Endogenous DNA is primarily found intracellularly in nuclei and mitochondria. However, extracellular, cell-free (cf) DNA, has been observed in several pathological conditions, including autoimmune diseases, prompting the interest of developing cfDNA as a potential biomarker. There is an upsurge in studies considering cfDNA to stratify patients, monitor the treatment response and predict disease progression, thus evaluating the prognostic potential of cfDNA for autoimmune diseases. Since the discovery of elevated cfDNA levels in lupus patients in the 1960s, cfDNA research in autoimmune diseases has mainly focused on the overall quantification of cfDNA and the association with disease activity. However, with recent technological advancements, including genomic and methylomic sequencing, qualitative changes in cfDNA are being explored in autoimmune diseases, similar to the ones used in molecular profiling of cfDNA in cancer patients. Further, the intracellular origin, e.g., if derived from mitochondrial or nuclear source, as well as the complexing with carrier molecules, including LL-37 and HMGB1, has emerged as important factors to consider when analyzing the quality and inflammatory potential of cfDNA. The clinical relevance of cfDNA in autoimmune rheumatic diseases is strengthened by mechanistic insights into the biological processes that result in an enhanced release of DNA into the circulation during autoimmune and inflammatory conditions. Prior work have established an important role of accelerated apoptosis and impaired clearance in leakage of nucleic acids into the extracellular environment. Findings from more recent studies, including our own investigations, have demonstrated that NETosis, a neutrophil cell death process, can result in a selective extrusion of inflammatory mitochondrial DNA; a process which is enhanced in patients with lupus and rheumatoid arthritis. In this review, we will summarize the evolution of cfDNA, both nuclear and mitochondrial DNA, as biomarkers for autoimmune rheumatic diseases and discuss limitations, challenges and implications to establish cfDNA as a biomarker for clinical use. This review will also highlight recent advancements in mechanistic studies demonstrating mitochondrial DNA as a central component of cfDNA in autoimmune rheumatic diseases.
Pyroptosis is an inflammatory form of programmed cell death following cellular damage or infection. It is a lytic process driven by gasdermin D-mediated cellular permeabilization and presumed osmotic forces thought to induce swelling and rupture. We found that pyroptotic cells do not spontaneously rupture in culture but lose mechanical resilience. As a result, cells were susceptible to rupture by extrinsic forces, such as shear stress or compression. Cell analyses revealed that all major cytoskeleton components were disrupted during pyroptosis and that sensitivity to rupture was calpaindependent and linked with cleavage of vimentin and loss of intermediate filaments. Moreover, while release of lactate dehydrogenase (LDH), HMGB1, and IL-1β occurred without rupture, rupture was required for release of large inflammatory stimuli-ASC specks, mitochondria, nuclei, and bacteria. Importantly, supernatants from ruptured cells were more immunostimulatory than those from nonruptured cells. These observations reveal undiscovered cellular events occurring during pyroptosis, define the mechanisms driving pyroptotic rupture, and highlight the immunologic importance of this event.
Objective. Neutrophils are key immune cells participating in host defense through several mechanisms, including the formation of neutrophil extracellular traps (NETs). This study was undertaken to investigate the role of neutrophils in juvenile dermatomyositis (JDM).Methods. Electron microscopy was used to identify neutrophils in tissue. NETs were also imaged using fluorescence microscopy and quantified using a myeloperoxidase-DNA enzyme-linked immunosorbent assay (ELISA) in plasma obtained from healthy children (n = 20), disease controls (n = 29), JDM patients (n = 66), and JDM patients with history of calcifications (n = 20). Clinical data included disease activity scores and complement C4 levels. Levels of immune complexes (ICs) and calprotectin were analyzed using ELISA.Results. Using electron microscopy, neutrophils were found to infiltrate affected muscle tissue, engulfing deposited calcium crystals. Uptake of the crystals led to neutrophil activation (P < 0.01) and subsequent phosphatidylinositol 3-kinase-and NADPH oxidase-dependent but peptidylarginine deiminase 4-independent formation of NETs, which contained mitochondrial DNA (P < 0.05), as confirmed in vivo (P < 0.001) and in vitro (P < 0.01). Peripheral NET levels were associated with calcinosis (P = 0.01), ICs (P = 0.008), and interleukin-8 levels (P = 0.004). Children with JDM had impaired NET clearance (P = 0.01), associated with autoantibody profiles including melanoma differentiationassociated protein 5 (P = 0.005), and depressed complement C4 levels (r = −0.72, P = 0.002). Furthermore, children with JDM showed evidence of neutrophil activation, with elevated levels of peroxidase activity (P = 0.02) and calprotectin (P < 0.01), which were associated with disease activity (P = 0.007), and dyslipidemia (odds ratio 4.7, P < 0.05).Conclusion. We found novel mechanisms of both calcium crystal-mediated neutrophil activation and cell death in JDM pathophysiology. Targeting this pathway may reduce the frequency and extent of calcinosis, as well as prevent long-term development of comorbidities, including atherosclerosis.
The surface glycoprotein hemagglutinin (HA) helps the influenza A virus to evade the host immune system by antigenic variation and is a major driving force for viral evolution. In this study, the selection pressure on HA of H5N1 influenza A virus was analyzed using bioinformatics algorithms. Most of the identified positive selection (PS) sites were found to be within or adjacent to epitope sites. Some of the identified PS sites are consistent with previous experimental studies, providing further support to the biological significance of our findings. The highest frequency of PS sites was observed in recent strains isolated during 2005–2007. Phylogenetic analysis was also conducted on HA sequences from various hosts. Viral drift is almost similar in both avian and human species with a progressive trend over the years. Our study reports new mutations in functional regions of HA that might provide markers for vaccine design or can be used to predict isolates of pandemic potential.
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