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Scooped by
Gilbert C FAURE
October 28, 2018 5:53 AM
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Abstract Anti‐Ro52 autoantibodies (Ro52‐autoAbs) appear in the sera of connective tissue disease (CTD) patients with interstitial lung disease (ILD). Studies using patient sera have shown a correlation between the generation of Ro52‐autoAbs and the clinical morbidity and severity of CTD with ILD. In this study, we used a single B‐cell manipulating technology and obtained 12 different monoclonal Ro52‐autoAbs (mRo52‐autoAbs) from the selected four patients suffering from severe ILD with a high titer of Ro52‐autoAbs in their sera. Western blot analysis revealed that 11 of 12 mRo52‐autoAbs bound to the coiled‐coil domain of Ro52. Competitive ELISA demonstrated that mRo52‐autoAbs competed with each other to bind to Ro52. Epitope mapping showed that two of them specifically bound to a peptide (PEP08) in the coiled‐coil domain. We then examined the titer of Ro52‐autoAbs in the sera of 192 CTD patients and assessed the relationship between the serum levels of Ro52‐autoAbs that were reactive to PEP08 peptide and the clinical morbidity and severity of ILD. Statistical analysis revealed that the production of PEP08‐reactive Ro52‐autoAbs correlated with the morbidity and severity of ILD in CTD. Assessment of the production of PEP08‐reactive Ro52‐autoAbs in autoimmune diseases is useful for predicting the clinical morbidity of ILD. Introduction Ro52/TRIM21 is known as an E3 ligase involved in the process of ubiquitination and plays a prominent regulatory role in inflammation 1-7. Ro52 is the common target of autoantibodies in connective tissue diseases (CTDs) 8, 9, such as systemic lupus erythematosus (SLE) 10-12, Sjögren's syndrome (SS) 13-17, polymyositis/dermatomyositis (PM/DM) 18, 19, and systemic sclerosis (SSc) 20. Ro52‐specific autoantibodies (Ro52‐autoAbs) have been detected in the sera of many patients with various CTDs 9, 21. Some of these patients develop severe interstitial lung disease (ILD), and several reports have indicated that the production of Ro52‐autoAbs in patient sera is related to the incidence of ILD in CTD 22, 23. In SSc patients, the generation of Ro52‐autoAbs has been correlated to the morbidity and severity of ILD 23. In mixed connective tissue disease (MCTD) patients, Ro52‐autoAbs have been detected in 50% of patients with lung fibrosis 22. The Ro52 protein consists of multi‐domains, including an N‐terminal RING finger domain with E3 ubiquitin ligase activity, a B‐box domain, a coiled‐coil (C‐C) domain involved in oligomerization, and the C‐terminal PRYSPRY domain that commonly determines functions by acting as a target 24, 25. Some epitopes of Ro52‐autoAbs in SLE and SS patients have been reported 26, most of which reside in the C‐C domain of Ro52 27-34. The ring and PRYSPRY domains are also targets of Ro52‐autoAbs 29, 35. These epitopes were determined in patient sera using polyclonal antibodies. Concerning monoclonal Ro52‐autoAbs (mRo52‐autoAbs), Mietzner et al. generated monoclonal antibodies (mAbs) from IgG memory B cells of SLE patients and showed that some of them reacted to Ro52 36. However, there have been no reports of epitopes for human mRo52‐autoAbs produced in patients with ILD. In this study, we used our single cell analysis method denoted the immunospot array assay on a chip (ISAAC) 37, 38 to obtain mRo52‐autoAbs from the PBMCs of four DM patients suffering from severe ILD. We obtained twelve human mRo52‐autoAbs and showed that eleven of them bound to the C‐C domain of Ro52. We further determined an epitope for two of these mRo52‐autoAbs. Finally, we examined the relationship between the production of Ro52‐autoAbs that were reactive to the determined epitope in patient sera as well as the clinical morbidity and severity of ILD in CTD. Results Isolation of mRo52‐autoAbs from DM patients with severe ILD using the ISAAC system First, we examined 192 CTD patients to determine the titer of Ro52‐autoAbs in their sera by EUROLINE scan. We also examined them by scanning high‐resolution computed tomography (HRCT) and found that 57% (109/192) of these patients had ILD. The results agreed with previous studies 22, 23. We then tried to analyze the correlation of Ro52‐autoAb‐production and ILD. To achieve this goal, we tried to obtain mRo52‐autoAbs from DM patients because they are frequently complicated with ILD 39. We randomly selected 4 DM patients with severe ILD who also suffered from anti‐synthetase syndrome and had anti‐SS‐A, anti‐Jo‐1, or anti‐PL‐7 autoantibodies as well as Ro52‐autoAbs in their sera (Supporting Information Table 1). One hundred six antibody‐secreting cells (ASCs) that produced Ro52‐autoAbs were detected in the PBMCs of these four DM patients using ISAAC and retrieved from the chip (Fig. 1A). The antibody cDNAs were cloned from the single cells, and mAbs were produced. Fifteen recombinant mAbs specific to recombinant Ro52 (rRo52) protein were obtained. Sequence analysis revealed that three of 15 mRo52‐autoAbs had the same amino acid sequence as another clone, which indicated that these clones were derived from the same B‐cell clone (Table 1). We eventually obtained 12 different mRo52‐autoAbs from four DM patients with ILD (Fig. 1B). Heavy‐chain Light‐chain Pt. No. Name of mRo52‐autoAb Subclass V D J CDR3‐IMGT Family V J CDR3‐IMGT 100,112 110,112‐20 IgG1 IGHV3 IGHD6 IGHJ4 AKDWAQFVLRYFFDH κ IGKV1 IGKJ1 QQYDSYPWT 100,112 110,112‐19a IgG1 IGHV3 IGHD6 IGHJ4 AKDWAQFVLRYFFDH κ IGKV1 IGKJ1 QQYDSYPWT 100,112 110,112‐26 IgG1 IGHV7 IGHD6 IGHJ4 ARQGMFSTGWYLAH κ IGKV1 IGKJ1 QNYKSYSRT 100,112 110,112‐29 IgG1 IGHV7 IGHD4 IGHJ5 ARGALTMPTRVDP κ IGKV1 IGKJ5 QQTYSTLPIT 55 55‐37 IgG1 IGHV1 IGHD3 IGHJ4 ARSFYFGSGSYPY κ IGKV3 IGKJ2 QQYGSSPYT 55 55‐34a IgG1 IGHV1 IGHD3 IGHJ4 ARSFYFGSGSYPY κ IGKV3 IGKJ2 QQYGSSPYT 150 150‐08 IgG1 IGHV3 IGHD1 IGHJ4 ARGKSWLDY κ IGKV3 IGKJ4 QQYGSSLT 150 150‐14 IgG1 IGHV1 IGHD2 IGHJ5 ARGVYRRSGGNSWGFDP κ IGKV2 IGKJ4 MQALQTPPT 150 150‐42 IgG1 IGHV3 IGHD1 IGHJ4 ARDLGGGRRIYYFDY κ IGKV3 IGKJ4 QQYGSSPL 150 150‐44 IgG1 IGHV3 IGHD3 IGHJ4 AKVIGITFGGVIFPFDY κ IGKV3 IGKJ1 QQYGTAPT 150 150‐45 IgG1 IGHV3 IGHD3 IGHJ4 AKGFGTTFGGVIFPFDY κ IGKV3 IGKJ1 QQYSTSPT 150 150‐46 IgG1 IGHV3 IGHD5 IGHJ4 ARGKYWLDY κ IGKV3 IGKJ4 QQYGSSLT 150 150‐60 IgG1 IGHV3 IGHD4 IGHJ3 ARARFRTTVTTYAFDI κ IGKV2 IGKJ1 MQALQTPPWT 150 150‐61 IgG1 IGHV3 IGHD2 IGHJ4 ARERGGFCSGGSCYSCDY κ IGKV2 IGKJ1 MQALQTPRT 150 150‐58a IgG1 IGHV3 IGHD2 IGHJ4 ARERGGFCSGGSCYSCDY κ IGKV2 IGKJ1 MQALQTPRT a same as the above‐mentioned mRo52‐autoAbs were obtained PBMCs from 4 patients (#55, #100, #112, and #150). PBMCs of 2 patients (#100, #112) were mixed since the number of PBMCs of these patients were too small for ISAAC. 15 mAbs were cloned and found to bind rRo52 protein in which 3 mAbs had the same amino acid sequence. Final number of mRo52‐autoAbs became 12. V, D, J gene segments and CDR3‐IMGT in heavy chain as well as V, J gene segments and CDR3‐IMGT in light chain are shown. Epitope mapping To determine the epitopes of mRo52‐autoAbs, we first synthesized the various truncated fragments of rRo52 and then examined the reactivity of each mRo52‐autoAb to the fragments (Fig. 2A and B). Eleven of 12 mRo52‐autoAbs reacted with the C‐C domain as well as the full‐length rRo52 protein. One mRo52‐autoAb (Ab100, 112–20) reacted with the full‐length protein but not with any of the other fragments. We then examined whether these eleven mRo52‐autoAbs competed for binding to a specific site of the C‐C domain using competitive ELISA as previously reported 40. mRo52‐autoAbs together with one of the peroxidase (POD)‐conjugated mRo52‐autoAbs were added to wells that were coated with rRo52 proteins. The bound enzyme activity was then measured after washing the samples. As shown in Fig. 3A, all eleven mRo52‐autoAbs, but not the control mAb, inhibited the binding of POD‐conjugated Ab55‐37 mAb. Similarly, all 11 mRo52‐autoAbs inhibited the binding of other POD‐conjugated mRo52‐autoAbs (Ab150‐61, Ab150‐42, and Ab150‐45). These results suggest that the mRo52‐autoAbs that were obtained from DM patients with severe ILD (DM‐ILD patients) bind to the same or juxtaposed epitopes of Ro52. We then synthesized the peptide library of the C‐C domain of Ro52 (Fig. 3B and Supporting Information Table 2) to determine the precise epitope for mRo52‐autoAbs and examined the binding of mRo52‐autoAbs to these peptides. Two of 11 mRo52‐autoAbs (Ab55‐37 and Ab150‐61) were used for the binding assay because they showed clear binding activity to the C‐C domain (Fig. 2B). Those two mRo52‐autoAbs reacted with a peptide (PEP08, RIHAEFVQQKNFLVEE) but not with the other peptides of the C‐C domain or the control HIV‐env peptide (Fig. 3C). It is notable that these two mRo52‐autoAbs were obtained from PBMCs of different patients (Table 1). These data show that PEP08 contains an epitope of the mRo52‐autoAbs. These data, together with the competitive‐binding data (Fig. 3) indicate that all the mRo52‐autoAbs obtained by the ISAAC system bind to this epitope or a juxtaposed epitope. Relationship between the production of PEP08‐reactive Ro52‐autoAb and the morbidity of ILD in CTD As described above, the production of Ro52‐autoAb was reported to be clinically associated with ILD in SSc and MCTD 22, 23. To investigate the relationship between the production of the Ro52‐autoAbs that bind to PEP08 in the sera of CTD patients and the incidence and severity of ILD, 192 CTD patients were selected from our hospital whose Ro52‐autoAb titer was examined by EUROLINE scan (Fig. 4A). Overall, 43% (82/192) of them were positive for Ro52‐autoAb, while the rest (110/192) were negative. The reactivity of Ro52‐autoAb sero‐positive sera to PEP08 was analyzed. The results showed that the Ro52‐autoAb from 45% (37/82) of the patients bound to PEP08, whereas that from 55% (45/82) of the patients did not (Fig. 4A and B). All the profiles of PEP08‐Ab‐positive, PEP08‐Ab‐negative, and Ro52‐autoAb‐negative patients with CTD are shown in Supporting Information Table 3. We then compared the morbidity and severity of ILD in 38 patients in the PEP08‐Ab‐positive group and 154 patients in the PEP08‐Ab‐negative group using an odds ratio and Fisher's exact test. In these analyses, we assumed that 110 patients who were Ro52‐autoAb‐negative were negative for PEP08‐Ab (Fig. 4A). The assumption was confirmed by analyzing 14 Ro52‐autoAb‐negative patients in Supporting Information Table 3. They were also negative for PEP08‐Ab (Fig. 4C). For the morbidity of ILD, 30/38 (79%) patients in the PEP08‐Ab‐positive group had ILD. In contrast, 80/154 (52%) patients in the PEP08‐Ab‐negative group had ILD (Fig. 4A and D, odds ratio = 3.47, P = 0.0031, CI: 1.50–8.05). We also compared the severity of ILD in the PEP08‐Ab‐positive group and the PEP08‐Ab‐negative group using the KL‐6 value that had been considered to be an indication of severity of ILD 41, 42.Overall, 26/33 (79%) patients in the PEP08‐Ab‐positive group had a high KL‐6 value (KL‐6 ≥ 500). In contrast, 82/143 (57%) patients in the PEP08‐Ab‐negative group had a high KL‐6 value (KL‐6 ≥ 500) (Fig. 4A and E, odds ratio = 2.65, P = 0.0285, CI: 1.13–6.78). There was no correlation between the titer of PEP08‐Ab and the KL‐6 value (Supporting Information Fig. 1). The results indicate that PEP08‐Ab‐positive CTD patients had a higher indication of morbidity and severity of ILD than PEP08‐Ab‐negative CTD patients. Discussion Ro52 is the common target of autoAbs in CTD, and the production of Ro52‐autoAbs in patient sera has been reported to be related to the incidence of ILD with CTD 22, 23; however, there have been no reports concerning the epitopes of Ro52 protein related to ILD. To gain an understanding of the relationship of the Ro52‐autoAb epitopes and ILD, we obtained 12 human mRo52‐autoAbs from peripheral blood B cells of DM‐ILD patients (Table 1 and Fig. 1) and determined their antigenic epitopes. We found that 11 of them reacted with the C‐C domain of the Ro52 protein (Fig. 2). Previous studies have also demonstrated that Ro52‐autoAbs reacts with the C‐C domain using serum of SS or SLE patients 27-34. The C‐C domain plays an important role in protein–protein interactions and is usually protruding from the main body of proteins. Thus, it tends to be recognized by B cells using their antigen receptors on the cell surface. We could not determine the epitopes of one mRo52‐autoAb, Ab100/112‐20, which reacted only with the full‐length protein, indicating that it may recognize the discontinuous conformational epitope formed by the two domains. In contrast, the remaining mRo52‐autoAbs may bind to linear epitopes since they recognized the antigen in immunoblotting with SDS‐denatured and linearized proteins. However, we could not exclude the possibility that they recognized refolded proteins during immunoblotting. Regarding the epitopes of Ro52‐autoAbs in various CTDs, their epitopes have been reported to be distributed in various rRo52 fragments (Supporting Information Table 4). In this study, by competitive ELISA (Fig. 3A) and epitope mapping using a peptide library (Fig. 3B and C), we found that two mRo52‐autoAbs from DM‐ILD patients bound to a Ro52 epitope (PEP08), and the remaining nine Abs bound to its juxtaposed epitopes. Interestingly, those two mRo52‐autoAbs were obtained from different patients. This outcome posed a strong possibility that PEP08 is one of the important epitopes for ILD in CTD, although we must consider that mRo52‐autoAbs obtained from DM patients may have biased the results. Wahlen et al. reported an epitope of Ro52‐autoAbs that was found to show production correlated with the onset of congenital heart block (CHB) named “P200” (aa200–239) 43. The maternal mRo52‐autoAbs of CHB patients did not react to the peptide named “P176” (aa176‐196). In contrast, our mRo52‐autoAbs from DM‐ILD patients reacted with the PEP08 (aa177‐192) involved in P176. Although all these peptides were included in the C‐C domain (aa133‐255), each epitope may play a different role in oligomerization and might be involved in the onset of different CTD. Concerning the functional role of Ro52‐autoAbs in the diseases, we investigated how the generation of PEP08‐specific Ro52‐autoAbs in the CTD patients affected the incidence and severity of ILD (Fig. 4). We showed that the PEP08‐Ab‐positive group had a higher morbidity of ILD (odds ratio = 3.47, p = 0.0031, CI: 1.50–8.05) (Fig. 4D). The group also exhibited a significant relationship to the severity of ILD (odds ratio = 2.76, p = 0.0285, CI: 1.13–6.78) (Fig. 4E). The results showed that the production of PEP08‐reactive Ro52‐autoAbs influenced the morbidity of ILD in CTD. Although the molecular mechanisms in which Ro52‐autoAbs induce a high risk of ILD are not known, further analysis of the in vivo effect of mRo52‐autoAbs using an animal model may facilitate further understanding of involvement of PEP08‐reactive Ro52‐autoAbs to the onset of ILD in CTD. This study has the following limitations: (1) The titer of KL‐6 was not assessed for some patients who did not develop ILD, as shown in Supporting Information Table 3). The epitopes of Ro52‐autoAbs produced in various CTDs were distributed on various Ro52 fragments (Supporting Information Table 4). Here, we determined the PEP08 epitope using mRo52‐autoAbs from DM‐ILD patients. Thus, the selection of DM patients may have biased the results. (3) In this study, we focused on anti‐Ro52 autoantibodies of CTD patients and investigated the clinical correlation of a mRo52‐autoAbs epitope to ILD. Although PEP08‐Ab‐positive CTD patients had a higher indication of morbidity and severity of ILD than PEP08‐Ab‐negative CTD patients, it remains unknown why many PEP08‐negative patients still had ILD and a high KL‐6 value. For the onset of ILD, multiple risk factors 41 should be considered to be involved. One of the risk factors is autoantibodies; however, it has been reported that ILD‐related autoantibodies are not only anti‐Ro52 autoantibodies but also anti‐PL‐7 and anti‐Jo‐1 autoantibodies 39, 44, among others. Thus, analysis of the involvement of anti‐PL‐7 and anti‐Jo‐1 autoantibodies in ILD may contribute to understanding the morbidity and severity of many PEP08‐negative patients. It was recently reported that the production of Ro52‐autoAbs in patient sera were related to undifferentiated connective tissue diseases (UCTD) 45, 46. Measurement of PEP08‐Ab in sera of UCTD patients may contribute to the prediction of ILD. In summary, we obtained twelve mRo52‐autoAbs from four DM‐ILD patients. Eleven of these mRo52‐autoAbs bound to the C‐C domain in immunoblotting, and they competed with each other to bind to the C‐C domain. We determined the binding site of mRo52‐autoAb using the peptide library and analyzed the relationship of the production of PEP08‐Ab and the clinical morbidity and severity of ILD. We showed that the PEP08‐Ab‐positive patients exhibited a higher incidence risk of ILD than PEP08‐Ab‐negative patients. Future studies using the mRo52‐autoAbs in vivo system may be required to understand the pathological function of Ro52‐Abs in ILD with CTD. Materials and methods Patients and their characterization Patients who visited the First Department of Internal Medicine at Toyama University Hospital and were diagnosed with CTD were included in the study (Fig. 4). Informed consent was obtained from all patients in accordance with a protocol approved by the Ethical Review Board of the University of Toyama. The characteristics of the patients at the time of enrollment are summarized in Supporting Information Table 3. The patients included 145 females (76%) and 47 males (24%). Their median age was 63 years. Sixty patients (31%) had PM/DM, 44 (23%) had SSc, 40 (21%) had rheumatoid arthritis, 17 (9%) had SS, 13 (7%) had MCTD, 10 (5%) had SLE, 6 (3%) had amyopathic dermatomyositis, and 2 (1%) had vasculitis syndrome. The Ro52‐autoAb‐titer in their sera was examined by a EUROLINE scan (Cosmic Corporation, Japan). In the EULOLINE scan, recombinant Ro‐52 (52 kDa) produced with the baculovirus system in insect cells is used to detect Ro52‐autoAbs. We defined the Ro52‐autoAb titer as “high” when the EUROLINE scan determined the titer as 2± or 3±. Diagnosis of ILD was performed by examining the interstitial markings in the lung in HRCT. The severity of ILD was examined by measuring the KL‐6 value (SRL, Tokyo, Japan). In this study, KL‐6 was used as a potential biomarker of ILD in CTD 41, 47, 48. KL‐6 was measured using electrochemiluminescence immunoassay as described previously 49. The sensitivity, specificity, and diagnostic accuracy were 93.9%, 96.3%, and 95.7% for KL‐6; 81.8%, 86.6%, and 85.2% for SP‐A; 69.7%, and 95.1%, and 87.8% for SP‐D; and 51.5%, 92.7%, and 80.9% for MCP‐1; respectively 41, 47, 48, indicating that KL‐6 is a superior serum biomarker for ILD compared with SP‐A or SP‐D. It has also been reported that KL‐6 is negatively well correlated with DLco and FVC 50 and positively correlated with the HRCT score 51 in cases of ILD with CTD. Severe ILD was defined when the KL‐6 value was greater than 500 U/mL. Detection of ASCs using the immunospot array assay on a chip (ISAAC) PBMCs were prepared from 10 mL of heparinized peripheral blood from patients with a high serum titer of Ro52‐autoAbs after centrifugation at 400 × g using a Ficoll‐Hypaque gradient. CD138+ cells were enriched with human CD138 MicroBeads (Miltenyi Biotec) using an autoMACS Pro‐separator (Miltenyi Biotec) according to the manufacturer's instructions. The ISAAC method is covered by patents that have been exclusively licensed to Valneva (Nantes, France). Details and instructions regarding the microwell array chip and the ISAAC method have been previously described 37, 38. Briefly, we coated the surface of the chip with 10 μg/mL recombinant Ro52 (rRo52) protein (ProSpec) in PBS by incubating the chip overnight at 4°C. After removing the antibody solution, we blocked the chip with 0.01% Biolipidure (NOF Corporation, Japan) for 30 min at room temperature (r.t.) and subsequently washed the chip with the culture medium (RPMI 1640 containing 10% fetal calf serum, 50 μM 2‐mercaptoethanol, 100 μg/mL streptomycin, and 100 U/mL penicillin). We then arrayed the CD138+ cells in culture medium on the chip and removed the residual cells outside the wells with gentle washing. We cultured the cells on the chip for 3 h at 37°C. After gentle washing, we added 1 μg/mL Cy3‐conjugated anti‐human IgG‐Fc antibody (Millipore) for 30 min. Finally, we stained the cells with 1 μM Oregon Green (Molecular Probes) for 5 min at r.t. After gentle washing, antigen‐specific antibody‐secreting cells were retrieved under a fluorescence microscope (BX51WI, Olympus) using a capillary with a micromanipulator. Amplification of antibody cDNA, production of antibody, and purification of antibody We amplified antibody cDNAs encoding the VH and VL fragments using a single‐cell 5’‐RACE method with primers for the γ, κ, and λ chains. We then inserted these cDNAs into expression vectors that contained cDNA encoding the whole immunoglobulin constant region for IgG1 or the light chains, as previously described 38, 52. Then, we co‐transfected Expi293FTM cells with both the heavy and light chain expression vectors using the Expi293FTM Expression System (Life Technologies). We then collected the culture supernatant and purified the antibody using a protein G column as previously described 38. The immunoglobulin gene repertoire was analyzed with the IMGT/V‐Quest tool (http://www.imgt.org/). Identification of mRo52‐autoAbs We coated the wells of 96‐well plates (Nunc) with 50 μL of 0.5 μg/mL rRo52 protein in PBS overnight at 4°C. The next day, we blocked the wells with 3% BSA in PBS containing 0.1% Tween 20 (PBST). After washing the wells, we added to the wells monoclonal antibodies (1 μg/mL) and patient sera in PBST and incubated the plate at r.t. for 1 h. After washing the wells, we added alkaline phosphatase‐conjugated anti‐human IgG Fc‐antibody (Sigma) and incubated the plate at r.t for 1 h. We washed the wells and detected the amount of bound antibodies using p−nitrophenylphosphate (Sigma). Subsequently, we measured the optical absorbance at 405 nm with an ELISA reader according to the manufacturer's instructions. Western blotting of mutant recombinant Ro52 (rRo52) proteins with mRo52‐autoAbs We conjugated peroxidase to mRo52‐autoAbs or an anti‐influenza nucleoprotein antibody 38, 53 (control antibody) using the Peroxidase Labeling Kit‐NH2 (Dojindo, Japan) according to the manufacturer's instructions. Full‐length or truncated rRo52 proteins were produced in vitro with the cell‐free protein synthesis system using purified wheat embryos as previously described 54. Briefly, we amplified cDNAs encoding full‐length or truncated rRo52 proteins by PCR and inserted them into a pEU‐E01 expression vector (CellFree Sciences, Ehime, Japan). All proteins were fused to a D1CE epitope tag at the C‐terminus 55. We then produced recombinant proteins using the WEPRO7240 Expression Kit (CellFree Sciences) according to the manufacturer's instructions. Next, we resolved the rRo52 proteins using SDS‐PAGE with 5–20% gradient acrylamide gel (BIO CRAFT, Tokyo, Japan) under reducing conditions and transferred them to an Immobilon‐P nylon membrane (Millipore). We treated the membrane with Blocking One (Nacalai Tesque, Kyoto, Japan) and probed it with peroxidase‐conjugated antibodies. Visualization was performed using the ECL detection system (GE Healthcare). To confirm the loading of recombinant proteins on the blotting membrane, we washed the membrane with PBST for a few minutes and inactivated the peroxidase of the peroxidase‐conjugated mRo52‐autoAb 0.01% sodium azide for 30 min followed by a wash with PBST for a few minutes. We then treated the membrane with Blocking One and probed it with peroxidase‐conjugated anti‐D1CE tag antibodies. Competitive ELISA assay with mRo52‐autoAbs We coated the wells of 96‐well Maxisorp plates (Nunc) with 50 μL of 5 μg/mL rRo52 in PBS and incubated them at 4°C overnight. The next day, we blocked the wells with 3% BSA in PBS at r.t. for 1 h. We prepared the solution containing 10 clones of mRo52‐autoAbs or control mAb at 10 μg/mL and one of the clones of mRo52‐autoAbs conjugated with peroxidase at 0.1 μg/mL. After washing the wells, we added 50 μL of a clone of unconjugated mRo52‐autoAb to each well with 50 μL of a clone of peroxidase‐conjugated mRo52‐autoAb and incubated them at r.t. for 1 h. After washing the samples, we added 50 μL of TMB solution (KPL) and incubated the samples for 30 min at r.t.; then, we added 50 μL of stop solution (KPL). We measured the optical absorbance at 450 nm with an ELISA reader (BMG LABTECH) according to the manufacturer's instructions. ELISA of mAbs with a peptide library We first prepared the peptide library for the C‐C domain of Ro52 (Eurofins Genomics). The amino acid sequences are shown in Supporting Information Table 2. This library had 21 peptides. Each peptide consisted of 16 amino acids and overlapped by eight amino acids with an adjacent peptide. We then coated the wells of 96‐well Maxisorp plates (Nunc) with 50 μL of 5 μg/mL peptides in PBS and incubated them at 4°C overnight. The next day, we blocked the wells with 3% BSA in PBS. After washing the samples, we added the peroxidase‐conjugated mRo52‐autoAb or control mAb to the wells and incubated them at r.t. for 1 h. After washing the cells, we detected the antibodies that were bound to antigens using a TMP detection system (KPL). Subsequently, we measured the optical absorbance at 450 nm with an ELISA reader (BMG LABTECH) according to the manufacturer's instructions. ELISA of patient serum samples with the peptides We coated the wells of 96‐well Maxisorp plates (Nunc) with 50 μL of 10 μg/mL peptide (PEP08: RIHAEFVQQKNFLVEE) or HIV env gp160 peptide (RYLRDQQLL) (MBL) in PBS and incubated the cells overnight at 4°C. The next day, we blocked the wells with 3% BSA in PBS. After washing the wells, we added the patient sera to the wells and incubated the plate at r.t. for 1 h. After washing the wells, we added alkaline phosphatase‐conjugated anti‐human IgG Fc‐antibody (Sigma) and incubated the plate at r.t for 1 h. We washed the wells and detected the amount of bound antibodies using p−nitrophenylphosphate (Sigma). Subsequently, we measured the optical absorbance at 405 nm with an ELISA reader according to the manufacturer's instructions. We performed an ELISA three times per patient. When the mean of the OD(PEP08)/OD(HIV) was higher than 1 + SD, we classified the patients in the “PEP08‐positive group.” When the mean of OD(PEP08)/OD(HIV) was less than 1 + SD, the patients were classified in the “PEP08‐negative group.” Acknowledgments We thank Sanae Hirota for technical assistance and Kaoru Hata for secretarial work. This research was supported by research grants from Chugai Pharmaceutical Co., LTD. (H.T.) and from the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from the Ministry of Education, Culture, Sports, Science (MEXT), Japan Agency for Medical Research and development (AMED) (A.M.); Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP18am0101077 (T.O.), and JSPS KAKENHI Grant Number 16H06499 (H.K.). All authors designed the experiments. M.O., T.O., Y.N., and H.K. performed the experiments. M.O., R.T., K.S., H.T., and K.T. acquired clinical data. M.O., T.O., H.K., and A.M. analyzed the experiments and wrote the manuscript. Conflicts of interest Okumura, Ozawa, Kishi, Taki, Tobe, and Muraguchi have a patent application pending for the Ro52 autoantibody (JP2016‐220793). The rest of the authors declare no commercial or financial conflict of interest. Supporting Information References Abbreviations ASC antibody‐secreting cell CTD connective tissue disease HRCT high‐resolution computed tomography ILD interstitial lung disease ISAAC immunospot array assay on a chip MCTD mixed connective tissue disease POD peroxidase SLE systemic lupus erythematosus UCTD undifferentiated connective tissue disease
The role of extrafollicular B cells in human systemic lupus is unknown. Jenks et al.
define the main components of this pathway and its prominence in severe disease. Its activation is mediated by hyper-responsiveness to Toll-like receptor-7 and leads to the generation of autoreactive...
Via Krishan Maggon
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Scooped by
Gilbert C FAURE
September 29, 2018 1:41 PM
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Conventional B cells express clonally specific antigen receptors, but a small subset of B cells from patients and mice with systematic lupus erythematosus simultaneously expresses two distinct antigen receptors.
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Scooped by
Gilbert C FAURE
August 20, 2018 1:55 AM
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Lupus patients can experience photosensitivity as manifested by joint pain, fatigue, and skin rashes. To better understand the mechanisms behind this photosensitivity, Shipman et al. examined patient skin samples and mouse models of lupus. They saw that, in healthy mice and human skin exposed to ultraviolet radiation, Langerhans cells protected keratinocytes from apoptosis through a mechanism involving the metalloproteinase ADAM17 and EGFR ligands. In the lupus mice and patient samples, the Langerhans cells were unable to do this. Stimulating EGFR in the skin may bring relief to photosensitive lupus patients.
Photosensitivity, or skin sensitivity to ultraviolet radiation (UVR), is a feature of lupus erythematosus and other autoimmune and dermatologic conditions, but the mechanistic underpinnings are poorly understood. We identify a Langerhans cell (LC)–keratinocyte axis that limits UVR-induced keratinocyte apoptosis and skin injury via keratinocyte epidermal growth factor receptor (EGFR) stimulation. We show that the absence of LCs in Langerin–diphtheria toxin subunit A (DTA) mice leads to photosensitivity and that, in vitro, mouse and human LCs can directly protect keratinocytes from UVR-induced apoptosis. LCs express EGFR ligands and a disintegrin and metalloprotease 17 (ADAM17), the metalloprotease that activates EGFR ligands. Deletion of ADAM17 from LCs leads to photosensitivity, and UVR induces LC ADAM17 activation and generation of soluble active EGFR ligands, suggesting that LCs protect by providing activated EGFR ligands to keratinocytes. Photosensitive systemic lupus erythematosus (SLE) models and human SLE skin show reduced epidermal EGFR phosphorylation and LC defects, and a topical EGFR ligand reduces photosensitivity. Together, our data establish a direct tissue-protective function for LCs, reveal a mechanistic basis for photosensitivity, and suggest EGFR stimulation as a treatment for photosensitivity in lupus erythematosus and potentially other autoimmune and dermatologic conditions.
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Scooped by
Gilbert C FAURE
August 2, 2018 2:25 AM
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Systemic lupus erythematosus (SLE) is an autoimmune disease characterised by a breakdown in immune tolerance that induces an attack on normal tissue
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Scooped by
Gilbert C FAURE
July 17, 2018 2:01 PM
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Lupus is a complex heterogeneous disease characterised by autoantibody production and immune complex deposition followed by damage to target tissues. Animal models of human diseases are an invaluable tool for defining pathogenic mechanisms and testing of novel therapeutic agents. There are perhaps more applicable murine models of lupus than any other human disease. There are spontaneous models of lupus, inducible models of lupus, transgenic-induced lupus, gene knockout induced lupus and humanised mouse models of lupus. These mouse models of lupus have contributed significantly to our knowledge of the pathogenesis of lupus and served as valuable preclinical models for proof of concept for new therapies. Despite their utility, mouse models of lupus have their distinct limitations. Although similar, mouse and human immune systems are different and thus one cannot assume a mechanism for disease in one is translatable to the other. Efficacy and toxicity of compounds can vary significantly between humans and mice, also limiting direct translation. Finally, the heterogeneous aspects of human lupus, both in clinical presentation, underlying pathogenesis and genetics, are not completely represented in current mouse models. Thus, proving a therapy or mechanism of disease in one mouse model is similar to proving a mechanism/therapy in a limited subset of human lupus. These limitations, however, do not marginalise the importance of animal models nor the significant contributions they have made to our understanding of lupus.
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Scooped by
Gilbert C FAURE
July 10, 2018 6:28 AM
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Autoantibodies targeting TLR and SMAD pathways define new subgroups in systemic lupus erythematosus
Journal of Autoimmunity, Volume 91
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Scooped by
Gilbert C FAURE
July 4, 2018 11:09 AM
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Scooped by
Gilbert C FAURE
June 19, 2018 4:02 AM
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Objective The type I interferon pathway is activated in many patients with systemic lupus erythematosus (SLE), and anti-double-stranded DNA (dsDNA) and anti-RNA binding protein autoantibodies are correlated with high interferon-α (IFNα) activity. We studied whether antiphospholipid (APL) antibodies, which should not stimulate Toll-like receptors, are also associated with high levels of IFNα activity.
Methods Serum IFNα activity was measured in patients with SLE using the WISH cell bioassay. IgG APL, anti-RBP and anti-dsDNA antibodies were measured in the clinical laboratory, and standard clinical cut-offs were used to define the positive results.
Results High IFNα activity was associated with anti-RBP and anti-dsDNA antibodies in all three ancestral backgrounds. Strikingly, African-American subjects with a positive APL antibody test had higher IFNα activity than those without IgG APL antibodies. This was not shared with other ancestral backgrounds. This finding was independent of other autoantibody profiles, and clinical features did not differ between IgG APL antibody positive versus negative African-American patients.
Conclusion The difference in association between IFNα activity and IgG APL status between ancestral backgrounds supports differences in molecular pathogenesis. This may suggest B cell hyperactivity in the setting of type I IFN in African-Americans and could suggest ways to individualise therapy.
AbstractObjective.We previously described the multiplex autoantibody SLE-key Rule-Out test, which detects a signature of autoantibody reactivity that distingu...
Via Krishan Maggon
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Suggested by
LIGHTING
May 11, 2018 12:23 PM
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Scooped by
Gilbert C FAURE
April 30, 2018 2:23 AM
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Objectives In a post-hoc analysis, we aimed to validate the Lupus Low Disease Activity State (LLDAS) definition as an endpoint in an systemic lupus erythematosus (SLE) Phase IIb randomised controlled trial (RCT) (MUSE [[NCT01438489][1]]) and then utilize LLDAS to discriminate between anifrolumab...
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Rescooped by
Gilbert C FAURE
from Laboratory Medicine
April 21, 2018 2:16 AM
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There are at least 100 different autoimmune diseases that can affect any organ of the body. In the USA, it is estimated that autoimmune diseases affect between 5% and 8% of the population (14.7–23.5 million people), but individual autoimmune diseases like Addison’s disease, autoimmune neuropathies and ACNA vasculitis can certainly be classified as rare (affecting less than 200,000 people [USA] or fewer than 1 in 2000 of the population [Europe]). Nevertheless, autoimmune diseases often require lifelong treatment, are considerably more prevalent among women and are among the 10 leading causes of mortalilty in women under 65 years. It is also interesting to compare the prevalence of autoimmune diseases to, for example, heart disease (22%) and cancer (9%), which seem to have a much higher public profile [1] and research support with funding quoted in billions of dollars rather than the millions for autoimmune diseases. The incidence of autoimmune diseases is increasing. For example, the International Diabetes Atlas reported that in 2017 there were 10 million more adults worldwide with type I diabetes that in 2015, and if this trend continues, by 2045, 67 million Europeans will be living with type I diabetes [2]. Over the last 20 years, our ability to detect and quantify the antibodies involved in autoimmune diseases has improved, and gradually such tests are becoming incorporated onto high throughput platforms often in Clinical Blood Sciences. However, autoantibody measurements can lack clinical specificity and sensitivity and show high variation between platforms and tests, and certified reference materials are only recently available for only a small number of autoantibodies. The number of requests is increasing far beyond what would be expected for a tests for rare conditions and with low pretest probabilities. Numerical reports give no impression of the “uncertainty” of the result, and the enthusiasm for automated analysis and reporting puts the responsibility on the requesting clinician to interpret the result, often without understanding of the limitations of the test. This issue of Clinical Chemistry and Laboratory Medicine highlights the diversity of autoimmune disease from organ-specific conditions to systemic multiorgan disease. It reminds us that there is significant overlap between Clinical Chemistry and Immunology and that we have an important role the diagnosis and management of a variety of autoimmune disease. We can clearly see how much work is being done to investigate, understand and improve autoantibody testing, but there is still much to be done to optimise the requesting, the analytical process and the interpretation of these complex assays. More than 90% of cases of Addison’s disease have an autoimmune aetiology with antibodies to adrenal tissue and more specifically antibodies to 21-hydroxylase being markers of the disease. The symptoms of adrenal insufficiency can be subtle, but patients may also present as an emergency in Addisonian crisis. It is also well known that Addison’s is associated with other autoimmune endocrinopathies, e.g. Grave’s disease, hypothyroidism or type I diabetes. Manso et al. [3] describe a patient with autoimmune hypothyroidism where the patient’s family history led to the finding of positive anti-adrenal antibodies. Further investigation confirmed the diagnosis of subclinical Addison’s and treatment was started. Their literature review showed that only 1% of patients with autoimmune thyroid disease had anti-adrenal-cortex antibodies. Use of a more sensitive ELISA-based assay increased the detection of antibodies to 21-hydroxylase in children with autoimmune thyroid disease to approximately 4 percent, although even at this frequency, it would only be worth checking for anti-adrenal antibodies in patient with clinical features or family history. Surprisingly, anti-adrenal-cortex antibodies are found in a much greater proportion of patients with premature ovarian failure and in chronic hypoparathyroidism, so screening for antibodies to 21-hydroxylase in such patients may be useful. Classically, adrenal antibodies have been detected by indirect immunofluorescence. This can be technically demanding, and the interpretation of the staining needs considerable training and experience. del Pilar Larosa et al. [4] have reported the development of an ELISA for 21-hydroxylase, the enzyme, which is the important antigen within the adrenal cortex. They report good specificity and sensitivity for Addison’s disease so this may be a better method for supporting a diagnosis of Addison’s than the qualitative immunofluorescence techniques. Type I diabetes also has an autoimmune aetiology, and in patients where the clinical and biochemical presentation is complex, antibodies to insulin, glutamic acid decarboxylase (GAD), insulinoma antigen (IA2) and zinc transporter 8 (ZnT8) may be measured to improve the diagnostic power. Censi et al. [5] report the case of a patient with high blood insulin concentrations, hypoglycaemia and a high serum concentration of antibodies to insulin. It is important that we are aware of these antibodies because they loosely bind to insulin, which is gradually released independent of the normal feedback mechanism generating hypoglycaemia. These circulating anti-insulin autoantibodies also have the capacity to interfere in analyses that may be used in the investigation of unexplained hypoglycaemia, e.g. insulin, C-peptide and proinsulin immunoassays. There has been a dramatic improvement in the understanding of autoimmune mediated neuropathies over the last 20 years with autoantibody responses to gangliosides being implicated in their pathology. These conditions can be acute or chronic; they can affect the motor, sensory or autonomic nerves; and the presenting features can be variable. Improved diagnosis with prompt treatment could prevent irreversible nerve damage and consequent disability. Klehmet et al. [6] describe a multiplex approach to detecting antibodies IgG and IgM antibodies to 11 ganglioside antigens in patients with autoimmune neuropathies, other neurological conditions and normal subjects. The IgM anti-sulphatyl, IgM anti-GM1 and IgM anti-GD1b antibodies showed a higher frequency in the inflammatory neuropathy patients compared to patients with other neurological conditions. This is early data, and the authors suggest that further investigation is warranted, but it does show the power of multiplex-type analysis in identifying markers for further directed investigation. The term “phospholipid” antibodies like the term “neurological” antibodies describes antibodies to a number of distinct antigenic targets. In both situations, the tissues where these antigens are expressed are throughout the body generating widespread symptoms. Phospholipids, including cardiolipin and β2 glycoprotein 1, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol, are an integral part of all cell membranes. Antibodies to these components are associated with arterial or venous thrombosis. There are well-defined diagnostic criteria for antiphospholipid syndrome, but only measurements of IgG and IgM antibodies to cardiolipin and/or β2 glycoprotein 1 are included alongside lupus anticoagulant (an adaptation of a clotting test). Antibodies to the other phospholipids have generally been considered to have limited diagnostic value; however, Zhang et al. [7] have evaluated the clinical relevance of antibodies to a phosphatidylserine-prothrombin complex. They found increased concentrations of IgG and IgM antibodies to phosphatidylserine-prothrombin complex in their patients with antiphospholipid syndrome, although in their study, cardiolipin antibodies showed the best clinical performance. Interestingly, antibodies to the phosphatidylserine-prothrombin complex were detected in patients with clinical features of antiphospholipid syndrome, but negative antibodies to the classical antigens, suggesting that although the majority of patients show cardiolipin antibodies, other antigenic specificities may be important in individual patients. Antinuclear antibodies (ANAs) is a commonly requested autoantibody with importance in the diagnosis of systemic autoimmune rheumatic diseases such as systemic lupus erythematosus, Sjogren syndrome, etc. ANAs have classically been detected by a manual indirect immunofluorescence method, which needs good laboratory skills and the ability to recognise the diverse and often complex patterns seen at the microscope reading of the slides. Samples with positive staining patterns have reflex testing by ELISA-based assay for antibodies to specific antigens. The deskilling of laboratories and the simple volume of requests are driving automation for ANA testing. Willems et al. [8] report a review of almost 10,000 patients who were tested for ANA by indirect immunofluorescence and an automated ANA “screen”. They report reasonable concordance and comparable receiver operator curves between the two methods. However, simply looking at the comparison does not tell the whole story. The data were also analysed by a requestor, who showed a significantly higher PPV for systemic autoimmune rheumatic diseases when testing was requested by clinicians specialised in systemic rheumatic disease. They concluded that the performance of immunoassay and indirect immunofluorescence depends on the specific disease being considered and that for optimum value both assay types are best used in combination and results interpreted in the context of the clinical picture. Antibodies to the extractable nuclear antigens Ro/SSA and La/SSB are found in a number of systemic connective tissue diseases, e.g. Sjogren syndrome, systemic lupus erythematosus, etc. These antibodies, if present, cross the placenta from mum to baby as part of the active IgG transport. The Ro and La antigens are expressed in foetal cardiac tissue from the 18th to the 24th week, particularly on the surface of cardiac myocytes, and in a proportion of patients, the antibodies cause inflammation, fibrosis and calcification of foetal cardiac conduction tissues leading to atrioventricular block. Antibodies to the p200 epitope of the Ro52/SSA antigen appear to have a critical pathogenic role the development of foetal heart block. Mattia et al. [9] have evaluated the performance of two “in-house” assays for anti-p200 antibodies in pregnant women positive for anti- SSA/Ro52 antibodies. They did find differences in assay performance, but further investigation may identify an analytical process for anti-p-200 antibodies that may help identify patients who are at a significantly increased risk future pregnancy being affected by foetal heart block. Antineutrophil cytoplasmic antibodies (ANCAs) are autoantibodies that are mainly directed against the cytoplasmic antigens proteinase 3 and myeloperoxidase of neutrophils and the monocytes. Their presence is associated with systemic vasculitides and in particular granulomatosis with polyangiitis, microscopic polyangiitis and Churg-Straus syndrome. Like ANAs, indirect immunofluorescence and ELISA-based assay are used together to detect and quantify these antibodies with a similar drive to replace the qualitative tests with automated quantitative analyses. Certified reference preparations for IgG anti-proteinase 3 and IgG anti-myeloperoxidase are only recently available and are not yet embedded in the analytical process, making comparison of results over time and between methods difficult. Borderline positive results, irrespective of the method used, present the greatest interpretative challenge with limited data on their significance. Watad et al. [10] present a retrospective study of ANCA investigations and conclude that patients with borderline-positive IgG anti-myeloperoxidase antibodies and positive ANCA staining by indirect immunofluorescence have a poorer outcome in terms of their renal function than patients with negative ANCA staining. Autoantibodies are complex analytes; the antigens that we use in the analysis vary subtly between companies, and even within a company between different lots, the antibodies that we are trying to detect can vary between patients and even within a patient during the disease course. The demand for autoantibody testing, particularly for the rare autoimmune diseases, is increasing beyond logic, but instead of educating users and optimising demand, we are increasing the capacity for measurement with high throughput automated systems. The analytical speed and detection systems introduce another source of variation into a group of tests where robust standardisation does not exist. The papers in this issue of Clinical Chemistry and Laboratory Medicine clearly show the relevance of autoantibody testing in a diverse range of disease. However, none of the analytes described in these eight papers were measured with respect to certified reference materials; as a scientific community, we need to embrace standardisation (or harmonisation) for autoantibody testing. Some of these papers also review large amounts of data, e.g. 14,555 ANCA results over a 10-year period [10] and 9856 ANA results over a 2-year period [8]. We consider the approximate incidence and prevalence of ANCA vasculitis (incidence 2/100,000 and prevalence 26/100,000) and SLE (incidence 5/100,000 and prevalence 52/100,000), but we may question why so many of these tests were requested when the pretest probability is so low. We have two enormous challenges – to ensure that test tests are requested appropriately and to improve the analysis and reduce the variability of autoantibody testing. References 1. Directorate General for Internal Policies, Policy Department A: Economic and Scientific policy. Workshop Autoimmune Diseases – Modern Diseases, Brussels 25th Sep 2017 Proceedings. Available at: http://www.europarl.europa.eu/cmsdata/133620/ENVI%202017-09%20WS%20Autoimmune%20diseases%20%20PE%20614.174%20(Publication).pdf. ; 2. International Diabetes Federation IDF Diabetes Atlas, 8th ed. Available at: http://www.diabetesatlas.org/resources/2017-atlas.html. ; 3. Manso J, Pezzani R, Scarpa E, Gallo N, Betterle C. The natural history of autoimmune addison’s disease with a non-classical presentation: a case report and review of literature. Clin Chem Lab Med 2018. doi: 10.1515/cclm-2017-1108. [Epub ahead of print]. PubMedGoogle Scholar 4. del Pilar Larosa M, Chen S, Steinmaus N, Macrae H, Guo L, Masiero S, et al. A new ELISA for autoantibodies to steroid 21-hydroxylase. Clin Chem Lab Med 2017. doi: 10.1515/cclm-2017-0456. [Epub ahead of print]. PubMedGoogle Scholar 5. Censi S, Albergoni MP, Gallo N, Plebani M, Boscaro M, Betterle C. Insulin autoimmune syndrome (Hirata’s disease) in an Italian patient: a case report and review of the literature. Clin Chem Lab Med 2017. doi: 10.1515/cclm-2017-0392. [Epub ahead of print]. Google Scholar 6. Klehmet J, Märschenz S, Ruprecht K, Wunderlich B, Büttner T, Hiemann R, et al. Analysis of anti-ganglioside antibodies by a line immunoassay in patients with chronic-inflammatory demyelinating polyneuropathies (CIDP). Clin Chem Lab Med 2018. doi: 10.1515/cclm-2017-0792. [Epub ahead of print]. PubMedGoogle Scholar 7. Zhang S, Wu Z, Zhang W, Zhao J, Norman GL, Zeng X, et al. Antibodies to phosphatidylserine/prothrombin (aPS/PT) enhanced the diagnostic performance in Chinese patients with antiphospholipid syndrome. Clin Chem Lab Med 2018. doi: 10.1515/cclm-2017-0811. [Epub ahead of print]. PubMedGoogle Scholar 8. Willems P, De Langhe E, Claessens J, Westhovens R, Van Hoeyveld E, Poesen K, et al. Screening for connective tissue disease-associated antibodies by automated immuno-assay. Clin Chem Lab Med 2018. doi: 10.1515/cclm-2017-0905. [Epub ahead of print]. Google Scholar 9. Mattia E, Hoxha A, Tonello M, Favaro M, Del Ross T, Calligaro A, et al. Detection of autoantibodies to the p200-epitope of SSA/Ro52 antigen. A comparison of two laboratory assays. Clin Chem Lab Med 2018. doi: 10.1515/cclm-2017-0704. [Epub ahead of print]. PubMedGoogle Scholar 10. Watad A, Bragazzi NL, Sharif K, Gilburd B, Yavne Y, McGonagle D, et al. Borderline positive anti-neutrophil cytoplasmic antibodies (ANCA)-PR3/MPO detection in a large cohort tertiary center: Lessons learnt from a real-life experience. Clin Chem Lab Med 2018. doi: 10.1515/cclm-2017-1053. [Epub ahead of print]. Google Scholar About the article Dr. Joanna Sheldon, Protein Reference Unit, St. George’s Hospital, Blackshaw Road, SW17 0NH, London, UK Published Online: 2018-04-17 Author contributions: The author has accepted responsibility for the entire content of this submitted manuscript and approved submission. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Citation Information: Clinical Chemistry and Laboratory Medicine (CCLM), 20180233, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/cclm-2018-0233. Export Citation ©2018 Walter de Gruyter GmbH, Berlin/Boston.
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Scooped by
Gilbert C FAURE
October 14, 2018 1:39 PM
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Nephrology (Carlton). 2018 Oct;23 Suppl 4:80-83. doi: 10.1111/nep.13469. Review
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Scooped by
Gilbert C FAURE
October 1, 2018 9:06 AM
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Objectives In patients with systemic lupus erythematosus (SLE), immune tolerance breakdown leads to autoantibody production and immune-complex glomerulonephritis. This study aimed to identify pathogenic plasma cells (PC) in the development of lupus nephritis.
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Scooped by
Gilbert C FAURE
September 18, 2018 11:43 AM
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Two variants of an autoimmune disease that affects thousands but is hard to diagnose are relatively common among black Africans, research shows.
Cochrane Database of Systematic Reviews Abstract available in Background Cyclophosphamide, in combination with corticosteroids, has been first‐line treatment for inducing disease remission for proliferative lupus nephritis, reducing death at five years from over 50% in the 1950s and 1960s to less than 10% in recent years. Several treatment strategies designed to improve remission rates and minimise toxicity have become available. Treatments, including mycophenolate mofetil (MMF) and calcineurin inhibitors, alone and in combination, may have equivalent or improved rates of remission, lower toxicity (less alopecia and ovarian failure) and uncertain effects on death, end‐stage kidney disease (ESKD) and infection. This is an update of a Cochrane review first published in 2004 and updated in 2012. Objectives Our objective was to assess the evidence and evaluate the benefits and harms of different immunosuppressive treatments in people with biopsy‐proven lupus nephritis. The following questions relating to management of proliferative lupus nephritis were addressed: 1) Are new immunosuppressive agents superior to or as effective as cyclophosphamide plus corticosteroids? 2) Which agents, dosages, routes of administration and duration of therapy should be used? 3) Which toxicities occur with the different treatment regimens? Search methods We searched the Cochrane Kidney and Transplant Specialised Register up to 2 March 2018 with support from the Cochrane Information Specialist using search terms relevant to this review. Studies in the Specialised Register are identified through searches of CENTRAL, MEDLINE, and EMBASE, conference proceedings, the International Clinical Trials Register (ICTRP) Search Portal and ClinicalTrials.gov. Selection criteria Randomised controlled trials (RCTs) and quasi‐RCTs comparing any immunosuppressive treatment for biopsy‐proven class III, IV, V+III and V+VI lupus nephritis in adult or paediatric patients were included. Data collection and analysis Data were abstracted and the risks of bias were assessed independently by two authors. Dichotomous outcomes were calculated as risk ratio (RR) and measures on continuous scales calculated as mean differences (MD) with 95% confidence intervals (CI). The primary outcomes were death (all causes) and complete disease remission for induction therapy and disease relapse for maintenance therapy. Evidence certainty was determined using GRADE. Main results In this review update, 26 new studies were identified, to include 74 studies involving 5175 participants overall. Twenty‐nine studies included children under the age of 18 years with lupus nephritis, however only two studies exclusively examined the treatment of lupus nephritis in patients less than 18 years of age. Induction therapy Sixty‐seven studies (4791 participants; median 12 months duration (range 2.5 to 48 months)) reported induction therapy. The effects of all treatment strategies on death (all causes) and ESKD were uncertain (very low certainty evidence) as this outcome occurred very infrequently. Compared with intravenous (IV) cyclophosphamide, MMF may have increased complete disease remission (RR 1.17, 95% CI 0.97 to 1.42; low certainty evidence), although the range of effects includes the possibility of little or no difference. Compared to IV cyclophosphamide, MMF is probably associated with decreased alopecia (RR 0.29, 95% CI 0.19 to 0.46; 170 less (129 less to 194 less) per 1000 people) (moderate certainty evidence), increased diarrhoea (RR 2.42, 95% CI 1.64 to 3.58; 142 more (64 more to 257 more) per 1000 people) (moderate certainty evidence) and may have made little or no difference to major infection (RR 1.02, 95% CI 0.67 to 1.54; 2 less (38 less to 62 more) per 1000 people) (low certainty evidence). It is uncertain if MMF decreased ovarian failure compared to IV cyclophosphamide because the certainty of the evidence was very low (RR 0.36, 95% CI 0.06 to 2.18; 26 less (39 less to 49 more) per 1000 people). Studies were not generally designed to measure ESKD. MMF combined with tacrolimus may have increased complete disease remission (RR 2.38, 95% CI 1.07 to 5.30; 336 more (17 to 1048 more) per 1000 people (low certainty evidence) compared with IV cyclophosphamide, however the effects on alopecia, diarrhoea, ovarian failure, and major infection remain uncertain. Compared to standard of care, the effects of biologics on most outcomes were uncertain because of low to very low certainty of evidence. Maintenance therapy Nine studies (767 participants; median 30 months duration (range 6 to 63 months)) reported maintenance therapy. In maintenance therapy, disease relapse is probably increased with azathioprine compared with MMF (RR 1.75, 95% CI 1.20 to 2.55; 114 more (30 to 236 more) per 1000 people (moderate certainty evidence). Multiple other interventions were compared as maintenance therapy, but patient‐outcome data were sparse leading to imprecise estimates. Authors' conclusions In this review update, studies assessing treatment for proliferative lupus nephritis were not designed to assess death (all causes) or ESKD. MMF may lead to increased complete disease remission compared with IV cyclophosphamide, with an acceptable adverse event profile, although evidence certainty was low and included the possibility of no difference. Calcineurin combined with lower dose MMF may improve induction of disease remission compared with IV cyclophosphamide, but the comparative safety profile of these therapies is uncertain. Azathioprine may increase disease relapse as maintenance therapy compared with MMF. Plain language summary available in Immunosuppressive treatment for people with proliferative lupus nephritis What is the issue? In lupus, the body’s immune system for fighting infection attacks different parts of the body, including the kidneys. About half of all people with lupus have kidney problems. An estimated one in every 10 people who have lupus kidney disease (lupus nephritis) can develop kidney failure. The goal of treatment is to protect kidney function and avoid side‐effects. While the life expectancy of patients who have lupus has dramatically improved, available treatments can cause serious side effects such as hair loss, serious infection, and infertility. It is important to know about which treatments help to treat lupus while causing the fewest side‐effects. What did we do? We searched the Cochrane Kidney and Transplant Specialised Register up to 2 March 2018 and we combined all studies testing treatments aimed to control the body’s immune system for lupus nephritis. What did we find? In this review update, 74 studies involving 5175 patients with lupus nephritis could be studied. Treatments included intravenous (given through a vein) cyclophosphamide, oral (tablets by mouth) mycophenolate mofetil (MMF), azathioprine, and tacrolimus (used alone or together with MMF). We also found studies of treatments called “biologic” therapies, that have been designed to change very specific parts of the body’s immune system that cause it to attack itself. We looked particularly at key outcomes such as whether treatment prevented patients from needing dialysis and controlled the lupus damage to the kidney tissue (called remission). We also looked at serious side‐effects including death, infection, infertility, and hair loss. After combining the available studies, compared with cyclophosphamide, MMF may be better at getting the lupus damage to the kidneys under control. However, the range where the actual effect may suggest that MMF may make little or no difference to disease remission compared to treatment with cyclophosphamide. MMF treatment given with tacrolimus may lead to more disease remission. MMF may result in less hair loss and worse diarrhoea, but we were not certain whether MMF reduces infertility or other serious side effects. MMF was better than azathioprine for preventing kidney disease in the longer term. None of the studies told us whether treatment had any effect on death or need for dialysis, and there was very low certainty of evidence for the use of biologics in patients with lupus nephritis. Conclusions Patients with lupus nephritis may have similar or slightly better outcomes when treated with MMF or MMF with tacrolimus compared to those patients who receive intravenous cyclophosphamide. We are still not certain which is the best treatment for lupus nephritis to protect against needing dialysis in the longer term. Unlock the full review
Via Krishan Maggon
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Scooped by
Gilbert C FAURE
July 19, 2018 2:25 AM
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Natural autoantibodies form a network that serves to protect the organism from outer and inner danger but also contributes to autoimmune disease. It is our view, that this critical component of the immune system deserves much greater attention than it currently attracts for the benefit of patients and for better understanding how immunity operates. Since the early days of immunology, pioneering work by Landsteiner and his group on haptens showed that immunization is a process that can raise highly specific antibodies. In the same era, Ehrlich put forward the dogma of “horror autotoxicus”, proclaiming the inability of the immune system to recognize self-constituents. Later on, in the 1950s, Burnet build upon these seminal observations and formulated the clonal selection theory which states that “cell clones produce highly specific antibodies for environmental constituents while the immune system is educated during ontogeny not to recognize self-constituents”. Nevertheless, other studies, contradicting the “dogma”, were mostly ignored. These studies showed that circulating antibodies in normal human sera could recognize various substances, including self-antigens, albeit with a low avidity 1. In the following years, additional experiments reinforced the notion of a persistent immune system reactivity against self e.g. in either normal or lupus-prone mice where the genetic background alone drives the immune system to generate IgM-producing cells that secret self-reactin
The choice of excipients is central in the drug development process. A bad choice of excipient can modify or masking the effect of the active substance. Some so-called excipients display bioactivity themselves, which can lead to unwanted effects in patients.
Via Krishan Maggon
Autoimmune diseases are chronic inflammatory pathologies that are characterized by the presence of antibodies against own epitopes in serum (autoantibodies). Systemic lupus erythematosus (SLE) is a common autoimmune pathology, characterized by the presence of antinuclear antibodies (ANAs).
Via Krishan Maggon
Understanding antibody repertoires and in particular, the properties and fates of B cells expressing potentially pathogenic antibodies is critical to define the mechanisms underlying multiple immunological diseases including autoimmune and allergic conditions as well as transplant rejection. Moreover, an integrated knowledge of the antibody repertoires expressed by B cells and plasma cells (PC) of different functional properties and longevity is essential to develop new therapeutic strategies, better biomarkers for disease segmentation, and new assays to measure restoration of B‐cell tolerance or, at least, of normal B‐cell homeostasis. Reaching these goals, however, will require a more precise phenotypic, functional and molecular definition of B‐cell and PC populations, and a comprehensive analysis of the antigenic reactivity of the antibodies they express. While traditionally hampered by technical and ethical limitations in human experimentation, new technological advances currently enable investigators to address these questions in a comprehensive fashion. In this review, we shall discuss these concepts as they apply to the study of Systemic Lupus Erythematosus.
Via Krishan Maggon
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Scooped by
Gilbert C FAURE
June 17, 2018 5:15 AM
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As the diagnosis and management of systemic lupus erythematosus (SLE) can be challenging, reclassification of SLE on the basis of biomarker data provides novel insights. Definitions of antinuclear antibodies and attention to SMAARTT criteria (specificity – sensitivity, measurable, actionable, added value, realistic, titres, timely) are needed to translate these new analytical frameworks into real-time clinical use.
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Scooped by
Gilbert C FAURE
May 30, 2018 2:00 PM
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Systemic lupus erythematosus (SLE) is associated with deficiencies in the complement protein C1q. Although C1q plays a role in the clearance of apoptotic cells, there are several redundant clearance pathways. Disruption of one pathway does not lead to an autoimmune defect. In a chronic graft-versus-host disease model of SLE, Ling et al. show that C1q dampens CD8+ T cell responses to self-antigens. C1q modulates metabolism through the mitochondrial cell-surface protein p32/gC1qR. The lack of C1q during a viral infection also enhances CD8+ T cell responses. Thus, C1q plays a role as a “metabolic rheostat” for effector CD8+ T cells.
Science , this issue p. [558][1]
[1]: /lookup/doi/10.1126/science.aao4555
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Scooped by
Gilbert C FAURE
May 5, 2018 2:57 PM
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Systemic lupus erythematosus (SLE) is associated with deficiencies in the complement protein C1q. Although C1q plays a role in the clearance of apoptotic cells, there are several redundant clearance pathways. Disruption of one pathway does not lead to an autoimmune defect. In a chronic graft-versus-host disease model of SLE, Ling et al. show that C1q dampens CD8+ T cell responses to self-antigens. C1q modulates metabolism through the mitochondrial cell-surface protein p32/gC1qR. The lack of C1q during a viral infection also enhances CD8+ T cell responses. Thus, C1q plays a role as a “metabolic rheostat” for effector CD8+ T cells.
Science , this issue p. [558][1]
[1]: /lookup/doi/10.1126/science.aao4555
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Suggested by
LIGHTING
April 25, 2018 12:04 PM
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Scoop.it!
