Synchronized turbo apoptosis induced by cold-shock.

In our research on the role of apoptosis in the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE), we aim to evaluate the effects of early and late apoptotic cells and blebs on antigen presenting cells. This requires the in vitro generation of sufficiently large and homogeneous populations of early and late apoptotic cells. Here, we present a quick method encountered by serendipity that results in highly reproducible synchronized homogeneous apoptotic cell populations. In brief, granulocytic 32Dcl3 cells are incubated on ice for 2 h and subsequently rewarmed at 37°C. After 30-90 min at 37°C more than 80-90% of the cells become early apoptotic (Annexin V positive/propidium iodide negative). After 24 h of rewarming at 37°C 98% of the cells were late apoptotic (secondary necrotic; Annexin V positive/propidium iodide positive). Cells already formed apoptotic blebs at their cell surface after approximately 20 min at 37°C. Inter-nucleosomal chromatin cleavage and caspase activation were other characteristics of this cold-shock-induced process of apoptosis. Consequently, apoptosis could be inhibited by a caspase inhibitor. Finally, SLE-derived anti-chromatin autoantibodies showed a high affinity for apoptotic blebs generated by cold-shock. Overall, cold-shock induced apoptosis is achieved without the addition of toxic compounds or antibodies, and quickly leads to synchronized homogeneous apoptotic cell populations, which can be applied for various research questions addressing apoptosis.

Amperometric complex-formation titrations at a dropping mercury electrode, employing normal pulse polarography.

Selective determinations of several metal ions by means of amperometric complex-formation titrations employing normal pulse polarography with a DME appear to be possible. Concentrations down to 3 x 10(-7)M have been determined with adequate accuracy. In alkaline medium deaeration is necessary; in acidic medium it can be omitted.

Amperometric complex-formation titrations with a dropping bismuth amalgam electrode in halide medium.

The use of the dropping bismuth amalgam electrode has been investigated for the selective determination of metal ions in the presence of large concentrations of halides by means of amperometric complex-formation titrations, using normal pulse polarography. Concentrations of metal ions down to 3 x 10(-7)M have been determined with adequate accuracy in the presence of about 0.1M chloride or 0.01M bromide. Calculated and experimental current-voltage curves have been compared and found to be in reasonable agreement.

Behaviour of solid electrodes in normal and differential pulse voltammetric methods.

In order to investigate the behaviour of solid electrodes in normal and differential pulse voltammetry, step functions have been applied to the electrochemical cell containing the electrodes to be tested, in the absence of electroactive species. The large residual current observed could be attributed to electrochemical reactions of the electrode material.

Anodic behaviour of a dropping lead amalgam electrode in halide medium in the presence of EDTA.

The behaviour of the dropping lead amalgam electrode has been studied. Calculated and experimental current-voltage curves have been compared and an explanation has been given for the observed differences. Selective determination of metal ions appears to be possible in the presence of saturated chloride, 1M bromide and 10(-2)M iodide by means of amperometric complex-formation titrations using normal pulse polarography with the dropping lead amalgam electrode.

Study of a glassy carbon electrode in amperometric detection using d.c., normal and differential pulse techniques.

The results of a comparative study on d.c., normal pulse and differential pulse techniques applied to anodic amperometric detection at a glassy carbon electrode in a voltammetric flow-through cell are presented. The important aspects examined are response time, linearity, limit of detection and selectivity. It is shown that the d.c. mode is the most favourable as long as no adsorption of oxidation products takes place. If strong adsorption occurs, normal pulse detection is recommended, although the limit of detection is somewhat larger.

Amperometric complex-formation titrations with a dropping indium amalgam electrode in halide medium.

The applicability of a dropping indium amalgam electrode for the determination of metal ions in the presence of large concentrations of halides by means of amperometric complex-formation titrations using normal pulse polarography has been investigated. Titrations appear to be possible in the presence of 4M potassium iodide, 1M potassium bromide and 1M potassium chloride.

The binding of lupus-derived autoantibodies to the C-terminal peptide (83-119) of the major SmD1 autoantigen can be mediated by double-stranded DNA and nucleosomes.

OBJECTIVES: To evaluate the binding of lupus-derived autoantibodies, double-stranded DNA and nucleosomes to the positively charged C-terminal SmD1(residues 83-119) peptide and the full-length SmD protein. METHODS: The binding of lupus-derived monoclonal antibodies, sera from patients with systemic lupus erythematosus, rheumatoid arthritis and systemic sclerosis, dsDNA and nucleosomes to the SmD1(83-119) peptide or the full-length SmD protein was determined using different ELISA methods. RESULTS: Monoclonal anti-dsDNA antibodies and the serum of patients with systemic lupus erythematosus that are positive for anti-dsDNA antibodies react with the SmD1(83-119) peptide in ELISA. However, DNaseI treatment of the blocking reagents leads to a decreased reactivity. Purified dsDNA and nucleosomes bind to the SmD1 peptide but not to the full-length SmD protein. CONCLUSIONS: The SmD1(83-119) peptide is able to bind dsDNA and nucleosomes, and dsDNA or nucleosomes in applied reagents lead to an apparent reactivity of anti-dsDNA, anti-histone or nucleosome-specific antibodies with the SmD1(83-119) peptide in ELISA.

Triggers for anti-chromatin autoantibody production in SLE.

The formation of autoantibodies against chromatin is the main feature of systemic lupus erythematosis (SLE), an autoimmune disease, which is T-cell dependent and autoantigen-driven. Historically, antibodies against dsDNA, one of the components of chromatin, are considered as a hallmark of SLE. However, dsDNA is poorly immunogenic. Nucleosome-specific T helper cells have been identified. These T cells propagate not only nucleosome-specific antibodies, but also anti-dsDNA antibodies. Nucleosomes are formed during apoptosis by cleavage of chromatin, and evidence of disturbed apoptosis has been found especially in certain murine models of lupus. In addition to an increased rate of apoptosis, autoimmunity against chromatin might also result from an impaired phagocytosis of apoptotic material, for which strong evidence has been provided by studies in certain knock-out mice (C1q, SAP, Dnase I). The induction of an immune response to nucleosomes could be enhanced by modifications of histones or DNA during apoptosis, altered presentation by antigen presenting cells or a viral infection. The release of nucleosomes and the formation of anti-chromatin autoantibodies result in formation of complexes, which bind to the glomerular basement membrane via heparan sulfate. This deposition incites glomerulonephritis, the most serious manifestation of SLE.

Decreased phagocytosis of apoptotic cells in diseased SLE mice.

Antibodies against nucleosomes are a serological hallmark of systemic lupus erythematosus (SLE). Apoptotic cells are the unique source of nucleosomes, which are formed through cleavage of chromatin by nucleases. These nucleosomes and other autoantigens targeted in SLE are expressed in apoptotic blebs or at the surface of apoptotic cells. Therefore, it is conceivable that circulating antibodies can influence apoptotic cell clearance. Using an in vitro phagocytosis assay, we analysed the phagocytic efficacy for apoptotic cells of resident peritoneal macrophages from pre-morbid and diseased lupus mice. The assay was carried out in the presence of autologous serum, using autologous apoptotic thymocytes as targets. Under these conditions macrophages from diseased MRL/lpr and NZBxNZW(F1) lupus mice, and from age-matched NZB mice showed a decreased phagocytic efficacy (decrease 47%, 48% and 37%, respectively compared to measurements in pre-morbid mice). The cause of this decrease resides in the serum, and is not due to an acquired defect of macrophages. In conclusion, during disease progression in murine SLE, apoptotic cell clearance becomes impaired, which might amplify further disease progression.