Molecular Cloning, Prokaryotic Expression, and Immunological Characterization of ß-Enolase from Grass Carp (Ctenopharyngodon idella).

ß-Enolase is a cross-allergen commonly found in fungi, plants, and aquatic products. Although studies on the allergenicity of fish enolase have been reported in recent years, they are still limited to a few species of marine fish. Therefore, the detection of freshwater fish in the food industry requires more studies of the molecular characterization as well as the allergenicity of enolase. In this study, the nucleotide sequence of ß-enolase from grass carp was obtained by molecular cloning technology. Structural domain analysis showed that it contained the characteristic structural domains of the enolase superfamily, and homology analysis indicated that enolases are highly conserved evolutionarily. Recombinant ß-enolase was obtained by prokaryotic expression, and its allergenicity was assessed by ß-enolase-sensitized mice, which confirmed the ability of ß-enolase to trigger an allergic response and cause a rise in Th1 and Th2 immune responses in mice. These results suggest that ß-enolase could be used as a characterizing substance for the detection of fish allergens in the food industry as well as the preparation of drugs for allergy-related studies.

In pancreatic cancer patients, chemotherapy reshapes the gene expression profile and antigen receptor repertoire of T lymphocytes and enhances their effector response to tumor-associated antigens.

Introduction: Pancreatic Ductal Adenocarcinoma (PDA) is one of the most aggressive malignancies with a 5-year survival rate of 13%. Less than 20% of patients have a resectable tumor at diagnosis due to the lack of distinctive symptoms and reliable biomarkers. PDA is resistant to chemotherapy (CT) and understanding how to gain an anti-tumor effector response following stimulation is, therefore, critical for setting up an effective immunotherapy. Methods: Proliferation, and cytokine release and TCRB repertoire of from PDA patient peripheral T lymphocytes, before and after CT, were analyzed in vitro in response to four tumor-associated antigens (TAA), namely ENO1, FUBP1, GAPDH and K2C8. Transcriptional state of PDA patient PBMC was investigated using RNA-Seq before and after CT. Results: CT increased the number of TAA recognized by T lymphocytes, which positively correlated with patient survival, and high IFN-γ production TAA-induced responses were significantly increased after CT. We found that some ENO1-stimulated T cell clonotypes from CT-treated patients were expanded or de-novo induced, and that some clonotypes were reduced or even disappeared after CT. Patients that showed a higher number of effector responses to TAA (high IFN-γ/IL-10 ratio) after CT expressed increased fatty acid-related transcriptional signature. Conversely, patients that showed a higher number of regulatory responses to TAA (low IFN-γ/IL-10 ratio) after CT significantly expressed an increased IRAK1/IL1R axis-related transcriptional signature. Conclusion: These data suggest that the expression of fatty acid or IRAK1/IL1Rrelated genes predicts T lymphocyte effector or regulatory responses to TAA in patients that undergo CT. These findings are a springboard to set up precision immunotherapies in PDA based on the TAA vaccination in combination with CT.

Impact of Enolase in Allergic Disease.

PURPOSE OF REVIEW: There is growing evidence that enolase is involved in allergy. This manuscript reviews the impact of enolase in allergic disease and describes several sources of this allergen including molds, plants, animals, and pollens, among others. IgE epitopes are carefully analyzed as they may account for cross-reactivity. RECENT FINDINGS: Enolase has been previously associated to food allergy and contact dermatitis. However, other groups and we have identified recently novel enolases derived from diverse pollens in patients suffering asthma and allergic rhinitis. Exposure to outdoor enolases may cause respiratory disease. Enolase has been identified across various species and its amino acid sequence is highly conserved among different sources of this allergen. The demonstration that enolase is involved in many allergic diseases including respiratory allergies, is of clinic relevance. Thus, the development of novel molecular-based diagnostic and therapeutic strategies may pave the way for improved diagnosis and therapeutics.

The molecular basis underlying T cell specificity towards citrullinated epitopes presented by HLA-DR4.

CD4+ T cells recognising citrullinated self-epitopes presented by HLA-DRB1 bearing the shared susceptibility epitope (SE) are implicated in rheumatoid arthritis (RA). However, the underlying T cell receptor (TCR) determinants of epitope specificity towards distinct citrullinated peptide antigens, including vimentin-64cit59-71 and α-enolase-15cit10-22 remain unclear. Using HLA-DR4-tetramers, we examine the T cell repertoire in HLA-DR4 transgenic mice and observe biased TRAV6 TCR gene usage across these two citrullinated epitopes which matches with TCR bias previously observed towards the fibrinogen ß-74cit69-81 epitope. Moreover, shared TRAV26-1 gene usage is evident in four α-enolase-15cit10-22 reactive T cells in three human samples. Crystal structures of mouse TRAV6+ and human TRAV26-1+ TCR-HLA-DR4 complexes presenting vimentin-64cit59-71 and α-enolase-15cit10-22, respectively, show three-way interactions between the TCR, SE, citrulline, and the basis for the biased selection of TRAV genes. Position 2 of the citrullinated epitope is a key determinant underpinning TCR specificity. Accordingly, we provide a molecular basis of TCR specificity towards citrullinated epitopes.

A novel quantitative double antigen sandwich ELISA for detecting total antibodies against Candida albicans enolase 1.

PURPOSE: This study aimed to develop a double antigen sandwich ELISA (DAgS-ELISA) method for more efficient, accurate, and quantitative detection of total antibodies against Candida albicans enolase1 (CaEno1) for diagnosing invasive candidiasis (IC). METHODS: DAgS-ELISA was developed using recombinant CaEno1 and a monoclonal antibody as the standard. Performance evaluation included limit of detection, accuracy, and repeatability. Dynamic changes in antibody levels against CaEno1 in serum from systemic candidiasis mice were analyzed using DAgS-ELISA. Patient serum samples from IC, Candida colonization, bacterial infections, and healthy controls were analyzed with DAgS-ELISA and indirect ELISA. RESULTS: DAgS-ELISA outperformed indirect ELISA in terms of linear range and test background. In systemic candidiasis mice, a distinctive 'double-peak' pattern in dynamic antibody levels was observed. Additionally, there was a high level of consistency in the positive rates of CaEno1 antibodies detected by both DAgS-ELISA and indirect ELISA. While the positivity rates differed among patient groups, no significant variations in antibody levels were detected among the various positive patient groups. CONCLUSIONS: DAgS-ELISA offers a reliable novel approach for IC diagnosis, enabling rapid, accurate, and quantitative detection of CaEno1 antibodies. Further validation and optimization are needed for its clinical application and effectiveness.

Label-Free Assessment of Neuron-Specific Enolase via Polydopamine over a Carbon-Nanotube-Based Flexible Immunosensor.

A label-free electrochemical immunosensor was developed for the rapid and sensitive detection of neuron-specific enolase (NSE). The electropolymerization of dopamine in conjunction with highly conductive carbon nanotubes offers a simple and quick platform for the direct anchoring of antibodies without the assistance of any coupling agent as well as a blocking agent. The developed immunosensor exhibited a wider detection range from 120 pM (9 ng mL-1) to 3 nM (200 ng mL-1) for NSE with a high sensitivity of 3.9 µA pM-1 cm-2 in 0.1 M phosphate-buffered saline (PBS) at physiological pH (7.4). Moreover, the short recognition time (15 min) for the antigen enabled the detection to be fast and less invasive. Additionally, the evaluation of a rate constant at various concentrations of NSE via feedback mode of scanning electrochemical microscopy (SECM) explained the profound effect of antigen concentration on the rate of flow of electrons. Therefore, the proposed immunosensor can be a promising tool for the early detection of small cell lung cancer in a very short period of time with consistent accuracy.

Characterization of chicken-derived antibody against Alpha-Enolase of Streptococcus pneumoniae.

Streptococcus pneumoniae is a clinically relevant pathogen notorious for causing pneumonia, meningitis, and otitis media in immunocompromised patients. Currently, antibiotic therapy is the most efficient treatment for fighting pneumococcal infections. However, an arise in antimicrobial resistance in S. pneumoniae has become a serious health issue globally. To resolve the problem, alternative and cost-effective strategies, such as monoclonal antibody-based targeted therapy, are needed for combating bacterial infection. S. pneumoniae alpha-enolase (spEno1), which is thought to be a great target, is a surface protein that binds and converts human plasminogen to plasmin, leading to accelerated bacterial infections. We first purified recombinant spEno1 protein for chicken immunization to generate specific IgY antibodies. We next constructed two single-chain variable fragments (scFv) antibody libraries by phage display technology, containing 7.2 × 107 and 4.8 × 107 transformants. After bio-panning, ten scFv antibodies were obtained, and their binding activities to spEno1 were evaluated on ELISA, Western blot and IFA. The epitopes of spEno1 were identified by these scFv antibodies, which binding affinities were determined by competitive ELISA. Moreover, inhibition assay displayed that the scFv antibodies effectively inhibit the binding between spEno1 and human plasminogen. Overall, the results suggested that these scFv antibodies have the potential to serve as an immunotherapeutic drug against S. pneumoniae infections.

Generation and characterization of avian single chain variable fragment against human Alpha-Enolase.

Overexpression of human alpha-enolase (hEno1)has been reported in a wide range of cancers and is tightly associated with poor prognosis, making it a remarkable biomarker and therapeutic target. In this study, polyclonal yolk-immunoglobulin (IgY) antibodies purified from hEno1-immunized chickens showed a noticeable specific humoral response. Phage display technology was used to construct two antibody libraries of IgY gene-derived single-chain variable fragments (scFvs) containing 7.8 × 107 and 5.4 × 107 transformants, respectively. Phage-based ELISA indicated that specific anti-hEno1 clones were significantly enriched. The nucleotide sequences of scFv-expressing clones were determined and classified into seven groups either in the short linker or the long linker. Moreover, higher mutation rates were revealed in the CDR regions, especially in the CDR3. Three distinguish antigenic epitopes were identified on the hEno1 protein. The binding activities of selected anti-hEno1 scFv on hEno1-positive PE089 lung cancer cells were confirmed using Western blot, flow cytometry, and immunofluorescence assay. In particular, hEnS7 and hEnS8 scFv antibodies significantly suppressed the growth and migration of PE089 cells. Taken together, these chicken-derived anti-hEno1 IgY and scFv antibodies have great potential to develop diagnostic and therapeutic agents for the treatment of lung cancer patients with high expression levels of hEno1 protein.

A Predictive Role of Autoantibodies Against the Epitope aa168-183 of ENO1 in the Occurrence of Miscarriage Related to Thyroid Autoimmunity.

Objective: The aim of the research is to study the association between the serum levels of autoantibodies against one important epitope (168FMILPVGAANFREAMR183, designated as P6) of α-enolase (ENO1-P6Abs) and miscarriage among euthyroid females with thyroid autoimmunity (TAI). Methods: Anti-ENO1-P6 total IgG was investigated in 432 euthyroid women, and its four subclasses were analyzed in 184 euthyroid women. The serum FT4, TSH, TgAb, and TPOAb levels were determined using an electrochemiluminescence immunoassay. The serum ENO1-P6Ab and anti-protein disulfide isomerase A3 autoantibody (PDIA3Ab) levels were determined using an enzyme-linked immunosorbent assay. Results: The serum levels of anti-ENO1-P6 total IgG, IgG2, IgG3, and IgG4 were significantly higher in euthyroid TAI females than in non-TAI controls. Additionally, anti-ENO1-P6 total IgG and its 4 subtypes were all markedly higher in euthyroid TAI females with pregnancy loss than those without miscarriage. Moreover, logistic regression analysis showed that highly expressed anti-ENO1-P6 total IgG, IgG1, IgG2, and IgG3 subtypes in the serum were all independent risk factors for euthyroid TAI-related miscarriage, and its IgG1 was also for non-TAI-related abortion. According to the trend test, the prevalence of miscarriage was increased in a titer-dependent manner with the raised levels of serum anti-ENO1-P6 total IgG and IgG1, IgG2, and IgG3 subtypes among euthyroid TAI females. The receiver operating characteristic curve analysis of anti-ENO1-P6 total IgG and IgG1, IgG2, and IgG3 subclass expressions in the serum for miscarriage prediction in euthyroid TAI females exhibited that the total areas under the curves were 0.773 ± 0.041, 0.761 ± 0.053, 0.827 ± 0.043, and 0.760 ± 0.050, respectively (all P <0.0001). Their corresponding optimal cut-off OD450 values were 0.68 (total IgG), 0.26 (IgG1), 0.97 (IgG2), and 0.48 (IgG3), with sensitivities of 70.8, 87.5, 83.3, and 85.4%, and specificities of 70.8, 59.1, 77.3, and 56.8%, respectively. There was an additive interaction between serum anti-ENO1-P6 and anti-PDIA3 total IgGs on the development of miscarriage (RERI = 23.6, AP = 0.79, SI = 5.37). Conclusion: The highly expressed ENO1-P6Abs may be important risk factors for euthyroid TAI-related miscarriage. The serum levels of ENO1-P6Abs may become good predictive markers for pregnancy loss in euthyroid TAI females, especially its IgG2 subclass expression.

A Sporothrix spp. enolase derived multi-epitope vaccine confers protective response in BALB/c mice challenged with Sporothrix brasiliensis.

Sporotrichosis is a cosmopolitan mycosis caused by pathogenic species of Sporothrix genus, that in Brazil is often acquired by zoonotic transmission involved infected cats with S. brasiliensis. Previous studies showed that the Sporothrix spp. recombinant enolase (rSsEno), a multifunctional protein with immunogenic properties, could be a promising target for vaccination against sporotrichosis in cats. Nevertheless, the considerable sequence identity (62%) of SsEno with its feline counterpart is a great concern. Here, we report the identification in silico, chemical synthesis and biological validation of six peptides of SsEno with low sequence identity to its cat orthologue. All synthesized peptides exhibit B-cell epitopes on the molecular surface of SsEno and proved to be highly reactive with the serum of infected mice with S. brasiliensis and sera of cats with sporotrichosis. Interestingly, our study revealed that anti-peptide sera did not react with the recombinant enolase from Felis catus (cats, rFcEno), thus, may not trigger autoimmune response in these felines if used as a vaccine antigen. The immunization with peptide mixture (PeptMix) formulated with Freund adjuvant (FA), induced high levels of antigen-specific IgG, IgG1 and IgG2b antibodies that conferred protection upon passive transference in infected BALB/c mice with S. brasiliensis. We also observed, that the FA+PeptMix formulation induced a Th1/Th2/Th17 cytokine profile ex vivo, associated with protecting effect against the experimental sporotrichosis. Our results suggest that the six SsEno-derived peptides here evaluated, could be used as safe antigens for the development of vaccine strategies against feline sporotrichosis, whether prophylactic or therapeutic.

E4 engages uPAR and enolase-1 and activates urokinase to exert antifibrotic effects.

Fibroproliferative disorders such as systemic sclerosis (SSc) have no effective therapies and result in significant morbidity and mortality. We recently demonstrated that the C-terminal domain of endostatin, known as E4, prevented and reversed both dermal and pulmonary fibrosis. Our goal was to identify the mechanism by which E4 abrogates fibrosis and its cell surface binding partner(s). Our findings show that E4 activated the urokinase pathway and increased the urokinase plasminogen activator (uPA) to type 1 plasminogen activator inhibitor (PAI-1) ratio. In addition, E4 substantially increased MMP-1 and MMP-3 expression and activity. In vivo, E4 reversed bleomycin induction of PAI-1 and increased uPA activity. In patients with SSc, the uPA/PAI-1 ratio was decreased in both lung tissues and pulmonary fibroblasts compared with normal donors. Proteins bound to biotinylated-E4 were identified as enolase-1 (ENO) and uPA receptor (uPAR). The antifibrotic effects of E4 required uPAR. Further, ENO mediated the fibrotic effects of TGF-ß1 and exerted TGF-ß1-independent fibrotic effects. Our findings suggest that the antifibrotic effect of E4 is mediated, in part, by regulation of the urokinase pathway and induction of MMP-1 and MMP-3 levels and activity in a uPAR-dependent manner, thus promoting extracellular matrix degradation. Further, our findings identify a moonlighting function for the glycolytic enzyme ENO in fibrosis.

Cloning and Characterization of Immunological Properties of Haemophilus influenzae Enolase.

Haemophilus influenzae is a common organism of the human upper respiratory tract; this bacterium is responsible of a wide spectrum for respiratory infections and can generate invasive diseases such as meningitis and septicemia. These infections are associated with H. influenzae encapsulated serotype b. However, the incidence of invasive disease caused by nontypeable H. influenzae (NTHi) has increased in the post-H. influenzae serotype b (Hib) vaccine era. Currently, an effective vaccine against NTHi is not available; due to this, it is important to find an antigen capable to confer protection against NTHi infection. In this study, 10 linear B cell epitopes and 13 CTL epitopes and a putative plasminogen-binding motif (252FYNKENGMY260) and the presence of enolase on the surface of different strains of H. influenzae were identified in the enolase sequence of H. influenzae. Both in silico and experimental results showed that recombinant enolase from H. influenzae is immunogenic that could induce a humoral immune response; this was observed mediating the generation of specific polyclonal antibodies anti-rNTHiENO that recognize typeable and nontypeable H. influenzae strains. The immunogenic properties and the superficial localization of enolase in H. influenzae, important characteristics to be considered as a new candidate for the development of a vaccine, were demonstrated.

A Chimera of Th1 Stimulatory Proteins of Leishmania donovani Offers Moderate Immunotherapeutic Efficacy with a Th1-Inclined Immune Response against Visceral Leishmaniasis.

Immunotherapy, a treatment based on host immune system activation, has been shown to provide a substitute for marginally effective conventional chemotherapy in controlling visceral leishmaniasis (VL), the deadliest form of leishmaniasis. As the majority of endemic inhabitants exhibit either subclinical or asymptomatic infection which often develops into the active disease state, therapeutic intervention seems to be an important avenue for combating infections by stimulating the natural defense system of infected individuals. With this perspective, the present study focuses on two immunodominant Leishmania (L.) donovani antigens (triosephosphate isomerase and enolase) previously proved to be potent prophylactic VL vaccine candidates, for generating a recombinant chimeric antigen. This is based on the premise that in a heterogeneous population, a multivalent antigen vaccine would be required for an effective response against leishmaniasis (a complex parasitic disease). The resulting molecule rLdT-E chimeric protein was evaluated for its immunogenicity and immunotherapeutic efficacy. A Th1 stimulating adjuvant BCG was employed with the protein which showed a remarkable 70% inhibition of splenic parasitic multiplication positively correlated with boosted Th1 dominant immune response against lethal L. donovani challenge in hamsters as evidenced by high IFN-γ and TNF-α and low IL-10. In addition, immunological analysis of antibody subclass presented IgG2-based humoral response besides considerable delayed-type hypersensitivity and lymphocyte proliferative responses in rLdT-E/BCG-treated animals. Our observations indicate the potential of the chimera towards its candidature for an effective vaccine against Leishmania donovani infection.

Split-Type Electrochemical Immunoassay System Triggering Ascorbic Acid-Mediated Signal Magnification Based on a Controlled-Release Strategy.

This research put forward a novel split-type electrochemical (EC) immunosensor which integrated the controlled-release strategy with EC detection for application in the field of biosensing. Concretely, ascorbic acid (AA) was packaged in a cadmium sulfide (CdS)-capped spherical mesoporous bioactive glass (SBG) nanocarrier (SBGCdS) on account of encapsulation technology. To reduce the complexity of the bioanalysis, the detection antibody-labeled SBGCdS-AA bioconjugate was applied in a 96-well microplate for the immunoreaction process, which is independent of the EC determination procedure. Thus, the immune interference and steric hindrance caused by the accumulation of nanomaterials on the electrode could be minimized. Subsequently, AA was released efficiently via the destruction effect of dithiothreitol on the disulfide bond. In addition, for the as-prepared FcAI/l-Cys/gold nanoparticles (GNPs)/porous BiVO4 (p-BVO)/ITO EC sensing platform in the detection solution, the synergetic catalysis of Fc and GNPs/p-BVO toward the oxidation of the released AA could be realized, which triggered AA-mediated significant signal magnification throughout this study. In particular, p-BVO with an ordered nanoarray structure could accelerate the electron transfer to assist in sensitivity improvement of this system. This novel biosensor was capable of assaying the neuron-specific enolase (NSE) biomarker sensitively, from which a linear range of 0.001-100 ng/mL was derived along with a low detection limit of 1.08 pg/mL. An innovative way could be paved in the bioanalysis of NSE and other biomarkers.

Evaluation of systemic lupus erythematosus disease activity using anti-α-enolase antibody and RDW.

The objective of the study was to investigate the value of anti-α-enolase antibody (Ab) combined with RDW in evaluating the activity of systemic lupus erythematosus (SLE). Levels of serum anti-α-enolase Ab and RDW were detected in 193 SLE patients and 98 healthy controls by ELISA and automatic blood cell counter (XN9000), respectively. Furthermore, the correlation between anti-α-enolase Ab and RDW in evaluating the activity of SLE was evaluated by correlation analysis. The level of anti-α-enolase Ab (9.16 ± 0.44 ng/mL in stable group and 10.26 ± 0.36 ng/mL in activity group) was significantly higher than that in the healthy control (7.05 ± 0.27 ng/mL). The level of RDW (12.92% ± 1.23% in stable group and 13.57% ± 2.12% in activity group) was significantly higher than that in the healthy control (12.46% ± 0.61%). The levels of anti-α-enolase Ab or RDW in SLE patients were positively correlated with SLEDAI-2 K score (r= 0.75, r = 0.73), respectively. Compared with the anti-α-enolase Ab (AUC: 78.0%) or RDW (AUC:80.0%) alone, anti-α-enolase Ab combined with RDW (AUC: 81.0%) had the best of the effectiveness of evaluating activity of SLE. These data suggested that combined anti-α-enolase Ab with RDW might be good biomarker to predict the activity of SLE in clinical.

Enolase-specific cross antibodies induce neutrophilic inflammation in the intestine.

The pathogenesis of ulcerative colitis (UC) is to be further investigated. House dust mites (HDM) are highly associated with the pathogenesis of immune inflammation in the body. This study aims to investigate the role of enolase (one of the HDM-derived proteins)-specific cross Abs in the induction of UC-like inflammation. The enolase specific IgG (EsIgG) was identified in UC patients by mass spectrometry. Mice were treated with EsIgG to induce inflammation in the colon mucosa. EsIgG was detected in the serum and the colon tissues of UC patients, which was positively correlated with the polymorphonuclear neutrophil (PMN) counts in the blood and colon tissues of UC patients. EsIgG formed immune complexes with the constitutive enolase in the UC colon epithelium that activated complement, induced epithelial cell apoptosis, compromised epithelial barrier functions, and resulted in UC-like inflammation in the mouse colon. In summary, UC patients have high serum levels of Abs against HDM-derived enolase and intestinal epithelial cell-derived enolase. These Abs attack the colonic epithelium to induce UC-like inflammation.

Serum IgG2 antibody multi-composition in systemic lupus erythematosus and in lupus nephritis (Part 2): prospective study.

OBJECTIVES: Circulating anti-ENO1 and anti-H2A IgG2 have been identified as specific signatures of LN in a cross-over approach. We sought to show whether the same antibodies identify selected population of patients with LN with potentially different clinical outcomes. METHODS: Here we report the prospective analysis over 36 months of circulating IgG2 levels in patients with newly diagnosed LN (n=91) and SLE (n=31) and in other patients with SLE recruited within 2 years from diagnosis (n=99). Anti-podocyte (ENO1), anti-nucleosome (DNA, histone 2 A, histone 3) and anti-circulating proteins (C1q, AnnexinA1-ANXA1) IgG2 antibodies were determined by home-made techniques. RESULTS: LN patients were the main focus of the study. Anti-ENO1, anti-H2A and anti-ANXA1 IgG2 decreased in parallel to proteinuria and normalized within 12 months in the majority of patients while anti-dsDNA IgG2 remained high over the 36 months. Anti-ENO1 and anti-H2A had the highest association with proteinuria (Heat Map) and identified the highest number of patients with high proteinuria (68% and 71% respectively) and/or with reduced estimated glomerula filtration rate (eGFR) (58% for both antibodies) compared with 23% and 17% of anti-dsDNA (agreement analysis). Anti-ENO1 positive LN patients had higher proteinuria than negative patients at T0 and presented the maximal decrement within 12 months. CONCLUSIONS: Anti-ENO1, anti-H2A and anti-ANXA1 antibodies were associated with high proteinuria in LN patients and Anti-ENO1 also presented the maximal reduction within 12 months that paralleled the decrease of proteinuria. Anti-dsDNA were not associated with renal outcome parameters. New IgG2 antibody signatures should be utilized as tracers of personalized therapies in LN. TRIAL REGISTRATION: The Zeus study was registered at https://clinicaltrials.gov (study number: NCT02403115).

Serum IgG2 antibody multicomposition in systemic lupus erythematosus and lupus nephritis (Part 1): cross-sectional analysis.

OBJECTIVES: Serum anti-dsDNA and anti-nucleosome IgGs have been proposed as signatures for SLE and LN in limited numbers of patients. We sought to show higher sensitivity and specificity of the same antibodies with the IgG2 isotype and included IgG2 antibodies vs specific intracellular antigens in the analysis. METHODS: A total of 1052 SLE patients with (n = 479) and without (n = 573) LN, recruited at different times from the beginning of symptoms, were included in the study. Patients with primary APS (PAPS, n = 24), RA (RA, n = 24) and UCTD (UCTD, n = 96) were analysed for comparison. Anti-nucleosome (dsDNA, Histone2A, Histone3), anti-intracellular antigens (ENO1), anti-annexin A1 and anti-C1q IgG2 were determined by non-commercial techniques. RESULTS: The presence in the serum of the IgG2 panel was highly discriminatory for SLE/LN vs healthy subjects. Serum levels of anti-dsDNA and anti-C1q IgG2 were more sensitive than those of IgGs (Farr radioimmunoassay/commercial assays) in identifying SLE patients at low-medium increments. Of more importance, serum positivity for anti-ENO1 and anti-H2A IgG2 discriminated between LN and SLE (ROC T0-12 months), and high levels at T0-1 month were detected in 63% and 67%, respectively, of LN, vs 3% and 3%, respectively, of SLE patients; serum positivity for each of these was correlated with high SLEDAI values. Minor differences existed between LN/SLE and the other rheumatologic conditions. CONCLUSION: Nephritogenic IgG2 antibodies represent a specific signature of SLE/LN, with a few overlaps with other rheumatologic conditions. High levels of anti-ENO1 and anti-H2A IgG2 correlated with SLE activity indexes and were discriminatory between SLE patients limited to the renal complication and other SLE patients. TRIAL REGISTRATION: The Zeus study was registered at https://clinicaltrials.gov, NCT02403115.

Multi-Autoantibody Signature and Clinical Outcome in Membranous Nephropathy.

BACKGROUND AND OBJECTIVES: Patients with membranous nephropathy can have circulating autoantibodies against membrane-bound (phospholipase A2 receptor 1 [PLA2R1] and thrombospondin type-1 domain containing 7A [THSD7A]) and intracellular (aldose reductase, SOD2, and α-enolase) podocyte autoantigens. We studied their combined association with clinical outcomes. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Serum levels of anti-PLA2R1, anti-THSD7A, anti-aldose reductase, anti-SOD2, and anti-α-enolase autoantibodies were determined in 285 patients at diagnosis and during follow-up using standardized and homemade assays. An eGFR>60 ml/min per 1.73 m2 and remission of proteinuria (<0.3/<3.5 g per d) after 12 months were the outcomes of interest. RESULTS: At diagnosis, 182 (64%), eight (3%), and 95 (33%) patients were anti-PLA2R1+, anti-THSD7A+, and double negative, respectively. The prevalence of a detectable antibody to at least one intracellular antigen was similarly distributed in patients who were anti-PLA2R1+ (n=118, 65%) and double negative (n=64, 67%). Positivity for anti-PLA2R1, anti-SOD2, and anti-α-enolase antibodies and higher titers at diagnosis were associated with poor clinical outcome independently to each other. Combined positivity for anti-PLA2R1, anti-SOD2, and anti-α-enolase was associated with highest risk of poor outcome (odds ratio, 5.5; 95% confidence interval, 1.2 to 24; P=0.01). In Kaplan-Meier analysis, patients who were anti-PLA2R1+/anti-SOD2+ or anti-PLA2R1+/anti-α-enolase+ had lower eGFR at 12 months compared with patients who were anti-PLA2R1+/anti-SOD2- or anti-α-enolase-. Predictive tests (net reclassification index and area under the curve-receiver-operating characteristic analysis) showed that combined assessment of antibodies improved classification of outcome in 22%-34% of cases for partial remission of proteinuria and maintenance of normal eGFR. For patients with nephrotic syndrome at diagnosis, anti-SOD2 positivity and high anti-PLA2R1 titer were associated with a lack of complete remission. Patients who were anti-PLA2R1-/anti-intracellular antigens- had the lowest proteinuria and the highest eGFR at diagnosis and the lowest risk of lower eGFR at 12 months. Epitope spreading was present in 81% of patients who were anti-PLA2R1+ and was associated with increased positivity for intracellular antigens and poor eGFR at diagnosis and 12 months. CONCLUSIONS: Combined serological analysis of autoantibodies targeting membrane-bound and intracellular autoantigens identifies patients with poor clinical outcomes.

Structural Insights into the Interactions of Candidal Enolase with Human Vitronectin, Fibronectin and Plasminogen.

Significant amounts of enolase-a cytosolic enzyme involved in the glycolysis pathway-are exposed on the cell surface of Candida yeast. It has been hypothesized that this exposed enolase form contributes to infection-related phenomena such as fungal adhesion to human tissues, and the activation of fibrinolysis and extracellular matrix degradation. The aim of the present study was to characterize, in structural terms, the protein-protein interactions underlying these moonlighting functions of enolase. The tight binding of human vitronectin, fibronectin and plasminogen by purified C. albicans and C. tropicalis enolases was quantitatively analyzed by surface plasmon resonance measurements, and the dissociation constants of the formed complexes were determined to be in the 10-7-10-8 M range. In contrast, the binding of human proteins by the S.cerevisiae enzyme was much weaker. The chemical cross-linking method was used to map the sites on enolase molecules that come into direct contact with human proteins. An internal motif 235DKAGYKGKVGIAMDVASSEFYKDGK259 in C. albicans enolase was suggested to contribute to the binding of all three human proteins tested. Models for these interactions were developed and revealed the sites on the enolase molecule that bind human proteins, extensively overlap for these ligands, and are well-separated from the catalytic activity center.