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W Emlen

Publications and source records attributed to W Emlen.

31 records · Page 2Linked to original sources
PubMed1988

Clearance and organ localization of small DNA anti-DNA immune complexes in mice.

DNA anti-DNA immune complexes (IC) play a major role in the pathogenesis of SLE. We studied the clearance and organ localization of small DNA anti-DNA IC formed at different Ag/antibody ratios in normal mice. IC formed at Ag excess, containing areas of "exposed" DNA not covered by IgG, showed rapid Ag-mediated clearance from the circulation by the liver. DNAse digestion of these IC in vitro yielded small IC devoid of exposed DNA that were cleared more slowly from the circulation. IC formed at antibody excess were cleared by an Ag-independent mechanism at rates proportional to the number of IgG in the IC. None of the IC studied bound significantly to complement receptors on circulating cells in vivo or in vitro. For all IC, after initial rapid clearance, 10 to 20% of the injected material persisted in the circulation. Analysis of these IC showed that they were processed in vivo to yield complexes similar to those generated by in vitro DNAse digestion. We conclude that IC containing exposed DNA are removed rapidly from the circulation by Ag-mediated clearance. However, in vivo processing of IC occurs to yield smaller IC that are cleared slowly. We propose that these IC containing small DNA may persist in the circulation and accumulate in tissues, thereby playing an important role in the pathogenesis of tissue injury in SLE.

Animals↗
PubMed1988

Hepatic binding of DNA is mediated by a receptor on nonparenchymal cells.

During cell death, nuclear material is released into the cellular environment and into the circulation. Studies in experimental animals have shown that circulating DNA is rapidly removed by the liver and broken down to oligonucleotides. The authors have used a perfused liver system in the mouse to study hepatic binding of single-stranded DNA. DNA binding to the liver was rapid and efficient, and did not require serum factors. Binding was saturable and temperature independent, suggesting a receptor-mediated process. Electron microscope autoradiography demonstrated DNA binding to sinusoidal lining cells, primarily Kupffer cells. In vitro studies with isolated cells confirmed that DNA bound to a trypsin-sensitive receptor on the adherent subset of hepatic nonparenchymal cells. The integrity of the perfused liver was confirmed by the demonstration of appropriate uptake and breakdown of asialoorosomucoid. Despite rapid binding of DNA, however, the perfused liver did not digest DNA or release DNA breakdown products. Infusion of DNAse at intervals after DNA perfusion demonstrated that significant amounts of DNA remained bound to the cell surface, and that serum nucleases were able to cleave this surface bound DNA. It is concluded that DNA binding to the liver is mediated by a receptor on Kupffer cells, but that DNA breakdown may occur at the cell surface or in circulation and may not require cell interiorization.

Animals↗
PubMed1987

Complement fixation by small, DNase-resistant DNA-anti-DNA immune complexes.

125I-ds DNA-anti-DNA immune complexes (IC) formed at antibody excess and containing DNA of 300-350 base pairs (bp) fixed complement, incorporated C3b and bound to the C3b receptors (CR1) on human red blood cells (RBC). When the IC were treated with DNase to generate small, DNase-resistant IC, some of the IC incorporated C3b, but did not bind to RBC. In order to examine C3b incorporation and RBC binding by IC of specific sizes, the DNase treated IC were fractionated by sucrose density gradient (SDG) ultracentrifugation. Small IC containing one, two, three or four IgG molecules per fragment of 125I-ds DNA were identified by autoradiography after electrophoresis of the SDG fractions on 3-12% linear polyacrylamide gradient gels. The SDG fractions were tested for C3b incorporation and RBC binding ability. There was neither C3b incorporation nor RBC binding activity in fractions which corresponded to 9-11S (containing IC with one IgG/DNA). Fractions which corresponded to 12-22S (containing IC with up to four IgG/DNA fragment) demonstrated increased C3b incorporation with increased size, but did not show significant RBC binding activity. Fractions with IC containing four or more IgGs (22-24S) incorporated C3b and bound to RBC at approximately the same level. It is concluded that DNase digested IC which contain three-four IgG/DNA fragment are large enough to activate complement and incorporate C3b, but are too small to bind to RBC CR1. These IC could therefore escape rapid clearance from the circulation via the erythrocyte CR1 clearance mechanism. Such IC could persist in the circulation and potentially elicit pathogenic effects in patients with systemic lupus erythematosus.

Antibodies, Antinuclear↗
PubMed1985

Effect of antibody excess on the size, stoichiometry, and DNAse resistance of DNA anti-DNA immune complexes.

The binding of antibodies to DNA was examined under conditions of increasing antibody excess. DNA anti-DNA immune complexes (IC) formed at increasing antibody to DNA ratios were digested with excess DNAse I, and the DNAse-resistant (protected) IC were analyzed. With increasing antibody excess, the size of the IC that were resistant to DNAse digestion increased, and the size of the protected DNA within the IC also increased. This suggested that IgG molecules could bind in close proximity along the DNA molecule, preventing access of DNAse to the DNA between adjacent IgG. To further define the binding of adjacent IgG, DNAse digested IC containing one or two IgG were isolated, and the DNA contained within these IC was analyzed on DNA sequencing gels. Binding of a single IgG to DNA resulted in the protection of a DNA fragment 35 to 45 base pairs (bp) long, corresponding to the distance between binding sites of a single IgG molecule. Binding of two IgG to DNA protected a DNA fragment 50 to 60 bp long, 1 1/2 times the size of the fragment protected by one IgG. These data suggest that in conditions of Ab excess, IgG molecules can interdigitate along the DNA molecule, resulting in small, stable, DNAse-resistant IC of high antibody density.

Antibodies, Antinuclear↗
PubMed1984

DNA-anti-DNA immune complexes. Antibody protection of a discrete DNA fragment from DNase digestion in vitro.

We examined the ability of DNase I to digest DNA that was contained with DNA-anti-DNA immune complexes. IgG isolated from the sera of 20 patients with systemic lupus erythematosus (SLE) and containing antibodies to DNA was incubated with double-stranded DNA to form immune complexes. Excess DNase was added, and digestion of DNA was monitored by the conversion of DNA to TCA soluble products. IgG from 8 of the 20 SLE patients protected DNA from degradation by DNase in direct proportion to the amount of DNA bound to IgG as measured in the Farr binding assay. Using IgG from these sera, we showed that the DNA protected from degradation remained bound to IgG during digestion and was 35-45 base pairs in size. The size of this fragment is the same as that which has been proposed to be the minimal size necessary for monogamous bivalent binding of IgG to DNA. We therefore compared the ability of F(ab')2 and Fab' to protect DNA from DNase digestion and demonstrated that the bivalent F(ab')2 fragments were protective, but that the univalent Fab' fragments were not. These results suggest that some antibodies to DNA that bind to DNA via monogamous bivalent binding can protect a 35-45-base pair DNA fragment from DNase digestion. The implications of this finding are discussed with regard to the in vivo behavior and potential pathogenicity of small DNA-anti-DNA immune complexes.

Animals↗
PubMed1984

Effect of DNA size and strandedness on the in vivo clearance and organ localization of DNA.

DNA-anti-DNA immune complexes play a major role in the pathogenesis of SLE. Evidence suggests that the DNA contained within these complexes, as well as free circulating DNA, is of small molecular weight and predominantly double stranded. Previous studies have shown that large DNA is cleared from circulation rapidly and efficiently. To examine if variations in the configuration of DNA itself affected its ability to persist in the circulation, we studied the clearance and organ uptake of single stranded DNA(ssDNA) and double stranded DNA(dsDNA) of different sizes in normal mice. Clearance of DNA from the circulation was described by two exponential components. The first component represented organ uptake, and was much more rapid for ssDNA than for dsDNA. The second component represented the excretion of breakdown products from the total body pool, and was the same for all DNA preparations. Regardless of its initial size, DNA larger than 15 bases did not persist in the circulation longer than 20 min for ssDNA, and longer than 40 min for dsDNA. Organ distribution studies showed that ssDNA was removed by the liver, but that dsDNA bound poorly to the liver and was distributed like oligonucleotide breakdown products. Our results suggest that dsDNA and ssDNA are removed from the circulation by different mechanisms. Although dsDNA remains in the circulation slightly longer than ssDNA, all DNA, regardless of its size or strandedness, is cleared from the circulation and broken down rapidly and efficiently.

Animals↗
PubMed1983

Purification of DNA antibodies using cibacron blue F3GA affinity chromatography.

Isolation of DNA antibodies by conventional affinity chromatography has been difficult, often requiring harsh eluting conditions and giving low yields. The triazene dye cibacron blue F3GA is a nucleotide analogue which, when bound to an agarose matrix, has been used to isolate numerous DNA-binding proteins. In this study, we have used cibacron blue chromatography to bind and purify another group of DNA-binding proteins, DNA antibodies. Greater than 90% of both ssDNA and dsDNA antibody activity from 4 SLE plasmas bound to the cibacron blue matrix. Between 30 and 65% of this antibody activity could be eluted from the column with 1.0 M NaCl, with a net 50--60-fold antibody purification from plasma. Studies with 2 monoclonal antibodies showed that a DNA antibody directed against the DNA phosphate backbone bound to cibacron blue, but a monoclonal antibody directed primarily against bases did not bind. Inhibition studies showed that DNA antibodies bound to the cibacron blue matrix at the antigen binding site, suggesting that cibacron blue does act as an antigen analogue. Although cibacron blue chromatography yields only partially purified DNA antibodies, this method should be useful in producing enriched DNA antibody preparations from ascites fluid, tissue culture supernatants, or serum.

Antibodies↗
PubMed1982

Clearance of circulating DNA-anti-DNA immune complexes in mice.

DNA-anti-DNA immune complexes, produced from single-stranded DNA and IgG from a patient with systemic lupus erythematosus were cleared from the circulation of normal mice extremely rapidly, at a rate similar to the clearance of DNA alone. The initial clearance of these complexes was more rapid than the clearance of aggregated IgG, a surrogate immune complex containing a comparable number of IgG molecules, suggesting that the antigen (DNA) in the complexes significantly altered the clearance kinetics of the complexes. Analysis of the late clearance component of these complexes showed that anti-DNA is released back into the circulation after initial removal, and is then cleared at a rate similar to monomeric IgG. Whether this anti-DNA represents free antibody, or antibody bound to small nuclease digested DNA fragments, awaits further study.

Animals↗
PubMed1980

Effect of preformed immune complexes on the clearance and tissue localization of single-stranded DNA in mice.

Recent studies have shown that DNA is cleared from the circulation extremely rapidly by the liver, and that normal individuals have low or immeasurable levels of circulating DNA. In some patients with SLE and in NZB/W mice, however, significant amounts of free DNA as well as DNA-anti-DNA immune complexes have been found in the circulation, suggesting a possible defect in DNA clearance in these conditions. To delineate factors which might contribute to the persistence of DNA in the circulation, we have assessed the effects of immune complexes on the clearance of single stranded DNA in normal C57Bl/6J mice. HSA-anti-HSA immune complexes at five-fold antigen excess were injected intravenously and after a variable, the clearance of single-stranded DNA was determined. Clearance of all doses of DNA was markedly suppressed 6 to 12 hr after the administration of immune complexes and returned to normal by 24 hr. Immune complexes decreased DNA clearance by blocking the hepatic uptake of DNA without altering the distribution of DNA to other organs. Histology and studies on the effect of immune complexes on the clearance of bromosulphophthalein (BSP) and sulphur colloid suggest that immune complexes affect DNA clearance by altering hepatic blood flow. The results obtained in this study suggest that circulating immune complexes in patients with SLE or in other conditions may suppress normal DNA clearance, and thereby contribute to the persistence of DNA in the circulation.

Animals↗
PubMed1978

Kinetics and mechanisms for removal of circulating single-stranded DNA in mice.

Clearance of exogenous ssDNA from circulation was rapid and occurred primarily through the liver. With higher doses of single-stranded DNA (ssDNA), both liver uptake and whole blood clearance approached a maximum, enabling larger amounts of ssDNA to persist in the circulation. The large molecular weight material (precipitable ssDNA) which remained in circulation was rapidly cleaved to 20,000-30,000 mol wt fragments by endonucleases, at least some of which could be demonstrated in plasma in vitro. Mononucleotide breakdown products appeared rapidly in circulation with no lag phase, suggesting that exonuclease activity was not dependent upon prior phagocytosis. Since no exonuclease activity could be demonstrated in plasma in vitro, it was postulated that breakdown of ssDNA by exonucleases occurs on the surface of hepatocytes of Kupffer cells.

Animals↗
PubMed1972

Thyroid state: effects on pre- and postsynaptic central noradrenergic mechanisms.

For hypothyroid rats, spontaneous motor activity was less than that in matched normal controls, and the specific activity of tyrosine hydroxylase in the midbrain was significantly greater than that in controls. Rats made hyperthyroid with thyroxine became hyperactive and showed increased sensitivity to the behaviorally activating effects of norepinephrine administered intraventricularly. In hyperthyroid rats, the specific activity of tyrosine hydroxylase in the midbrain remained within the normal range. These results are consonant with studies that suggested both receptor "tuning" and feedback regulation of activity of enzymes involved in biosynthesis of presynaptic neurotransmitter as methods of regulation of the central catecholamine synapse. These results may also help explain the reported potentiation by thyroid hormone of the antidepressant effects of imipramine.

Animals↗