Reactivity in ELISA with DNA-loaded nucleosomes in patients with proliferative lupus nephritis

Item does not contain fulltextAutoantibodies against nucleosomes are considered a hallmark of systemic lupus erythematosus (SLE). We compared in patients with proliferative lupus nephritis the diagnostic usefulness of a dsDNA-loaded nucleosome ELISA (anti-dsDNA-NcX) with ELISAs in which dsDNA or nucleosomes alone were coated. First, we analysed whether DNA loading on nucleosomes led to masking of epitopes by using defined monoclonal anti-DNA, anti-histone and nucleosome-specific autoantibodies to evaluate the accessibility of nucleosomal epitopes in the anti-dsDNA-NcX ELISA. Second, autoantibody levels were measured in these 3 ELISAs in 100 patients with proliferative lupus nephritis (LN) before immunosuppressive treatment and in 128 non-SLE disease controls. In patients with LN inter-assay comparisons and associations with clinical and serological parameters were analysed. The panel of monoclonal antibodies revealed that all epitopes were equally accessible in the anti-dsDNA-NcX ELISA as in the two other ELISAs. Patients with proliferative lupus nephritis were positive with dsDNA-loaded nucleosomes in 86%, with DNA in 66% and with nucleosomes in 85%. In the non-lupus disease control group these frequencies were 1.6% (2 out of 128) for both the anti-dsDNA-NcX and the anti-dsDNA ELISA and 0% in the anti-nucleosome ELISA. The levels in the anti-dsDNA-NcX ELISA were high in a group of patients with LN that showed absent reactivity in the anti-DNA or low levels in the anti-nucleosome ELISA. Anti-dsDNA-NcX positivity was associated with higher SLEDAI scores within this group. Within nucleosome-based ELISAs, we propose the anti-dsDNA-NcX ELISA as the preferred test system

CTNSmRNA as a potential treatment for nephropathic cystinosis

Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to correct the genetic defect in nephropathic cystinosis using synthetic mRNA. Cystinosis is a prototype disorder of proximal tubular dysfunction caused by mutations in the CTNS gene, encoding the lysosomal cystine-H+ symporter cystinosin, and leading to cystine accumulation in all cells of the body. The kidneys are the first and most severely affected organs, presenting glomerular and proximal tubular dysfunction. Cysteamine is the current therapeutic standard that reduces cellular cystine levels, but has many side effects and does not restore kidney function. Here, we show that synthetic mRNA is safe and effective to reintroduce functional cystinosin using lipofection in CTNS-/- kidney cells and following direct injection in ctns-/- zebrafish larvae. CTNS mRNA therapy results in prompt lysosomal expression of the functional protein and decreases cellular cystine accumulation for up to 14 days. In the ctns-/- zebrafish, CTNS mRNA therapy improves proximal tubular reabsorption, reduces proteinuria, and restores brush border expression of the multi-ligand receptor megalin. We propose that mRNA-based therapy, if sufficient kidney targeting can be achieved, may be a new approach to treat cystinosis

A review of ecological gradient research in the Tropics: identifying research gaps, future directions, and conservation priorities

The Tropics are global centers of biodiversity. Ecological and land use gradients play a major role in the origin and maintenance of this diversity, yet a comprehensive synthesis of the corresponding large body of literature is still missing. We searched all ISI-listed journals for tropical gradient studies. From the resulting 1023 studies, we extracted study-specific information, and analyzed it using descriptive analytical tools and GLMs. Our results reveal that dry tropical areas are vastly understudied compared to their humid counterparts. The same holds true for large parts of Africa, but also the Philippines and the South Asian region. However, we also found that (applied) research output of developing tropical countries is nowadays on par with the output of developed countries. Vegetation and elevation were the most studied response variable and gradient, respectively. By contrast, inconspicous organisms such as oribatid mites and edaphic gradients were largely missing in the literature. Regarding biodiversity, tropical gradient studies dealt extensively with species richness and ecosystem diversity, but much less with genetic diversity. We encourage a wider use of modern statistical learning tools such as non-linear (spatio-temporal) regression and classification techniques, and simulations. Finally, we would embrace an even further development of synergies between applied and basic research and between researchers based in developed and in tropical countries. Keywords Synthesis Tropical ecology Environmental gradient relationships Biodiversit

Autoantibodies against Modified Histone Peptides in SLE Patients Are Associated with Disease Activity and Lupus Nephritis.

Persistent exposure of the immune system to death cell debris leads to autoantibodies against chromatin in patients with systemic lupus erythematosus (SLE). Deposition of anti-chromatin/chromatin complexes can instigate inflammation in multiple organs including the kidney. Previously we identified specific cell death-associated histone modifications as targets of autoantibodies in SLE. In this study we addressed, in a large cohort of SLE patients and controls, the question whether plasma reactivities with specific histone peptides associated with serology and clinical features. Plasma from SLE patients with and without lupus nephritis, disease controls, and healthy controls, were tested in ELISA with histone H4 peptide acetylated at lysines 8, 12 and 16 (H4pac), H2B peptide acetylated at lysine 12 (H2Bpac), H3 peptide trimethylated at lysine 27 (H3pme), and their unmodified equivalents. SLE patients displayed a higher reactivity with the modified equivalent of each peptide. Reactivity with H4pac showed both a high sensitivity (89%) and specificity (91%) for SLE, while H2Bpac exhibited a high specificity (96%) but lower sensitivity (69%). Reactivity with H3pme appeared not specific for SLE. Anti-H4pac and anti-H2Bpac reactivity demonstrated a high correlation with disease activity. Moreover, patients reacting with multiple modified histone peptides exhibited higher SLEDAI and lower C3 levels. SLE patients with renal involvement showed higher reactivity with H2B/H2Bpac and a more pronounced reactivity with the modified equivalent of H3pme and H2Bpac. In conclusion, reactivity with H4pac and H2Bpac is specific for SLE patients and correlates with disease activity, whereas reactivity with H2Bpac is in particular associated with lupus nephritis

A prospective study of anti‐chromatin and anti‐C1q autoantibodies in patients with proliferative lupus nephritis treated with cyclophosphamide pulses or azathioprine/methylprednisolone

Contains fulltext : 52040.pdf (publisher's version ) (Closed access)OBJECTIVE: To study the prevalence and course of anti-chromatin (anti-nucleosome, anti-double-stranded (ds) DNA and anti-histone) and anti-C1q autoantibodies in patients with proliferative lupus nephritis (LN), treated in a randomised controlled trial with either cyclophosphamide or azathioprine plus methylprednisolone. METHODS: Autoantibody levels were measured and analysed in 52 patients with proliferative LN, during their first year of treatment. Levels in both treatment arms were compared and associations with clinical, serological and outcome parameters were studied. RESULTS: At study entry, prevalences for anti-nucleosome, anti-dsDNA, anti-histone and anti-C1q autoantibodies were 81%, 96%, 23% and 65%, respectively. Anti-chromatin autoantibodies correlated with each other, but not with anti-C1q levels. If patients were divided for their autoantibody titre at the start of treatment above or below the median, the only significant differences were higher SLE disease activity index with higher anti-nucleosome, and higher creatinine with higher anti-C1q autoantibodies. During the first year, a comparable rapid decline in the levels of anti-nucleosome, anti-dsDNA and anti-C1q autoantibodies was seen in both treatment arms. Anti-histone autoantibody levels were low and did not change. Renal flares were not preceded by rises in autoantibody titres. CONCLUSIONS: These results indicate that measurement of anti-chromatin and anti-C1q autoantibodies is useful for diagnosing LN, but not for monitoring disease course

Impact of left atrial appendage morphology on thrombus formation after successful left atrial appendage occlusion: Assessment with cardiac-computed-tomography

Abstract A standardized imaging algorithm by cardiac computed tomography angiography (cCTA) (LOVE-view) was used in 30 patients to evaluate the influence of different left atrial appendage (LAA) morphologies on development of thrombosis in the LAA 6 months after implantation of an occlusion device (Watchman or Amplatzer-Cardiac-Plug) in patients with non-valvular atrial fibrillation, CHA2DS2-VASc-Score >1 and a contraindication for oral anticoagulation. The distribution of different LAA morphologies was 40% windsock, 17% broccoli and 43% chicken wing type. There was no significant difference in the level of thrombosis regarding LAA morphology or the type of chosen occlusion device. The rates of complete LAA thrombosis was 40% in broccoli type, 33% in windsock and 15% in chicken wing type. Independently of LAA type, 13% had none and 60% incomplete thrombosis. The ratio of density (LA/LAA) was 0.14 in patients with complete thrombosis and 0.67 in those with none or incomplete thrombosis. cCTA and the LOVE-view-imaging-algorithm were shown to be a valuable method for standardized imaging in clinical routine in a greater set of patients. Surprisingly thrombosis of the occluded LAA was still in progress in most cases at 6 months, whereas further studies are needed defining its clinical consequences, especially for the selection of the optimal post-procedural antithrombotic treatment strategy

Circulating apoptotic microparticles in systemic lupus erythematosus patients drive the activation of dendritic cell subsets and prime neutrophils for NETosis

\u3cp\u3eOBJECTIVE: Circulating chromatin-containing apoptotic material and/or neutrophil extracellular traps (NETs) have been proposed to be an important driving force for the antichromatin autoimmune response in patients with systemic lupus erythematosus (SLE). The aim of this study was to determine the exact nature of microparticles in the circulation of SLE patients and to assess the effects of the microparticles on the immune system.\u3c/p\u3e\u3cp\u3eMETHODS: We analyzed microparticles isolated from the plasma of patients with SLE, rheumatoid arthritis (RA), and systemic sclerosis (SSc), as well as from healthy subjects. The effects of the microparticles on blood-derived dendritic cells (DCs) and neutrophils were assessed by flow cytometry, enzyme-linked immunosorbent assay, and immunofluorescence microscopy.\u3c/p\u3e\u3cp\u3eRESULTS: In SLE patients, we identified microparticles that were highly positive for annexin V and apoptosis-modified chromatin that were not present in healthy subjects or in RA or SSc patients. These microparticles were mostly CD31+/CD45- (endothelial), partly CD45+/CD66b+ (granulocyte), and negative for B and T cell markers. Microparticles isolated from the plasma of SLE patients increased the expression of the costimulatory surface molecules CD40, CD80, CD83, and CD86 and the production of proinflammatory cytokines interleukin-6, tumor necrosis factor, and interferon-α by blood-derived plasmacytoid DCs (PDCs) and myeloid DCs (MDCs). SLE microparticles also primed blood-derived neutrophils for NETosis. Microparticles from healthy subjects and from RA or SSc patients exhibited no significant effects on MDCs, PDCs, and NETosis.\u3c/p\u3e\u3cp\u3eCONCLUSION: Circulating microparticles in SLE patients include a population of apoptotic cell-derived microparticles that has proinflammatory effects on PDCs and MDCs and enhances NETosis. These results underline the important role of apoptotic microparticles in driving the autoimmune response in SLE patients.\u3c/p\u3

Evaluation of the efficacy of cystinosin supplementation through CTNS mRNA delivery in experimental models for cystinosis

Abstract Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to treat the genetic defect in cystinosis using synthetic mRNA in cell models and ctns −/− zebrafish embryos. Cystinosis is a prototype lysosomal storage disorder caused by mutations in the CTNS gene, encoding the lysosomal cystine-H+ symporter cystinosin, and leading to cystine accumulation in all cells of the body. The kidneys are the first and the most severely affected organs, presenting glomerular and proximal tubular dysfunction, progressing to end-stage kidney failure. The current therapeutic standard cysteamine, reduces cystine levels, but has many side effects and does not restore kidney function. Here, we show that synthetic mRNA can restore lysosomal cystinosin expression following lipofection into CTNS −/− kidney cells and injection into ctns −/− zebrafish. A single CTNS mRNA administration decreases cellular cystine accumulation for up to 14 days in vitro. In the ctns −/− zebrafish, CTNS mRNA therapy improves proximal tubular reabsorption, reduces proteinuria, and restores brush border expression of the multi-ligand receptor megalin. Therefore, this proof-of-principle study takes the first steps in establishing an mRNA-based therapy to restore cystinosin expression, resulting in cystine reduction in vitro and in the ctns −/− larvae, and restoration of the zebrafish pronephros function