Search

Background Antiglomerular basement membrane (anti-GBM) disease is associated with HLA-DRB1*1501 (the major predisposing genetic factor in the disease), with α3127-148 as a nephritogenic T and B cell epitope. Although the cause of disease remains unclear, the association of infections with anti-GBM disease has been long suspected. Methods To investigate whether microbes might activate autoreactive T and B lymphocytes via molecular mimicry in anti-GBM disease, we used bioinformatic tools, including BLAST, SYFPEITHI, and ABCpred, for peptide searching and epitope prediction. We used sera from patients with anti-GBM disease to assess peptides recognized by antibodies, and immunized WKY rats and a humanized mouse model (HLA-DR15 transgenic mice) with each of the peptide candidates to assess pathogenicity. Results On the basis of the critical motif, the bioinformatic approach identified 36 microbial peptides that mimic human α3127-148. Circulating antibodies in sera from patients with anti-GBM recognized nine of them. One peptide, B7, derived from Actinomyces species, induced proteinuria, linear IgG deposition on the GBM, and crescent formation when injected into WKY rats. The antibodies to B7 also targeted human and rat α3127-148. B7 induced T cell activation from human α3127-148-immunized rats. T cell responses to B7 were detected in rats immunized by Actinomyces lysate proteins or recombinant proteins. We confirmed B7's pathogenicity in HLA-DR15 transgenic mice that developed kidney injury similar to that observed in α3135-145-immunized mice. Conclusions Sera from patients with anti-GBM disease recognized microbial peptides identified through a bioinformatic approach, and a peptide from Actinomyces induced experimental anti-GBM GN by T and B cell crossreactivity. These studies demonstrate that anti-GBM disease may be initiated by immunization with a microbial peptide.

The major epitopes recognized by autoantibodies in anti-glomerular basement membrane (GBM) disease are found in the α3-subunit non-collagenous domain of type IV collagen [α3(IV)NC1], which is present in the glomerular and alveolar basement membranes. These epitopes are structurally cryptic, owing to the hexamer formation of the non-collagenous domain of α3, α4, and α5 subunits and are expressed by the dissociation of the hexamer. Anti-GBM disease usually manifests as a single attack (SA), and we rarely see patients who repeatedly relapse. We recently treated a patient with anti-GBM disease who exhibited repeated relapse (RR). Here, we conducted immunohistochemistry of formalin-fixed paraffin-embedded normal kidney sections and immunoblotting using recombinant human α3(IV)NC1 to compare the epitopes recognized by anti-GBM antibodies in the RR patient and SA patients. Although a clear staining of GBM especially in the connecting basement membrane of Bowman's capsule was observed when IgGs of SA patients were used as primary antibodies, such staining was not obtained when IgG of the RR patient was employed. In immunoblotting of α3(IV)NC1 using the IgG of the RR patient as a primary antibody, an 18-kDa band was detected besides the 56.8-kDa band corresponding to the whole-size α3(IV)NC1. Whereas the 56.8-kDa band disappeared after digestion of the recombinant α3(IV)NC1 by protease, the 18-kDa band remained. Furthermore, the 18-kDa band was not detected by a commercially available anti-α3(IV)NC1 monoclonal antibody. These findings suggest that the IgG of the RR patient recognizes the epitope distinct from that recognized by the anti-α3(IV)NC1 monoclonal antibody.

Background Anti-glomerular basement membrane disease (GBM) is an autoimmune disease caused by the deposition of circulating anti-GBM antibodies. Non-collagen region of α3 chain of type IV collagen (α3(IV)NC1) is one of the main target antigens, in which EA and EB are the most classical antigen epitopes. It has been reported that anti-GBM antibodies can be detected in HIV patients; however, its immunological characteristics are still unclear. Objectives In this study, the positive rate of the anti-GBM antibodies in HIV and the immunological characteristics of the target antigens were clarified. Methods A total of 93 HIV patients diagnosed in Beijing Youan Hospital from November 2017 to January 2018 were included. Enzyme-linked immunosorbent assay was used to measure the serum IgG autoantibodies specifically against GBM in these patients, as well as their subtypes and antigen spectra. Results It was found that five out of the 93 patients with HIV had low to moderate levels of anti-GBM antibodies. However, these patients presented with no clinical manifestation of any kidney injury or pulmonary hemorrhages. Compared with HIV patients with negative antibodies, there were no significant differences in gender, age, CD4+T cell count and HIV viral load. All sera of five patients recognized non-collagenous domain1 (NC1) of alpha 3 chain of type IV collagen [(α3(IV)NC1] as classic anti-GBM patients, followed by α5(IV)NC1. The antibodies against α3(IV)NC1 were IgG3 predominant, while these antibodies did not react with either of the classic epitopes on α3 (EA and EB). Conclusion These data suggest a distinct immunological profile of anti-GBM antibodies in patients with HIV, and might explain the non-pathogenic features of HIV associated anti-GBM antibodies.

AIM Anti-glomerular basement membrane (GBM) disease is an autoimmune disorder with rapidly progressive glomerulonephritis and alveolar haemorrhage. Fever symptoms and prodromal infections have been reported in many cases, but still not been elucidated. METHODS Our study enrolled 140 consecutive patients with anti-GBM disease and retrospectively analyzed the characteristics of fever symptoms and the possible reasons. RESULTS Among the 140 patients, 94 (67.1%) patients presented with fever (over 37.5°C) prior to admission or within 48 h of hospitalization. Among those with fever, 74 (78.7%) patients had infections, 15 (16.0%) patients had positive serum anti-neutrophil cytoplasmic antibodies, all towards myeloperoxidase, which was comparable to the patients without fever (17.4%, P = 0.830). There were 93/140 patients suffered from infections, with 47.3% in lungs and 31.2% on upper respiratory tract. In some cases, we identified the microbes of infections, including Candida albicans, Escherichia coli, Acinetobacter baumannii, Enterococcus faecalis, Klebsiella pneumoniae, Hemolytic staphylococci, Pseudomonas aeruginosa and Citrobacter braakii. Patients with fever had higher levels of serum anti-GBM antibodies (154.9 ± 58.4 vs. 106.0 ± 63.2 IU/mL, P < 0.001), higher serum creatinine (733.4 ± 402.5 vs. 580.6 ± 368.1 μmol/L, P = 0.032), higher percentage of crescents (87.0 ± 15.6 vs. 67.4 ± 37.6%, P = 0.021), and higher frequency of progression to end stage renal disease (ESRD) (80.9% vs. 60.9%, P = 0.011). CONCLUSION We concluded that fever is a common symptom in anti-GBM disease and associates with more severe glomerulonephritis. The majority of patients at presentation had fever with respiratory tract infections, which needs further investigation to reveal their role in the pathogenesis of anti-GBM disease.

Myeloperoxidase (MPO) is a common target antigen of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis and is recognized in one-third of patients with anti–glomerular basement membrane (GBM) disease. Our previous study identified over 60% of patients with anti-GBM disease recognizing linear peptides of MPO heavy chain. Here we tested whether aberrant glycosylation alters MPO antigenicity through exposure of neo-epitopes on MPO molecules. Atypical glycosylated MPO molecules, including all possible glycosylation types, were prepared by exoglycosidase and endoglycosidase treatments. Antibodies were detected from the sera of 40 patients with anti-GBM disease without the coexistence of MPO-ANCA. Circulating antibodies against aberrant glycosylated MPO existed in 21 of these patients. Non-glycan MPO and MPO with only N-acetylglucosamine had high frequencies of recognition (16 and 15 patients, respectively). Antibodies binding to aberrant glycosylated MPO could not be inhibited by intact MPO or GBM antigen. When applied to ethanol-fixed neutrophils from normal individuals, these antibodies yielded a typical cytoplasmic staining pattern (c-ANCA). Antigen specificity was detected in 90% of the antibodies using five peptides containing one of the five N-glycosylation sites each, mostly on N323, N355, and N391. The antibodies were restricted to IgG1 subclass, could activate complement, and induce neutrophil degranulation in vitro . Thus, aberrant glycosylated MPO exposed neo-epitopes and was recognized by half of the patients with anti-GBM disease. Their antibodies possessed pathogenic characteristics and may be associated with kidney injury.

In a substantial number of patients with crescentic glomerulonephritis, both anti-glomerular basement membrane (GBM) antibodies and anti-neutrophil cytoplasmic antibodies (ANCA) are detected simultaneously. ANCA is presumed to be the initial event but the mechanism is unknown. In the present study, we investigated the antibodies against linear epitopes on Goodpasture autoantigen in sera from patients with ANCA-associated vasculitis, aiming to reveal the mechanisms of the coexistence of the two kinds of autoantibodies. Thirty-one patients with ANCA-associated vasculitis were enrolled in this study. Twenty-four overlapping linear peptides were synthesized across the whole sequence of Goodpasture autoantigen. Serum antibodies against linear peptides were detected by ELISA and their associations with clinical features were further analyzed. Twenty-five out of the thirty-one (80.6%) sera from patients with ANCA-associated vasculitis possessed antibodies against linear peptides on Goodpasture autoantigen. These antibodies could be detected in 50% of patients with normal renal function (Scr ≤ 133 μmol/L), 70% of patients with moderate renal dysfunction (133 μmol/L 600 μmol/L) (P = 0.032). The highest recognition frequencies were found for peptides P4 (51.6%), P14 (54.8%), and P24 (54.8%), which contained the sequences that constitute the conformational epitopes of EA (P4) and EB (P14) recognized by anti-GBM antibodies. The level of anti-P4 antibodies was positively correlated with the percentage of crescents in glomeruli (r = 0.764, P = 0.027). Patients with anti-P24 antibodies had a significantly higher prevalence of renal dysfunction on diagnosis (88.2 vs. 42.9%, P = 0.018). Antibodies against linear epitopes on Goodpasture autoantigen could be detected in sera of patients with ANCA-associated vasculitis, which might mediate the production of antibodies towards the conformational epitopes on Goodpasture autoantigen, namely, the anti-GBM antibodies.

Objective Alzheimer's disease (AD) is along with cognitive decline due to amyloid-β (Aβ) plaques, tau hyperphosphorylation, and neuron loss. Shenqi Xingnao Granules (SQXN), a traditional Chinese medicine, significantly ameliorated the cognitive function and daily living abilities of patients with AD. However, till date, no study has investigated the mechanism of action of SQXN on AD. The present study aimed to verify the effects of SQXN treatment on cognitive impairments and AD-like pathologies in APP/PS1 mice. Methods Four-month-old APP/PS1 transgenic (Tg) mice were randomly divided into a model group and SQXN-treated (3.5, 7, 14 g/kg per day) groups. Learning-memory abilities were determined by Morris water maze and object recognition test. All mice were sacrificed and the brain samples were collected after 75 d. The soluble Aβ contents were detected by Elisa kit; The levels of expression of NeuN, APP, phosphorylated tau and related protein were measured by Western blotting; The inflammation factors were detected by the proinflammatory panel kit. Results Four-month-old APP/PS1 mice were administered SQXN by oral gavage for 2.5 months. Using the Morris water maze tests and Novel object recognition, we found that SQXN restored behavioral deficits in the experimental group of Tg mice when compared with the controls. SQXN also inhibited neuronal loss (NeuN marker). SQXN treatment decreased soluble Aβ42 through inhibiting the expression of sAPPβ and BACE-1 without regulating full-length amyloid precursor protein (FL APP). Insulin degrading enzyme (IDE), the Aβ degrading enzyme, were increased by SQXN. In addition, SQXN reduced hyperphosphorylated tau protein levels and prevented excessive activation of p-GSK-3β in the brain of APP/PS1 mice. Compared with APP/PS1 transgenic negative mice, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-12p70, KC/GRO and TNF-α were not obviously changed in the brain of 6.5-month-old APP/PS1 transgenic (Tg) mice. However, SQXN could inhibited the expression of IL-2. Conclusion These results demonstrate that SQXN ameliorates the cognitive impairments in APP/PS1 mice. The possible mechanisms involve its inhibition of neuronal loss, soluble Aβ deposition, tau hyperphosphorylation and inflammation.

Background In Goodpasture disease, the noncollagenous domain 1 of the α3 chain (α3NC1) of type IV collagen is the main target antigen of antibodies against glomerular basement membrane (GBM). We previously identified a nephritogenic epitope, P14 (α3127-148), that could induce crescentic nephritis in WKY rats, and defined its core motif. Designing a modified peptide, replacing critical pathogenic residues with nonpathogenic ones (on the basis of homologous regions in α1NC1 chain of type IV collagen, known to be nonpathogenic), might provide a therapeutic option for anti-GBM GN. Methods We synthesized a modified peptide, replacing a single amino acid, and injected it into α3-P14-immunized rats from day 0 (the early-treatment group) or a later-treatment group (from days 17 to 21). A scrambled peptide administrated with the same protocol served as a control. Results The modified peptide, but not the scrambled peptide, attenuated anti-GBM GN in both treatment groups, and halted further crescent formation even after disease onset. Kidneys from the modified peptide-treated rats exhibited reductions in IgG deposits, complement activation, and infiltration by T cells and macrophages. Treatment also resulted in an anti-inflammatory cytokine profile versus a proinflammatory profile for animals not receiving the modified peptide; it also reduced α3-P14-specific T cell activation, modulated T cell differentiation by decreasing Th17 cells and enhancing the ratio of Treg/Th17 cells, and inhibited binding of α3-P14 to antibodies and MHC II molecules. Conclusions A modified peptide involving alteration of a critical motif in a nephritogenic T cell epitope alleviated anti-GBM GN in a rat model. Our findings may provide insights into an immunotherapeutic approach for autoimmune kidney disorders such as Goodpasture disease.

Autoantibody against glomerular basement membrane (GBM) plays a direct role in the initiation and development of Goodpasture’s (GP) disease. The principal autoantigen is the non-collagenous domain 1 (NC1) of α3 chain of collagen IV, with two immunodominant epitopes, EA-α3 and EB-α3. We recently demonstrated that antibodies targeting α5NC1 are bound to kidneys in GP patients, suggesting their pathogenic relevance. In the present study, we sought to assess the pathogenicity of the α5 autoantibody with clinical and animal studies. Herein, we present a special case of GP disease with circulating autoantibody reactive exclusively to the α5NC1 domain. This autoantibody reacted with conformational epitopes within GBM collagen IV hexamer and produced a linear IgG staining on frozen sections of human kidney. The antibody binds to the two regions within α5NC1 domain, EA and EB, and inhibition ELISA indicates that they are targeted by distinct sub-populations of autoantibodies. Sequence analysis highlights five residues that determine specificity of antibody targeting EA and EB epitopes of α5NC1 over homologous regions in α3NC1. Furthermore, immunization with recombinant α5NC1 domain induced crescentic glomerulonephritis and alveolar hemorrhage in Wistar-Kyoto rats. Thus, patient data and animal studies together reveal the pathogenicity of α5 antibodies. Given previously documented cases of GP disease with antibodies selectively targeting α3NC1 domain, our data presents a conundrum of why α3-specific antibodies developing in majority of GP patients, with α5-specific antibodies emerged in isolated cases, the answer for which is critical for understanding of etiology and progression of the GP disease.

Cell-mediated autoimmunity, particularly that involving autoreactive T cells, participates in mediating anti-glomerular basement membrane (GBM) disease. However, direct kidney injury mediated by renal infiltrated T cells has not been clearly elucidated in humans. The T cell profile (CD3, CD4, CD8, IL-17, and foxp3) and macrophage (CD68) were examined by immunohistochemistry on renal biopsy tissues from 13 patients with anti-GBM disease. The correlation between cell infiltration and clinical data was also analyzed. We found that the distribution of T cell infiltration was predominant in the peri-glomerular and interstitial areas. CD3+ T cell infiltratrion around the glomeruli with cellular crescent formations was significantly higher than that around the glomeruli with mild mesangial proliferation. CD8+ T cells significantly accumulated around the glomeruli with cellular crescents without IgG deposits compared to those with IgG deposits. The prevalence of infiltrating CD8+ T cells was correlated with the percentage of ruptured Bowman's capsules. In conclusion, cellular immunity may play a crucial role in the inflammatory kidney injury in anti-GBM patients. The periglomerular infiltration of T cells, especially CD8+ T cells, may participate in the pathogenic mechanism of glomerular damage.

Approximately 20%-30% of patients with anti-glomerular basement membrane disease present coexisting anti-myeloperoxidase (MPO) autoantibodies. We previously showed the recognition of a linear fragment of the MPO heavy chain N-terminus ((1)H, MPO279-409) in plasma from most double-positive patients. Herein, we investigated the frequency of autoantibodies against overlapping (1)H-derived linear peptides in plasma from patients with anti-glomerular basement membrane disease.We synthesized 13 overlapping linear peptides ((1)H-1 to (1)H-13) covering MPO279-409. We retrospectively collected plasma samples from 67 patients with anti-glomerular basement membrane disease from 1996 to 2012, and we screened them for IgG autoantibodies by ELISA using intact human MPO and the overlapping peptides as antigens, and we further investigated the clinical significance. Autoantibody binding to the linear MPO structure was confirmed by Western blotting.We followed up the 67 patients until 2015, with a median follow-up time of 10.0 (2.3-36.0) months, and 56 ESRD events occurred among the 67 patients with follow-up data. Plasma from 23.9% (16) of the patients recognized intact human MPO, whereas 62.7% (42) plasma samples recognized MPO279-409 linear peptides. Of the 13 linear peptides, (1)H-4 (44.8%, 30 patients) and (1)H-12 (40.3%, 27 patients) exhibited the highest recognition frequencies. Patients with autoantibodies against (1)H-11 or (1)H-12 (MPO371-400) were older (46.1±18.8 versus 34.1±16.6 years; P0.01), had higher serum creatinine upon diagnosis (median 7.8 mg/dl, interquartile range 4.9-12.6 mg/dl versus median 5.4 mg/dl, interquartile range 2.4-7.3 mg/dl; P=0.02), and had a higher probability of progressing to ESRD; however, multivariate Cox regression analysis showed that (1)H-11 or 12 reaction was not an independent risk factor for renal failure (hazard ratio, 1.2; 95% confidence interval, 0.8 to 2.8; P=0.19).Autoantibodies against linear peptides of MPO can be detected in the majority of patients with anti-glomerular basement membrane disease, and several are associated with disease severity. The potential common pathogenic mechanism between anti-glomerular basement membrane antibodies and anti-MPO autoantibodies in anti-glomerular basement membrane disease requires further investigation.

Ubiquitous contamination of microplastics and arsenic in soil ecosystems can induce many health issues to nontarget soil organisms, and will also cause many potential threats to the gut bacterial communities of soil fauna. However, the changes in the gut bacterial communities of soil fauna after exposure to both microplastics and arsenic remain unknown. In this study, the toxicity and effects on the gut microbiota of earthworm Metaphire californica caused by the combined exposure of microplastics and arsenic were examined by using arsenic species analysis and high throughput sequencing of gut microbiota. Results showed that total arsenic and arsenic species in the earthworm gut and body tissues after exposure to combination of microplastics with arsenate (As(V)) were significantly different from that treated with As(V) alone. Microplastics lessened the accumulation of total arsenic and the transformation rate of As(V) to arsenite (As(III)). Microplastics alleviated the effect of arsenic on the gut microbiota possibly via adsorbing/binding As(V) and lowering arsenic bioavailability, thus prevented the reduction of As(V) and accumulation of total arsenic in the gut which resulted in a lower toxicity on the earthworm. The study broadens our understanding of the ecotoxicity of microplastics with other pollutants on the soil animals and on their gut microbiota.

Patients with both anti–glomerular basement membrane (anti-GBM) disease and Castleman disease have been rarely reported. In this study, we report 3 patients with this combination. They had immunologic features similar to patients with classic anti-GBM disease. Sera from the 3 patients recognized the noncollagenous (NC) domain of the α3 chain of type IV collagen (α3(IV)NC1) and its 2 major epitopes, EA and EB. All 4 immunogloblin G (IgG) subclasses against α3(IV)NC1 were detectable, with predominance of IgG1. In one patient with lymph node biopsy specimens available, sporadic plasma cells producing α3(IV)NC1-IgG were found, suggesting a causal relationship between the 2 diseases. One patient, who achieved remission with antibody clearance and normalization of serum creatinine and interleukin 6 concentrations after plasma exchange and 3 cycles of chemotherapy, experienced recurrence of anti-GBM antibodies and an increase in interleukin 6 concentration after chemotherapy discontinuation because of adverse effects, but both returned to normal after another cycle of chemotherapy. This clinical course and the pathologic findings support the hypothesis that the Castleman disease–associated tumor cells are the source of the anti-GBM autoantibodies.

Infection has long been suspected as a trigger of autoimmune diseases, and molecular mimicry mechanism was hypothesized in this study. Microbe originated peptides were searched from the Uniprot database based on a previous defined critical amino acid motif within α3129-150, isoleucine137, tryptophan140, glycine142, phenylalanine 143 and phenylalanine 145. 23826 microbial peptides were identified using our searching strategy, among which seven were related with human infections. Circulating IgG and IgM antibodies against the seven microbial peptides were detected using ELISA in 76 patients with anti-GBM disease. Four peptides were recognized by both IgG and IgM antibodies, and one peptide was recognized by IgG antibodies only. Peptides from Bacteroides, Saccharomyces cerevisiae, and Bifidobacterium thermophilum possessed the highest recognition frequency with the prevalence of 73.7%, 61.8% and 67.1% for IgG, 56.6%, 44.7% and 67.1% for IgM in anti-GBM patients. Patients with antibodies against these microbial peptides showed more severe kidney injury, including higher serum creatinine and higher percentage of crescent formation. In conclusion, antibodies against microbial peptides were identified in the circulation of anti-GBM patients, implying its etiological role in eliciting autoimmune response against α3(IV)NC1 through molecular mimicry.

Goodpasture's disease is closely associated with HLA, particularly DRB1*1501. Other susceptible or protective HLA alleles are not clearly elucidated. The presentation models of epitopes by susceptible HLA alleles are also unclear. We genotyped 140 Chinese patients and 599 controls for four-digit HLA II genes, and extracted the encoding sequences from the IMGT/HLA database. T-cell epitopes of α3(IV)NC1 were predicted and the structures of DR molecule-peptide-T-cell receptor were constructed. We confirmed DRB1*1501 (OR = 4·6, P = 5·7 × 10-28 ) to be a risk allele for Goodpasture's disease. Arginine at position 13 (ARG13) (OR = 4·0, P = 1·0 × 10-17 ) and proline at position 11 (PRO11) (OR = 4·0, P = 2·0 × 10-17 ) on DRβ1, encoded by DRB1*1501, were associated with disease susceptibility. α134-148 (HGWISLWKGFSFIMF) was predicted as a T-cell epitope presented by DRB1*1501. Isoleucine137 , tryptophan140 , glycine142 , phenylalanine143 and phenylalanine145 , were presented in peptide-binding pockets 1, 4, 6, 7 and 9 of DR2b, respectively. ARG13 in pocket 4 interacts with tryptophan140 and forms a hydrogen bond. In conclusion, we propose a mechanism for DRB1*1501 susceptibility for Goodpasture's disease through encoding ARG13 and PRO11 on MHC-DRβ1 chain and presenting T-cell epitope, α134-148 , with five critical residues.

Summary Background and objectives Linear epitopes on the Goodpasture autoantigen involved in human anti-glomerular basement membrane (GBM) disease are not fully defined. This study investigated the linear epitopes recognized by circulating antibodies in anti-GBM patients, aiming to identify the potential nephrogenic linear epitopes and their clinical significance. Design, setting, participants, & measurements Sixty-eight patients with anti-GBM disease were enrolled. Twenty-four overlapping linear peptides were synthesized across the whole sequence of the human Goodpasture autoantigen. ELISA detected circulating antibodies against linear epitopes. Their associations with clinical features were further analyzed. Results Antibodies against linear peptides were detected in sera from 55 patients (80.9%). Three major epitopes with high frequencies were identified: P14 (41%), P16 (36.8%), and P18 (57%). P14, a formerly defined T cell epitope, was a mutual B cell epitope. Antibodies against P14 were frequently detected in patients with positive antineutrophil cytoplasmic antibodies (39.3% versus 12.5%; P=0.01). Patients with anti-P16 antibodies presented with higher serum creatinine on diagnosis (665.5±227.2 versus 443.7±296.8 μmol/L; P=0.001) and worse renal outcome during follow-up (hazard ratio, 2.10; 95% confidence interval, 1.10–3.90; P=0.02). The level of anti-P18 antibodies positively correlated with the percentage of crescents in glomeruli (r=0.54; P=0.008). Recognition of P22 was an independent predictor for patient death (hazard ratio, 3.02; 95% confidence interval, 1.20–7.57; P=0.02). Conclusions Antibodies against linear epitopes on the Goodpasture autoantigen could be detected in human anti-GBM disease and were associated with kidney injury. P14 was a mutual T and B cell epitope, implying its nephrogenic role in disease initiation.

Aim: Cases with anti-glomerular basement membrane (GBM) disease have been reported with linear deposit of immunoglobulin G (IgG) along GBM, but have undetectable anti-GBM antibodies in circulation by enzyme linked immunosorbent assays (ELISA). We speculated that the structure of the antigens recognized by these antibodies may contribute to the negative results of ELISA. Methods: Sera from four patients were collected, with typical linear deposit of IgG along GBM but no anti-GBM reactivity by commercial ELISA kits. Circulating anti-GBM antibodies were detected by indirect immunofluorescence. Antigen specificity and its conformational structure was investigated by western-blot analysis, using recombinant human α1–α5(IV)NC1 and chimeric proteins EA and EB as antigens. Results: The presence of circulating anti-GBM antibodies were confirmed by indirect immunofluorescence with linear deposit of IgG towards cryptic epitopes along GBM on normal kidney sections. These antibodies did not recognize recombinant human α1, α2, α4 or α5(IV)NC1, but could blot α3(IV)NC1 under non-reducing non-boiling conditions on western-blot analysis, when the conformational epitope(s) on α3(IV)NC1 were thought to be preserved. When α3(IV)NC1 was prepared under reducing conditions with β-mercaptoethanol and/or boiled to destroy the disulfide bonds, the binding with the antibodies disappeared. Moreover, these antibodies recognized neither EA nor EB, indicating their distinct epitope repertoire. Conclusion: Circulating anti-GBM antibodies undetectable by ELISA could recognize cryptic and conformation-dependent epitopes restricted on α3(IV)NC1, distinct from EA and EB. Indirect immunofluorescence was necessary for antibody detection and treatment monitoring under such circumstances.

Desulfitobacterium hafniense strain DH is a sulfate-reducing species. Here, we report the draft genome sequence of strain DH, with a size of 5,368,588 bp, average G+C content of 47.48%, and 5,296 predicted protein-coding sequences.

Linear deposition of IgG and complement 3 (C3) along glomerular basement membrane (GBM) is generally revealed in the kidneys of human anti-GBM disease. Our recent studies demonstrated the pathogenic role of complement activation in renal damage of this disease. However, the pathways of complement activation were still paradoxical. In this study, renal biopsy tissues from 10 patients with anti-GBM disease were used to investigate the pathways of complement activation by detecting the deposition of various complement components, including C1q, factor B, factor P (properdin), mannose-binding lectin (MBL), C3d, C4d and C5b-9, using immunohistochemistry and immunofluorescence. We found that C1q, factor B, properdin, C3d, C4d and C5b-9 were detected in all the glomeruli of our patients, along GBM with a linear and/or granular staining pattern. Furthermore, C1q, factor B and properdin co-localized well with C5b-9. The properdin also co-localized well with C3d. However, the deposition of MBL was diffusive in mesangium, GBM, Bowman's capsule and within crescents and was not co-localized with C5b-9 but partially co-localized with C4d. The intensity of factor B deposition (3.3 vs. 1.2, P

Background Anti-glomerular basement membrane (GBM) disease is a well-known antibody-induced autoimmune disease. A few patients have glomerular C1q deposition, but it is usually absent on renal histopathology. The role of C1q deposition in kidney injury is unclear. Recently, anti-C1q antibodies are demonstrated to be pathogenic in the target organ damage of many autoimmune diseases, by facilitating C1q deposition and enhancing complement activation via classical pathway. In the current study, we investigated the associations between anti-C1q antibodies in sera and C1q deposition in kidney of patients with anti-GBM disease. Results It was shown that the severity of kidney injury was comparable between patients with and without C1q deposition, including the prevalence of oliguria/auria, the median percentage of crescents in glomeruli and the mean concentration of serum creatinine. Serum anti-C1q antibodies were detected in 15/25 (60%) patients with a low titer. The prevalence of C1q deposition in kidney was comparable between patients with and without serum anti-C1q antibodies (26.7% vs. 30.0%, p > 0.05). No association was found between anti-C1q antibodies and the severity of kidney injury. Conclusions The classical pathway of complement may not play a pathogenic role in the kidney injury of human anti-GBM disease. Anti-C1q antibodies could be detected in more than half of patients, which need further investigations.

Summary Background and objectives Antiglomerular basement membrane autoantibodies are pathogenic in antiglomerular basement membrane disease with two major epitopes, EA and EB, on α3 chain of type IV collagen. This study investigated the epitope spectrum of antiglomerular basement membrane autoantibodies, aiming to identify the association between epitope specificity and kidney injury. Design, setting, participants, & measurements All 108 patients with antiglomerular basement membrane disease and complete clinical data were divided into three groups according to renal dysfunction: mild group (n=20) with serum creatitine≤1.5 mg/dl; moderate group (n=22) with serum creatinine=1.5–6.8 mg/dl; severe group (n=66) with serum creatitine≥6.8 mg/dl. Epitope spectrums of antibodies were determined by ELISA, and their associations with kidney damage were analyzed. Sequential serum samples in 40 patients were examined during disease courses. Results EA and EB were recognized in 79.6% and 72.2% of patients, respectively. EA and EB reactions were the lowest in the mild group and higher in the moderate group (EA: 35.0% versus 81.8%, P=0.002; EB: 15.0% versus 68.2%, P=0.001). They were the highest in the severe group (EA: 92.4%, P=0.31; EB: 90.9%, P=0.02). Close association was observed between renal injury and EA and EB reactions. Multivariate Cox regression analysis showed that EB reaction was an independent risk factor for renal failure (hazard ratio=6.91, P=0.02). The recognition for non-EAB remained low among groups. No augmentation of epitope spectrum was shown in serial serum samples. Conclusions Intramolecular epitope spreading might occur before the onset of human antiglomerular basement membrane disease. The autoimmunity to EA and EB, especially EB, was crucial for kidney dysfunction.

The association of anti-glomerular basement membrane (GBM) disease, also known as Goodpasture's disease, with membranous nephropathy (MN) has been well documented. However, little is known about the clinical and immunological features of patients with such a combination. This study was designed to investigate the clinical and immunological features of anti-GBM patients with MN and to provide insight into the pathogenesis of this rare entity. Eight patients with combined anti-GBM disease and MN were found to have significantly lower levels of serum creatinine, a significantly lower proportion of oliguria/anuria, and significantly better renal outcomes compared with 30 patients with classical anti-GBM disease. Antibody levels against the EB conformational epitope of anti-α3(IV)NC1 were significantly lower in these patients, as was their levels of anti-α3(IV)NC1 immunoglobulin G1 (IgG1) and IgG3. Serum antibodies against the M-type phospholipase A2 receptor were undetectable in anti-GBM patients with MN but presented in 13 of the 20 patients with primary MN. Thus, patients with combined anti-GBM disease and MN have distinct clinical features and a different immunological profile of MN.

Background The pathophysiological significance of variable region glycosylation of autoantibodies is still unclear. In the current study, the influence of the variable region N-linked oligosaccharides on the reactivity of three autoantibody specificities was investigated with Sambucus nigra agglutinin (SNA), which mainly binds to oligosaccharides with terminal α2, 6-linked sialic acid on the variable region of IgG. Methods Twenty-seven patients with serum positive anti-neutrophil cytoplasmic autoantibodies (ANCA) against myeploperoxidase (MPO) or proteinase 3 (PR3), or autoantibodies against glomerular basement membrane (GBM) were included. Total IgG was isolated and separated into non-SNA-binding and SNA-binding fractions with SNA affinity chromatography. Antigen-specific IgG was purified by immunoaffinity chromatography. Results At the same concentration of IgG, the antigen binding level of non-SNA-binding IgG was significantly lower than that of SNA-binding IgG for MPO-ANCA (absorbance value at 405 nm, 0.572 ± 0.590 vs. 0.962 ± 0.670, P < 0.001) and for PR3-ANCA (0.362 ± 0.530 vs. 0.560 ± 0.531, P = 0.003). The antigen binding level of non-SNA-binding IgG was significantly higher than that of SNA-binding IgG for anti-GBM antibodies (1.301 ± 0.594 vs. 1.172 ± 0.583, P = 0.044). The level of variable region glycosylation of total IgG was significantly lower than that of affinity-purified MPO-ANCA (1.021 ± 0.201 vs. 1.434 ± 0.134, P = 0.004). The level of variable region glycosylation of total IgG was significantly higher than that of affinity-purified anti-GBM antibodies (1.034 ± 0.340 vs. 0.734 ± 0.333, P = 0.007). The SNA-binding fraction of MPO-ANCA-containing IgG and PR3-ANCA-containing IgG induced higher levels of neutrophil oxygen radical production than the corresponding non-SNA-binding fractions (P < 0.001 and P = 0.043, respectively). The level of variable region glycosylation of affinity-purified MPO-ANCA was higher in active AAV than the same patients in remission (P = 0.001). Conclusion Characteristics of variable region glycosylation of ANCA and anti-GBM antibodies were different from that of total IgG, which might influence the antigen-binding ability of these antibodies. Variable region glycosylation of ANCA might influence the effect of ANCA-induced neutrophils respiratory burst.

Anti-glomerular basement membrane (anti-GBM) disease and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis both could cause rapidly progressive glomerulonephritis. The coexistence of ANCAs and anti-GBM antibodies was known as “double positive,” which was extremely rare in children. We report a pediatric case with coexistence of ANCAs and anti-GBM antibodies. A 6-year-old girl presented with acute renal failure, hematuria, proteinuria, and oliguria. She was double positive of ANCAs specific to myeloperoxidase, and anti-GBM antibodies. Kidney biopsy confirmed linear immunoglobulin (Ig)G deposit along GBM and 100% of crescent formation in glomeruli; among them 83.3% were cellular crescents. Human leukocyte antigen (HLA) gene typing showed DRB1∗1501, an allele strongly associated with anti-GBM disease, and DRB1∗0405, an independent risk factor for renal failure in patients with ANCA-associated vasculitis. The titer of anti-GBM antibodies was 1:800, and the predominant IgG subclass was IgG1, which was closely related with severe kidney injury and worse outcome. The target antigen of anti-GBM antibodies was restricted on the noncollagen domain 1 of the α3 chain of type IV collagen (α3[IV]NC1), with recognitions to both epitopes, EA (α317–31) and EB (α3127–141). This is the first reported pediatric case with coexistence of ANCAs and anti-GBM antibodies, in which the HLA typing and immunologic characters of autoantibodies were identified. The findings on this early-onset patient are meaningful for understanding the mechanisms of both anti-GBM disease and ANCA-associated vasculitis.

Background The autoantigen of anti-glomerular basement membrane (GBM) disease has been identified as the non-collagenous domain 1 of α3 chain of type IV collagen, α3(IV)NC1. Our previous study revealed a peptide on α3(IV)NC1 as a major linear epitope for B cells and potentially nephrogenic, designated as P14 (α3129-150). This peptide has also been proven to be the epitope of auto-reactive T cells in anti-GBM patients. This study was aimed to further characterize the critical motif of P14. Methods 16 patients with anti-GBM disease and positive anti-P14 antibodies were enrolled. A set of truncated and alanine substituted peptides derived from P14 were synthesized. Circulating antibodies against the peptides were detected by enzyme linked immunosorbent assay (ELISA). Results We found that all sera with anti-P14 antibodies reacted with the 13-mer sequence in the C-terminus of P14 (P14c) exclusively. The level of antibodies against P14 was highly correlated with the level of antibodies against P14c (r=0.970, P

54 human genes were selected as test targets for parallel cloning, expression, purification and crystallization. Proteins from these genes were selected to have a molecular weight of between 14 and 50 kDa, not to have a high percentage of hydrophobic residues (i.e. more likely to be soluble) and to have no known crystal structures and were not known to be subunits of heterocomplexes. Four proteins containing transmembrane regions were selected for comparative tests. To date, 44 expression clones have been constructed with the Gateway cloning system (Invitrogen, The Netherlands). Of these, 35 clones were expressed as recombinant proteins in Escherichia coli strain BL21 (DE3)-pLysS, of which 12 were soluble and four have been purified to homogeneity. Crystallization conditions were screened for the purified proteins in 96-well plates under oil. After further refinement with the same device or by the hanging-drop method, crystals were grown, with needle, plate and prism shapes. A 2.12 A data set was collected for protein NCC27. The results provide insights into the high-throughput target selection, cloning, expression and crystallization of human genomic proteins.

Goodpasture antigen, the non‐collagenous domain of α3 chain of type IV collagen [α3(IV)NC1], is the target antigen of anti‐glomerular basement membrane (GBM) antibodies. The pathogenicity of T cell epitopes is not elucidated clearly. In this study, we aim to define the nephritogenic T cell epitopes and its critical amino acid residues. Twenty‐four overlapping linear peptides were synthesized covering the whole sequence of human α3(IV)NC1. Wistar–Kyoto rats were immunized with linear peptides, and experimental autoimmune glomerulonephritis was evaluated. Critical amino acid was identified by the loss of nephritogenic function after each amino acid substitution by alanine. Of the 24 peptides, P14 (α3127‐148) could induce 90.5% (19/21) of WKY rats developing anti‐GBM glomerulonephritis with proteinuria, elevated serum urea and creatinine, IgG linear deposit on GBM and substantial (in average 82.4 ± 5.6%) crescent formation in glomeruli. Lymphocytes of immunized rats proliferated in response to α3127‐148 and α3(IV)NC1 in vitro. Sera of these rats recognized α3127‐148 and later on together with intact human α3(IV)NC1. Antibodies towards α3127‐148 and intact α3(IV)NC1 could also be detected from the kidney elutes. These antibodies showed no cross‐reaction with each other, which implies intramolecular epitope spreading during disease progress. After sequential amino acid substitution, the α3127‐148 with substitution of tryptophan136, isoleucine137, leucine139 or tryptophan140 lost its nephritogenicity. Human α3127‐148 is a nephritogenic T cell epitope in WKY rats, with the critical amino acids as W136I137xL139W140. These findings might facilitate future investigation on microbial aetiology and potential specific immunotherapy of anti‐GBM disease.