted only weakly with a restricted range of peptides of type I collagen. Sera from women with RA reacted strongly with multiple peptides of type II, whereas sera from women with silicon implants or SLE reacted only weakly. The reactivity of women silicon implants suggests that silicone or its biodegradation products can act as adjuvants in situ to enhance the immunogenicity of type I collagen, or protein-silicone conjugates.     

New Automated Chemiluminescence Immunoassay for Simultaneous but Separate Detection of Human Immunodeficiency Virus Antigens and Antibodies

The recently launched Liaison XL Murex HIV Ab/Ag assay (DiaSorin S.p.A) uses chemiluminescence immunoassay technology for the combined qualitative determination of p24 antigen of HIV-1 and specific antibodies to both HIV-1 and HIV-2. We studied 571 serum samples from those submitted to our laboratory for HIV screening. The samples were divided into 3 subsets: subset A, 365 samples collected prospectively during 1 week; subset B, 158 samples from confirmed HIV-positive patients; and subset C, 48 samples with a positive screening result but a negative or indeterminate confirmatory test result. Our standard screening/confirmatory algorithm was used as a reference. In subset A (prospective), 5 samples were positive and 360 negative by the standard procedure. Liaison XL Murex HIV Ab/Ag correctly identified all 5 positive samples (100%) and 357 negative samples (99.2%). In subset B (confirmed positive), all 158 positive samples were in total agreement in both procedures. In subset C (screen positive only), Liaison XL Murex HIV Ab/Ag yielded accurate results in 42 out of 48 samples (87.5%). Global sensitivity and specificity for Liaison XL Murex HIV Ab/Ag (all subsets included) were 98.3% and 98.5%, respectively. Considering only nonselected prospective samples and confirmed positive samples (subsets A and B), the corresponding sensitivity and specificity values were 100% and 99.2%, respectively. The new fully automated HIV screening test showed high sensitivity and specificity compared to our standard algorithm. Its added advantage of being able to detect HIV-1 and HIV-2 antibodies and p24 antigen separately could prove useful in the diagnosis of early infections.