No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Apoptosis and nucleosomes
Abstract
Drs Berden and Koutouzov presented evidence that nucleosomes are antigens in lupus pathogenesis and that apoptotic cells are the source of nucleosomes. Berden's group measured persistence of circulating nucleosomes and nucleosome-antibody complexes in autoimmune mice and demonstrated nucleosomal deposition in skin of SLE patients as well as in renal lesions. Koutouzov reported that anti-nucleosomes are among the earliest autoantibodies in MRL+/+ mice, appearing several weeks before anti-DNA antibodies. Treatment of the mice with a pro-apoptotic drug, taxotere, accelerated autoantibody production and development of lesions. Herrmann proposed that persistent immunization results from reduced dead cell clearance and reduced production of immunosuppressive cytokines by defective scavenger macrophages. He also described accumulation of apoptotic cells in germinal follicles in SLE patients and attachment of nuclear antigens that are produced in apoptosis to the surface of follicular dendritic cells. Apoptosis-derived nucleosomes may be important in both the immunizing and effector arms of pathogenesis.
... This was further confirmed by our in vitro studies using the human breast cancer cell line MCF-7, where fragment-size profiling was indicative of active release, whereas exposure to the demethylating agent 5-AZA-CR induced the release of additional shorter fragments, indicative of apoptosis (see below) [34] Nuclear originating ccfDNA is liberated in the bloodstream either as free DNA (unbound DNA) or bound to protein or lipoprotein complexes (nucleosomes, vitrosomes, fragments of cellular membranes) [119][120][121] or enclosed in EVs such as exosomes, apoptotic bodies and microvesicles (MVs) [122,123]. DNA that is enclosed in exosomes is called exosomal DNA (exoDNA), while apoptotic bodies usually contain nucleosomes, protecting them from DNAses and RNAses [124][125][126]. ccfDNA of mitochondrial origin (cf mtDNA) also circulates in the bloodstream, either free or bound to fragments of mitochondrial membranes [127]. ...
... DNA that is bound to nucleosomes is protected from degradation and nucleosomes are circulating as mono or oligo-nucleosome fragments [128] giving a specific DNA pattern (166 or multiples). Often, nucleosomes are enclosed in apoptotic bodies and engulfed from macrophages [124] and they have been shown to be able to cross the cellular membrane [125]. ...
Simple Summary
Our research focuses in the elucidation of the nature of circulating cell-free DNA (ccfDNA) as a biological entity and its exploitation as a liquid biopsy biomaterial. Working on breast cancer, it became clear that although a promising biosource, its clinical exploitation is burdened mainly by gaps in knowledge about its biology and specific characteristics. The current review covers multiple aspects of ccfDNA in breast cancer. We cover key issues such as quantity, integrity, releasing structures, methylation specific changes, release mechanisms, biological role. Machine learning approaches for analyzing ccfDNA-generated data to produce classifiers for clinical use are also discussed.
Abstract
Breast cancer (BC) is a leading cause of death between women. Mortality is significantly raised due to drug resistance and metastasis, while personalized treatment options are obstructed by the limitations of conventional biopsy follow-up. Lately, research is focusing on circulating biomarkers as minimally invasive choices for diagnosis, prognosis and treatment monitoring. Circulating cell-free DNA (ccfDNA) is a promising liquid biopsy biomaterial of great potential as it is thought to mirror the tumor’s lifespan; however, its clinical exploitation is burdened mainly by gaps in knowledge of its biology and specific characteristics. The current review aims to gather latest findings about the nature of ccfDNA and its multiple molecular and biological characteristics in breast cancer, covering basic and translational research and giving insights about its validity in a clinical setting.
... A nucleosome is a basic unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound in sequence around eight histone protein cores. Under physiological conditions these complexes are packed in apoptotic particles and engulfed by macrophages [64]. ...
Circulating tumor DNA (ctDNA) in the blood of cancer patients contains much information on genetic and epigenetic profiles associated with cancer development, progression, and response to therapy. Analysis of ctDNA provides an opportunity for non-invasive sampling of tumor DNA repetitiously and therefore advance precision medicine. Recent development in massively parallel sequencing and digital genomic techniques support the analytical and clinical validity of ctDNA as a promising 'liquid biopsy' in human cancer. In this review, we discussed the current status of cell-free ctDNA including ctDNA biology, recently developed techniques for ctDNA detection, breast cancer specific detecting strategies, with a focus on clinical applications of ctDNA-based biomarkers in breast oncology.
... Nuclear fragments that are not sequestered within apoptotic bodies are a source of antigens in autoimmune diseases such as systemic lupus erythematosus (SLE). 47,48 Until the early 1990s, comprehensive characterization of the execution phase of apoptosis remained elusive because of the asynchronous nature of apoptotic cell death. 49,50 Caspases play major roles both in the commitment phase and in the execution phase of apoptosis through their cleavage of numerous protein substrates. ...
Apoptosis, also known as programmed cell death, is a fundamental homeostatic mechanism essential for normal embryonic development and the maintenance of healthy adult tissues. The processes that affect cell membrane dynamics during the tightly regulated apoptotic program have attracted considerable interest over the years. Distinct biochemical and structural alterations to plasma membrane composition and topography contribute to the efficient removal of cellular corpses. In this review, we will discuss these membrane alterations and their importance in maintaining cell and tissue homeostasis.
... Docetaxel targets dividing cells and acts to stabilize microtubule assembly, which leads to G2/M phase cell cycle arrest followed by apoptosis [15]. Apoptotic products can then in turn incite an immunogenic response against DNA-histone and RNA-protein complexes [16]. Therefore, in an individual with a pre-existing autoimmune disorder in which antibodies against nucleoproteins already exist (such as anti-SSA/Ro), increased levels of apoptotic products from chemotherapy could stimulate an exacerbated immune response, and manifest as severe cutaneous lesions [17]. ...
Background
Drug-induced subacute cutaneous lupus erythematosus is an uncommon disorder associated with the use of pharmacological agents including systemic chemotherapy.
Case presentation
We report a case of docetaxel-induced subacute cutaneous lupus erythematosus in a 60-year-old Caucasian female with Sjögren’s syndrome diagnosed 2 months after receiving docetaxel as part of the adjuvant FEC-D (5-fluorouracil, epirubicin, cyclophosphamide, docetaxel) chemotherapy protocol for early stage breast cancer. Although the exact mechanisms behind the autoimmune response elicited by docetaxel are unclear, the involvement of anti-SSA/Ro antibodies has been implicated.
Conclusion
This case highlights the symptom severity and clinical course of docetaxel-induced subacute cutaneous lupus erythematosus, and highlights the importance of recognizing this uncommon but potentially severe chemotherapy-associated cutaneous reaction.
... In the apoptotic cell, the disintegrated nucleus is found within blebs and packaged into membrane-clad apoptotic bodies that facilitate uptake by neighboring cells or by specialized phagocytic cells. The release of nuclear fragments from apoptotic cells is believed to be the source of antigens in autoimmune diseases such as systemic lupus erythematosus (Rosen and Casciola-Rosen, 1999; Stollar and Stephenson, 2002). ...
Membrane blebbing during the apoptotic execution phase results from caspase-mediated cleavage and activation of ROCK I. Here, we show that ROCK activity, myosin light chain (MLC) phosphorylation, MLC ATPase activity, and an intact actin cytoskeleton, but not microtubular cytoskeleton, are required for disruption of nuclear integrity during apoptosis. Inhibition of ROCK or MLC ATPase activity, which protect apoptotic nuclear integrity, does not affect caspase-mediated degradation of nuclear proteins such as lamins A, B1, or C. The conditional activation of ROCK I was sufficient to tear apart nuclei in lamin A/C null fibroblasts, but not in wild-type fibroblasts. Thus, apoptotic nuclear disintegration requires actin-myosin contractile force and lamin proteolysis, making apoptosis analogous to, but distinct from, mitosis where nuclear disintegration results from microtubule-based forces and from lamin phosphorylation and depolymerization.
... Elevated concentrations of circulating nucleosomes have been found in SLE 27 . Recently, much attention has been drawn to the hypothesis that deficient handling of apoptotic material, including circulating nucleosomes, is an important etiological factor in SLE [28][29][30][31] and is reflected by abnormal formation of ANA, including ANSA. It has been suggested that ANSA can serve as a valuable diagnostic marker of SLE or be used to measure lupus disease activity [32][33][34] . ...
To evaluate antinuclear antibody (ANA) tests in established cases of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) by indirect immunofluorescence microscopy (F-ANA) and enzyme-immunoassays detecting antinucleosomal antibodies (ANSA-EIA).
Sera from 50 patients with SLE and 65 patients with RA were analyzed regarding abnormal concentrations of F-ANA (serum dilution>or=1:200=95th percentile among 300 healthy blood donors). The sera were also analyzed with 2 commercial ANSA-EIA kits.
An abnormal F-ANA titer occurred in 76% of the SLE sera compared to 23% in RA, and was not related to present use of antirheumatic drugs. At dilution 1:50, 84% of the SLE sera were F-ANA-positive compared to 20% of healthy women. Forty percent and 56%, respectively, of the SLE sera tested positive in the 2 ANSA-EIA kits. By the most sensitive assay, 96% of the ANSA-positive SLE sera produced a homogenous (chromosomal) F-ANA staining pattern compared to 18% of the ANSA-negative SLE sera. Ten of the 15 F-ANA-positive RA sera (63%) generated homogenous F-ANA staining and 13 (20%) tested positive in the most sensitive ANSA-EIA, but with no correlation to the F-ANA staining pattern.
The sensitivity of F-ANA at an abnormal titer was surprisingly low (76%) in established cases of SLE. ANSA occurred in 56% of the SLE sera, but also in a fair number (20%) of RA sera. Practically all ANSA-positive SLE sera were identified by chromosomal F-ANA staining. We conclude that the antigen-specific antinucleosomal EIA does not have high enough diagnostic specificity to justify use of this analysis for routine diagnostic purposes.
... The generation of nucleosomes requires apoptosis [33][34][35] . A major step towards understanding the development of autoimmune response in SLE has come from the finding that cells undergoing apoptosis constitute a unique reservoir of SLE autoantigens. ...
Lupus nephritis (LN) is a prototypic autoimmune disease. Its immunopathogenesis is characterized by the loss of self-tolerance. In this article, we review our current understanding of the disease mediators of LN. There is ample evidence to suggest a pathogenic role of nephritogenic autoantibodies. These antibodies cross react with nucleosomal epitopes, and the in vivo generation of nucleosomes requires apoptosis. Furthermore, there is an intriguing and paradoxical relationship between complement and systemic lupus erythematosus (SLE). Immune complex-mediated activation of complement through the classic pathway is traditionally believed to be a major mechanism by which tissue injury occurs. In contrast, hereditary deficiencies of complement components increase the risk of SLE. Finally, the roles of reactive nitrogen and oxygen species are emphasized.
Chemotherapy has been recognized for years as a significant source of iatrogenic injury to the skin. Cutaneous side effects from conventional chemotherapeutic agents are well described, but the emergence of targeted drugs has led to new patterns of cutaneous toxicity. Since drug-induced subacute cutaneous lupus erythematosus (DI-SCLE) was first described in 1985, a continually expanding list of medications has been associated with its development. This article is protected by copyright. All rights reserved.
I read with interest the recent case by Haus et al about a patient who developed a couple of lesions of lymphomatoid papulosis (LyP) on the red parts of a tattoo [1]. Their report deserve however a few comments. The wide number of reported pseudolymphoma on tattoos are T-cell or mixed T and B-cell and not “mainly” B-cell as wrote in the manuscript [2,3].This article is protected by copyright. All rights reserved.
Pachyonychia congenita (PC) is a rare genodermatosis transmitted as an autosomal dominant trait, caused by mutations in the differentiation-specific keratin genes KRT6a (52%), KRT6b (3%), KRT16 (28%) or KRT17 (17%) which are expressed in the nails, skin, oral mucosa, larynx, hair and teeth. Approximately 1000 patients with PC have been identified, of which 400 have been confirmed genetically. Two sub-types have been classically described: PC-1 (Jadassohn-Lewandowsky type, OMIM#167200), caused by KRT6a or KRT16 mutations, with predominant oral leucokeratosis and palmoplantar keratoderma, and PC-2 (Jackson-Lawler type, OMIM#167210) resulting from KRT6b or KRT17 mutations, with neonatal teeth, pili torti and multiple cysts.2,3 The presence of multiple sebaceous cysts (steatocystoma multiplex (SM)) at puberty has been proposed to differentiate PC-2 from PC-12,3, but it is now recognized that there is a considerable overlap between the two classical sub-types of PC, and a new classification based on the mutated keratin gene has now been proposed (PC-6a, PC-6b, PC-16 and PC-17).This article is protected by copyright. All rights reserved.
Background
It has been demonstrated that Fasmediated apoptosis participates in the physiopathology of lupus nephritis, although it is not clear whether it contributes to the development of the tissue damage.Since YY-1 down regulates Fas in cancer cell lines, it is reasonable to consider that this transcription factor may control Fas expression in lupus nephritis. The objective was to determine the correlation between YY-1 and Fas expression in renal biopsies from children with type IV lupus nephritis, and their association with the clinical condition of the patients.
Importance:
Several chemotherapeutic agents have been reported to induce subacute cutaneous lupus erythematosus (SCLE). To our knowledge, this is the first report to date of SCLE induced by monotherapeutic gemcitabine hydrochloride and includes a comprehensive review of all published cases of chemotherapeutic drug-induced SCLE.
Observations:
We describe a patient who developed a SCLE-like eruption after being administered gemcitabine and discuss 16 other published cases of chemotherapeutic drug-induced SCLE.
Conclusions and relevance:
This case and a review of the literature call attention to gemcitabine and other chemotherapeutic agents that have been reported to cause drug-induced SCLE. We also discuss the clinical features of the disease.
Drug-induced subacute cutaneous lupus erythematosus (SCLE) is associated with use of the following classes of medications: anti-hypertensives, anti-cholesterolemia, anti-psychotics, and anti-inflammatory drugs. Docetaxel is an anti-neoplastic agent, which is widely used for treatment of non-small cell lung cancer. Few cases of docetaxel-induced SCLE have been reported in the medical literature. Here, we report the case of a 58-year-old female patient who developed drug-induced SCLE after administration of docetaxel. After 4 cycles of chemotherapy with docetaxel and cisplatin, erythematous skin eruptions developed on the patient's face. Skin biopsies of the eruptions were remarkable for interfacing dermatitis with basement membrane thickening. Immunofluorescent study revealed characteristic features of SCLE, including granular deposition of IgM, C3, and apoptotic bodies along the basement membrane. The skin eruptions resolved gradually after cessation of drug and with the use of topical corticosteroids.
Simulating antigen-antibody interactions is essential for elucidating antigen-antibody mechanics. Proteins interactions are vital for elucidating antibody-ssDNA associations in immunology. Therefore, this study investigated the dissociation of the human systemic lupus erythematosus antibody-ssDNA complex structure. Dissociation (i.e. the distance between the center of mass of the ssDNA and the antibody) is also studied using the potential of mean force calculations based on molecular dynamics and the explicit water model. The MM-PBSA method is also used to prove our dissociation simulations. With 605 nanosecond molecular dynamics simulations, the results indicate that the 8 residues (i.e. Gly44 (HCDR2), Asn54 (HCDR2), Arg98 (HCDR3), Tyr100 (HCDR3), Asp101 (HCDR3), Tyr32 (LCDR1), Tyr49 (LCDR2) and Asn50 (LCDR2)), and the five inter-protein molecular hydrogen bonds may profoundly impact the antibody-ssDNA interaction, a finding which may be useful for protein engineering of this antibody-ssDNA structure. Experimental binding affinity of this antibody-ssDNA complex equals 7.00 kcal mol(-1). Our dissociation binding affinity is 7.96 ± 0.33 kcal mol(-1) and MM-PBSA binding affinity is 9.12 ± 1.65 kcal mol(-1), which is close to the experimental value. Additionally, the 8 residues Gly44 (HCDR2), Asn54 (HCDR2), Arg98 (HCDR3), Tyr100 (HCDR3), Asp101 (HCDR3), Tyr32 (LCDR1), Tyr49 (LCDR2) and Asn50 (LCDR2) may play a more significant role in developing bioactive antibody analogues.
Introduction:
Carcinogenesis is accompanied by deregulated tumor cell death and changes in proliferative processes. Apoptotic cells release different components, such as nucleosomes and caspases, into the blood circulation that can be detected by minimally invasive assays. Caspases belong to a large family of proteases, which are frequently overexpressed in various cancer entities and involved in metastases. One critical event of tumor invasion that signals the initiation of the metastatic cascade is the degradation of basement membrane components by protease supporting invasive cell migration and the dissemination of tumor cells.
Areas covered:
In consideration of PubMed publications, the current review article specifically focuses on the clinical utility of circulating nucleosomes along with protease and caspase activities and discusses the quantification of these parameters as potential, minimally invasive assay.
Expert opinion:
The quantification of these circulating cell death products is a promising marker for the pathogenesis of malignant diseases and monitoring of anticancer therapies. The measurement of circulating protease activities and tumor cells in the blood may provide additional information on tumor progression and metastases.
It has been demonstrated that Fasmediated apoptosis participates in the physiopathology of lupus nephritis, although it is not clear whether it contributes to the development of the tissue damage.Since YY-1 down regulates Fas in cancer cell lines, it is reasonable to consider that this transcription factor may control Fas expression in lupus nephritis. The objective was to determine the correlation between YY-1 and Fas expression in renal biopsies from children with type IV lupus nephritis, and their association with the clinical condition of the patients.
Eighteen biopsies from children with type IV lupus nephritis and 5 controls were studied. Fas and YY-1 expression were determined by immunochemistry and quantified by densytometric analysis. The clinical conditions at the moment the biopsy were obtained from the clinical records and the results were analyzed through a one-way ANOVA with p<0.005.
The results of the densytometric analysis showed an inverse relationship between YY-1 and Fas expression. YY-1 was grouped according to the intensity of expression in low, moderate and high and compared with the expression of Fas. The lupus nephritis biopsies, which revealed high expression of YY-1, corresponded to patients with less number of clinical complications,better outcome and fewer alterations on renal function.In contrast, low expression of YY-1 correlated with high Fas expression and worst clinical conditions.
The present study suggests that YY-1regulates Fas expression in lupus nephritis and that it is associated with the clinical outcome of the patients,although further studies are necessary to determine weather it factor may serve as a prognosis factor. This is the first evidence of YY-1 participation in the physiopathology of lupus nephritis.
DNA, mRNA and microRNA are released and circulate in the blood of cancer patients. Changes in the levels of circulating nucleic acids have been associated with tumour burden and malignant progression. In the past decade a wealth of information indicating the potential use of circulating nucleic acids for cancer screening, prognosis and monitoring of the efficacy of anticancer therapies has emerged. In this Review, we discuss these findings with a specific focus on the clinical utility of cell-free nucleic acids as blood biomarkers.
The objective of this study is to investigate the presence of anti-nucleosome (anti-NCS) and anti-chromatin (anti-CRT) antibodies in patients with cutaneous lupus erythematosus (CLE) compared with active and inactive systemic lupus erythematosus (SLE). A total of 154 subjects were evaluated: 54 patients presenting CLE, 66 patients with active SLE and 34 with inactive SLE. Lupus activity was assessed using the disease activity index (SLEDAI). Anti-NCS and anti-CRT antibodies were detected by enzyme-linked immunosorbent assay (ELISA). Only one of 54 patients with CLE tested positive for both anti-NCS and anti-CRT antibodies. The prevalence of anti-CRT antibodies was significantly higher in active SLE (84.8%) when compared with inactive SLE (26.4%) and CLE (1.8%) (P < 0.001). Anti-NCS antibodies were also more prevalent in active SLE patients (74.2%) than inactive SLE (11.7%) and CLE patients (1.8%) (P < 0.001). The presence of anti-CRT and anti-NCS antibodies was correlated to disease activity in patients with SLE (r = 0.4937, r = 0.5621, respectively). Furthermore, the detection of both antibodies was correlated with disease activity in patients with SLE who tested negative for anti-dsDNA antibodies (r = 0.4754 for anti-NCS and r = 0.4281 for anti-CRT). The presence of these two auto-antibodies was strongly associated with renal damage in patients with SLE (OR = 13.1, for anti-CRT antibodies and OR = 25.83, for anti-NCS antibodies). The anti-NCS and anti-CRT antibodies were not found in CLE. In patients with SLE, there is a correlation of these antibodies with disease activity and active nephritis. When compared with anti-dsDNA antibodies, anti-NCS and anti-CRT antibodies were more sensitive in detecting disease activity and kidney damage in lupus patients.
Apoptosis in atherosclerotic lesions is triggered by inflammatory processes, both via cell-cell contact and by cytokines and oxidized lipids. The role of apoptosis in atherogenesis is dual, depending on the stage of the plaque: In early stages, apoptotic death of smooth muscle--and inflammatory cells, such as lymphocytes and macrophages--may delay atherosclerotic process. However, once the plaque is formed, apoptosis may lead to plaque rupture and thrombosis.
We describe 4 patients who developed subacute cutaneous lupus erythematosus (SCLE)-like photodistributed eruptions after ingestion of docetaxel (Taxotere). The development of SCLE-like cutaneous eruptions has been associated with the intake of drugs including thiazide diuretics, calcium channel blockers, angiotensin converting-enzyme inhibitors, phenytoin, etanercept, antihistaminics, interferons, statins, and terbinafine. Docetaxel, a chemotherapeutic drug used in breast cancer therapy, has not to our knowledge been reported to cause SCLE.
Skin biopsies were obtained from 4 patients with photodistributed rashes while taking docetaxel.
In all patients, skin biopsies were remarkable for an atrophying interface dermatitis associated with mucin deposition. Immunofluorescent testing revealed the characteristic pattern of SCLE, namely, granular epidermal keratinocyte deposition of IgG and C5b-9. The eruptions resolved following cessation of the drug.
Pathogenetically, docetaxel may evoke a lupus-like eruption through its proapoptotic effects on replicating cells, which could in turn provoke the release of nucleosomes postulated to be target antigens in LE. It seems reasonable to postulate that the rapidly replicating keratinocyte, when subjected to the cytotoxic effects of docetaxel, would also manifest nucleosome release followed by a local autoimmune reaction in a genetically predisposed host.
Autoantibody production and leukocytopenia may be linked in patients with lupus erythematosus (LE). Unclear is the ability of different autoantibody species to induce apoptosis and cell loss. Laboratory routine analyses (white blood cell counts, autoantibody detection), and flow cytometry (annexin V, CD3, CD4, CD8) have been performed in 126 consecutive LE-patients. Nuclei of PBMC were investigated flow cytometrically for the presence of the 85 kDa poly-(ADP-ribose)-polymerase (PARP) fragment. Peripheral total white blood cells (WBC), lymphocytes, T-cells, CD3+ CD4+, and CD3+ CD8+ cells were significantly decreased in patients with LE (P from 1.2 x 10(-14) to P < .0008). In the presence of either antinuclear (P from 1.2 x 10(-14) to P < .0008) or anti-dsDNA antibodies (P from 2.9 x 10(-12) to P < .007) were significantly diminished. Differences in cell numbers in LE patients with versus without anti-Ro/SSA were less pronounced: significant differences could be only obtained in lymphocytes and T-cells (P < .02). Anti-La/SSB antibodies were accompanied by significant increased leukocytes (P < .02). PARP cleavage (85 kDa) in nuclei was preferentially observed in cases with nuclear targeting autoantibodies. These results indicate that nuclear targeting autoantibodies are associated to lower peripheral blood cells counts than Ro/SSA, and La/SSB cytoplasmic targeting autoantibodies. This provides an explanation for the pathogenesis of cytopenias associated with SLE.
To evaluate pathogenetic and clinical significance of autoantibodies (AAB) with catalytic activity in the serum of patients with autoimmune myocarditis (AM).
The study was made on the sera from 99 patients with AM of different course: malignant, benign, myocardiosclerosis (MCS). In addition to standard immunological parameters, the study was made of serum levels of anticardiomyosine-antiCM (protabzymes) and anti-DNA (DNA-abzymes) of AAB. After obtaining anti-CM and anti-DNA IgG-AT, we determined non-specific and specific proteolytic activity of anti-CM.
Maximal specific activity of protabzymes was seen in 73% patients with malignant AM, it correlated with blood levels of anti-CM AAB, DNA-abzymes activity was very high in 45% patients. In MCS proteolytic activity of autoAT was absent in 61% patients. In benign AM occurrence of protabzymes was confirmed in 35% cases. Elevated DNA-hydrolyzing activity of DNA-abzymes occurred in 13% cases. The activity had no significant correlation with serum titers of AB. In MCS proteolytic activity of AAB was absent in 61% cases, but high activity of anti-CM AAB was in 28%. The activity of DNA-abzymes in 44% ranged considerably which, in seropositive cases, detected significant correlation with serum titers of DNA-binding autoAT.
Evaluation of catalytic activity of AAB may be considered as a criterial test assessing the stage, clinical variants and severity of AM. It also permits formulation of the disease prognosis and its possible outcomes.
Reported in this study are the initial results from studies to develop rabbit models of systemic lupus erythematosus (SLE) by immunizations using two distinct peptides on branched polylysine backbones (multiple Ag peptide)-peptides. Eleven rabbits received a peptide from the Sm B/B' spliceosomal complex previously shown to be immunogenic in rabbits, and 13 rabbits received a peptide from the rabbit N-methyl-d-aspartate receptor NR2b. All 24 animals in different generations of pedigreed, noninbred rabbits produced peptide-specific responses. Anti-nuclear autoantibody responses, including anti-dsDNA, were seen in 17 of 24 rabbits. To date, two rabbits have been observed to have seizure-like events and a third nystagmus. A model for eliciting development of SLE in genetically related yet heterogeneous rabbits may more closely resemble development of human SLE than do some models in inbred mice. Through selective breeding, it may also ultimately provide additional information about the genetics and etiology of SLE and serve as a model for assessing new treatment options.
We investigated in vivo the relationship between the degree of peripheral blood lymphocyte apoptosis and circulating dendritic cells (DC) as well as the immunological status in 45 patients with systemic lupus erythematous (SLE). Apoptosis was detected by phosphatidylserine externalization and assays to detect caspase activation. The total DC count (tDC) and their myeloid, mDC1 (BDCA1+) and mDC2 (BDCA3+), and plasmacytoid, pDC (BDCA3+), subtypes were assessed. Moreover, several immunological parameters, such as complement proteins, interferons (IFN), tumor necrosis factor (TNF)-alpha, interleukin (IL)-4, and IL-6 levels were assessed. There were no significant differences in both the intensity of apoptosis and DC counts between active and inactive SLE as well as between untreated patients and those treated with steroids. The incidence of lymphocyte apoptosis correlated positively with T-cell count, both T-helper (p=0.004) and cytotoxic T cells (p=0.001), but not with B or natural killer (NK) cells. The intensity of apoptosis enhanced along with the increase in complement C3 (p=0.016) and decrease in IFN-gamma (p=0.040) levels. The apoptotic cell count correlated with tDC (p=0.031), mDC1 (p=0.007), and pDC (p=0.039) counts. Both tDC and mDC1 counts correlated positively with antinuclear antibody (ANA) titers (p=0.017 and 0.032, respectively). Moreover, tDC correlated with C4 (p=0.039) and pDC with both C3 (p=0.032) and C4 (p=0.007) levels. The DC counts correlated inversely with IFN-gamma (tDC, p=0.038; mDC1, p=0.009), IL-6 (mDC2, p=0.031), or serum IgG levels (tDC, p=0.006; mDC1, p<0.001; mDC2, p=0.001). We found a positive correlation between lymphocyte apoptosis and peripheral blood DC count as well as the level of complement proteins in patients with SLE. The enhanced lymphocyte apoptosis was accompanied by the decrease in concentration of some cytokines, such as IFN-gamma or IL-6, as well as serum IgG level. This finding may reflect pathogenetic events during development of the disease, which include a persistent signal derived from circulating apoptotic lymphocytes, mobilizing the complement system, and attracting peripheral blood DC.
To develop a conceptual model of using catalytic autoantibodies as diagnostic and monitoring tools in organ-specific autoimmune disorders.
A total of 99 patients (56 males and 43 females aged 21-52 years) with autoimmune myocarditis (AM) and 198 patients (77 males and 121 females aged 8-79 years) with autoimmune uveitis (A U) participated in the study. AM patients were examined for anticardiomyosin and anti-DNA autoantibodies (ACM, ADNAab), AU patients - for autoantibodies to S-antigen, IRBP, redopsin, phosphocine, autoDNA.
AM patients had double level of DNA-binding autoantibodies. In 1/3 of them there was hydrolysing DNA and cytotoxic activity. In AU patients maximal titers were in Behcet's disease, sympathic ophthalmia, generalized uveitis and viral uveitis.
Autoantibodies with different specificity and function including DNA-abzymes can be additional diagnostic and prognostic markers.
Under various conditions, noxious stimuli damage mitochondria, resulting in mitochondrial fragmentation; however, the mechanisms by which fragmented mitochondria are eliminated from the cells remain largely unknown. Here we show that cytoplasmic vacuoles originating from the plasma membrane engulfed fragmented mitochondria and subsequently extruded them into the extracellular spaces in undergoing acute tumor necrosis factor alpha-induced cell death in a caspase-dependent fashion. Notably, upon fusion of the membrane encapsulating mitochondria to the plasma membrane, naked mitochondria were released into the extracellular spaces in an exocytotic manner. Mitochondrial extrusion was specific to tumor necrosis factor alpha-induced cell death, because a genotoxic stress-inducing agent such as cisplatin did not elicit mitochondrial extrusion. Moreover, intact actin and tubulin cytoskeletons were required for mitochondrial extrusion as well as membrane blebbing. Furthermore, fragmented mitochondria were engulfed by cytoplasmic vacuoles and extruded from hepatocytes of mice injected with anti-Fas antibody, suggesting that mitochondrial extrusion can be observed in vivo under pathological conditions. Mitochondria are eliminated during erythrocyte maturation under physiological conditions, and anti-mitochondrial antibody is detected in some autoimmune diseases. Thus, elucidating the mechanism underlying mitochondrial extrusion will open a novel avenue leading to better understanding of various diseases caused by mitochondrial malfunction as well as mitochondrial biology.
Objective
To evaluate the effect of disease progression and lipopolysaccharide (LPS) administration on the presence of nucleosomes, antinucleosome reactivity, and nucleosome–Ig complexes in the circulation of MRL and control mice.Methods
Plasma samples from lupus-prone (MRL/lpr and MRL/+) and control (CBA, Swiss, and BALB/c) mice were tested in enzyme-linked immunosorbent assays for the presence of nucleosomes, antinucleosome antibodies, and nucleosome–Ig complexes. Nucleosome kinetics, apoptosis induction, and phagocytosis of apoptotic cells were also analyzed in MRL/lpr, MRL/+, and CBA control mice after a single injection of LPS or phosphate buffered saline.ResultsNucleosomes were found in the circulation of MRL/lpr and MRL/+ mice from week 4 onward. Nucleosomes were also detected in young control mice, but with increasing age, the nucleosomes disappeared. Antinucleosome antibodies, nucleosome–Ig complexes, and albuminuria were found only in the MRL/lpr mice. LPS administration led to a significant increase in circulating nucleosomes (3–8-fold) in all strains tested. In only the MRL/lpr mice was this increase followed by a significant decrease in antinucleosome titers and an increase in nucleosome–Ig complexes. The number of apoptotic cells in the thymus after LPS was significantly higher in the MRL/lpr mice than in the MRL/+ and CBA control mice. LPS caused a profound reduction (50–70%) of the phagocytosis of apoptotic cells by peritoneal macrophages, which was comparable for all strains.Conclusion
In MRL lupus-prone mice, nucleosomes are persistently present in the circulation, whereas in control mice, nucleosomes are present only at a young age. The formation of antinucleosome antibodies and nucleosome–Ig complexes is a characteristic feature of MRL/lpr mice. LPS administration increases systemic nucleosome release due to an enhancement of apoptosis and a decrease in the clearance of apoptotic cells.
The phagocytosis of dying cells is an integral feature of apoptosis and necrosis. There are many receptors involved in recognition of dying cells, however, the molecular mechanisms of the scavenging process remain elusive. The activation by necrotic cells of complement is well established, however, the importance of complement in the scavenging process of apoptotic cells was just recently described. Here we report that the complement components C3 and C4 immediately bound to necrotic cells. The binding of complement was much higher for lymphocytes compared to granulocytes. In case of apoptotic cell death complement binding was a rather late event, which in lymphocytes was preceded by secondary necrosis. Taken together complement binding is an immediate early feature of necrosis and a rather late event during apoptotic cell death. We conclude that complement may serve as an opsonin for fragments of apoptotic cells that have escaped regular scavenging mechanisms.
Objective
To describe new assays for the detection and quantification of antibodies to RNPs in rheumatic diseases, using soluble nuclear antigens synthesized de novo in reticulocyte lysates.
Methods
Sera from 381 patients with various rheumatic diseases, including 212 patients with systemic lupus erythematosus (SLE), were analyzed in order to evaluate the sensitivity and specificity of serum autoantibody reactivities to several recombinant soluble autoantigens: U1‐A RNP, Sm‐B, SSA/Ro 52 and SSA/Ro 60, SSB/La, and Ku. Radioligand assays (RLAs) were performed following the in vitro transcription and translation of each autoantigen from the corresponding complementary DNA, labeled with ³⁵S‐methionine. The radiolabeled protein was then bound by the specific serum autoantibody, forming immune complexes that were captured by protein A–Sepharose beads and quantified by counting the radioactivity.
Results
Among the SLE patients, 44% were positive for anti–U1‐A RNP activity, 34% for anti–Sm‐B, 44% for anti‐SSA (32% for Ro 52 and 46% for Ro 60), 32% for anti‐SSB/La, and 11% for anti‐Ku reactivities. SSA antibodies had a high frequency in patients with mixed connective tissue disease (MCTD) (80%); 65% of these patient sera reacted with Ro 52, 45% with Ro 60, and 45% with U1‐A RNP. Twenty percent of the MCTD patients also exhibited antibodies to Sm‐B and Ku. In patients with Sjögren's syndrome, anti‐SSA was the main anti‐RNP antibody (63%), together with SSB/La antibodies (44%). Among patients with inflammatory myopathy, only antibodies against Ro 52 (36%) and Ro 60 (36%) were present. These new RLA allowed observation of a strong correlation (P < 0.0001) between Sm‐B antibody levels and the severity of SLE (as measured by the SLE Disease Activity Index), and establishment of a correlation between anti–U1‐A RNP antibodies and the occurrence of SLE nephritis (P < 0.02). All RLAs were highly specific for the antigen tested and displayed, in the disease groups studied, a higher sensitivity than conventional immunodiffusion assays.
Conclusion
These highly sensitive, specific, and quantitative RLAs represent new tools for the detection of autoantibodies to RNP antigens in rheumatic diseases, and may be useful for (differential) diagnosis in clinical practice.
To investigate the fate of apoptotic cells in the germinal centers (GCs) of patients with systemic lupus erythematosus (SLE).
The cardinal feature of systemic lupus erythematosus is the formation of anti-nuclear antibodies. In recent years, it has become clear that the nucleosome is a major autoantigen that drives this T cell-dependent autoimmune response, as exemplified by the presence of nucleosome-specific T helper cells and the high prevalence of nucleosome-specific autoantibodies. The only way to generate nucleosomes in vivo is by the process of apoptosis. There is growing evidence that in systemic lupus erythematosus apoptosis is disturbed, leading to the release of nucleosomes. Moreover, apoptosis-induced modifications of these autoantigens may render them more immunogenic, especially if the removal of apoptotic cells is insufficient. The first indications for the impaired clearance of apoptotic cells in systemic lupus erythematosus are emerging. Nucleosomes are also important for mediating tissue lesions, especially glomerulonephritis. In lupus nephritis nucleosomes, nucleosome-specific antibodies and nucleosome/IgG complexes have been identified in the glomerular immune deposits. Via their cationic histone part nucleosomes mediate the binding of anti-nuclear antibodies to intrinsic constituents of the glomerular basement membrane, such as the anionic heparan sulfate and collagen IV. Appreciation of this binding mechanism may lead to new treatment strategies, as shown for non-coagulant heparinoids.
To describe new assays for the detection and quantification of antibodies to RNPs in rheumatic diseases, using soluble nuclear antigens synthesized de novo in reticulocyte lysates.
Sera from 381 patients with various rheumatic diseases, including 212 patients with systemic lupus erythematosus (SLE), were analyzed in order to evaluate the sensitivity and specificity of serum autoantibody reactivities to several recombinant soluble autoantigens: U1-A RNP, Sm-B, SSA/Ro 52 and SSA/Ro 60, SSB/La, and Ku. Radioligand assays (RLAs) were performed following the in vitro transcription and translation of each autoantigen from the corresponding complementary DNA, labeled with 35S-methionine. The radiolabeled protein was then bound by the specific serum autoantibody, forming immune complexes that were captured by protein A-Sepharose beads and quantified by counting the radioactivity.
Among the SLE patients, 44% were positive for anti-U1-A RNP activity, 34% for anti-Sm-B, 44% for anti-SSA (32% for Ro 52 and 46% for Ro 60), 32% for anti-SSB/La, and 11% for anti-Ku reactivities. SSA antibodies had a high frequency in patients with mixed connective tissue disease (MCTD) (80%); 65% of these patient sera reacted with Ro 52, 45% with Ro 60, and 45% with U1-A RNP. Twenty percent of the MCTD patients also exhibited antibodies to Sm-B and Ku. In patients with Sjögren's syndrome, anti-SSA was the main anti-RNP antibody (63%), together with SSB/La antibodies (44%). Among patients with inflammatory myopathy, only antibodies against Ro 52 (36%) and Ro 60 (36%) were present. These new RLA allowed observation of a strong correlation (P < 0.0001) between Sm-B antibody levels and the severity of SLE (as measured by the SLE Disease Activity Index), and establishment of a correlation between anti-U1-A RNP antibodies and the occurrence of SLE nephritis (P < 0.02). All RLAs were highly specific for the antigen tested and displayed, in the disease groups studied, a higher sensitivity than conventional immunodiffusion assays.
These highly sensitive, specific, and quantitative RLAs represent new tools for the detection of autoantibodies to RNP antigens in rheumatic diseases, and may be useful for (differential) diagnosis in clinical practice.
Nucleosomes play a central role in the antinuclear antibody response in lupus. Lupus anti-dsDNA and antihistone antibodies directed toward nucleosomes belong together with nucleosome-specific antibodies, to a broad antinucleosome antibody family. Besides anti-dsDNA, nucleosome-specific antibodies have a major role in the pathophysiology of systemic lupus erythematosus (SLE) and emphasize the role of nucleosome-antinucleosome immune complexes. Antinucleosome immunoglobulin G antibodies are a more sensitive marker of SLE than anti-dsDNA, and are almost exclusively found in lupus, scleroderma, and mixed connective tissue diseases. An understanding of the key role of the nucleosome will likely make possible new therapeutic interventions in SLE, such as a tolerance induction to the subnucleosomal particles.
To investigate the fate of apoptotic cells in the germinal centers (GCs) of patients with systemic lupus erythematosus (SLE).
Lymph node biopsy specimens obtained from 7 SLE patients with benign follicular hyperplasia, 5 non-SLE patients with benign follicular hyperplasia (non-SLE), 5 patients with malignant follicular lymphoma, and 3 patients with dermatopathic lymphadenitis were stained with monoclonal antibodies against macrophages (CD68) and follicular dendritic cells (CR2/CD21). TUNEL staining and transmission electron microscopy were performed to detect apoptotic cells. Confocal microscopy was used to evaluate the in vivo capacity of tingible body macrophages to remove apoptotic cell material.
In a subgroup of patients with SLE, apoptotic cells accumulated in the GCs of the lymph nodes. The number of tingible body macrophages, which usually contained engulfed apoptotic nuclei, was significantly reduced in these patients. In contrast to what was observed in all controls, TUNEL-positive apoptotic material from SLE patients was observed to be directly associated with the surfaces of follicular dendritic cells (FDCs).
Our findings suggest that in a sub-group of SLE patients, apoptotic cells are not properly cleared by tingible body macrophages of the GCs. Consequently, nuclear autoantigens bind to FDCs and may thus provide survival signals for autoreactive B cells. This action may override an important control mechanism for B cell development, resulting in the loss of tolerance for nuclear antigens.