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PubMed1990

The disappearance of macromolecules from the peritoneal cavity during continuous ambulatory peritoneal dialysis (CAPD) is not dependent on molecular size.

The transport of macromolecules from the circulation to the peritoneal cavity is a size-selective restricted process, while the transport of these solutes from the peritoneal cavity is probably mainly by lymphatic absorption. If so, it should be independent of molecular size. Therefore, we studied with a clearance technique the disappearance of intraperitoneally administered inulin and polydisperse dextran 70 in nine continuous ambulatory peritoneal dialysis (CAPD) patients and compared the results with the simultaneously measured appearance clearance of serum proteins. Using gel permeation chromatography 18 dextran fractions with different molecular radii could be analyzed. Inulin clearance (2.94 mL/min) was higher than total dextran clearance (1.30 mL/min). The maximal dextran concentration in all dialysate samples was found in the 50.4 A fraction. The clearances of the dextran fractions were the same of different molecular sizes. All disappearance clearances were higher than the appearance clearances: the protein/dextran clearance ratio ranged from 0.15 for albumin/36 A to 0.04 for alpha 2-macroglobulin/91 A. This confirms that the appearance of a macromolecule, but not its disappearance is dependent on molecular size. It is concluded that the disappearance of macromolecules from the peritoneal cavity is mainly a size independent convective process, possibly by lymphatic uptake. This implies that total dextran 70 clearance can be used for measurement of lymphatic absorption in CAPD patients.

Adult↗
PubMed1991

Do gamma delta T cells play an important role in autoimmune disease?

A T cell subpopulation using an alternative receptor heterodimer (gamma delta T cells) contributes 0.5-10% to the T cell population in the normal peripheral blood. One subset of gamma delta T cells (V gamma 9+/V delta 2+/ C gamma 1) preferentially recognizes mycobacterial antigens, which are the triggering antigens in the rat adjuvant arthritis (AA), an animal model with some similarities with rheumatoid arthritis (RA). Here we summarize data on the prevalence of gamma delta T cells in peripheral blood, synovial fluid and synovial membranes of RA patients which show that gamma delta T cells, in particular the subset reactive with mycobacterial antigens in normal individuals, are rare in all studied compartments. Furthermore, there was no enrichment of gamma delta T cells in the peripheral blood of patients with ankylosing spondylitis or systemic lupus erythematosus, autoimmune diseases with involvement of the joints. In the AA model depletion of the "conventional" alpha beta T cells completely prevented the induction of the disease and very effectively improved ongoing arthritis even though substantial numbers of gamma delta T cells were present in these animals. Taken together, our data do not support the notion of a significant contribution of gamma delta T cells to chronic joint inflammation in the autoimmune diseases studied or in adjuvant arthritis.

Animals↗
PubMed1990

Protein components of a cytochrome P-450 linalool 8-methyl hydroxylase.

The cytochrome P-450 heme-thiolate monooxygenases that hydroxylate monoterpene hydrocarbon groups are effective models for the cytochrome P-450 family. We have purified and characterized the three proteins from a P-450-dependent linalool 8-methyl hydroxylase in Pseudomonas putida (incognita) strain PpG777. The proteins resemble the camphor 5-exohydroxylase components in chemical and physical properties; however, they show neither immunological cross-reactivity nor catalytic activity in heterogenous recombination. These two systems provide an excellent model to probe more deeply the heme-thiolate reaction center, molecular domains of substrate specificity, redox-pair interactions, and the regulation of the reaction cycle.

Acyclic Monoterpenes↗
PubMed1990

The primary structure of the 32-kDa subunit of human replication protein A.

Replication protein A (RP-A) is a complex of three polypeptides of molecular mass 70, 32, and 14 kDa, which is absolutely required for simian virus 40 DNA replication in vitro. We have isolated a cDNA coding for the 32-kDa subunit of RP-A. An oligonucleotide probe was constructed based upon a tryptic peptide sequence derived from whole RP-A, and clones were isolated from a lambda gt11 library containing HeLa cDNA inserts. The amino acid sequence predicted from the cDNA contains the peptide sequence obtained from whole RP-A along with two sequences obtained from tryptic peptides derived from sodium dodecyl sulfate-polyacrylamide gel-purified 32-kDa subunit. The coding sequence predicts a protein of 29,228 daltons, in good agreement with the electrophoretically determined molecular mass of the 32-kDa subunit. No significant homology was found with any of the sequences in the GenBank data base. The protein predicted from the cDNA has an N-terminal region rich in glycine and serine along with two acidic and two basic segments. Monoclonal antibodies have been raised against the 70- and 32-kDa subunits of RP-A. The cloned cDNA has been overexpressed in bacteria using an inducible T7 expression system. The protein made in bacteria is recognized by a monoclonal antibody that is specific for the 32-kDa subunit of RP-A. This monoclonal antibody against the 32-kDa subunit inhibits DNA replication in vitro.

Amino Acid Sequence↗
PubMed1985

Association between the Ro and La antigenic determinants: immunodiffusion analysis of human spleen extract.

The Ro (SS-A) and La (SS-B) antigenic determinants appear to be related to one another because of the frequent coincidence of spontaneous anti-Ro and anti-La in the same autoimmune sera, and because of a tendency of the Ro and La immunoprecipitin lines in double immunodiffusion analysis to fuse. We have developed an enzyme immunodiffusion staining (EIS) procedure that permitted us to identify the specific antigenic determinants found in an immunoprecipitin line. By using this technique with human spleen extract, we showed that the Ro and La particles are found together as a complex, as well as individually. The EIS technique insured that our results were not confounded by lack of monospecificity of our autoantibody and antigenic reagents. Ro-La antigenic complexes exist at physiologic pH, and are dissociated by high ionic strength. They may be formed in vivo either intracellularly or extracellularly. Such Ro-La complexes could be immunogenic, and thereby might account for the frequent coincidence of the anti-La and anti-Ro autoantibody specificities.

Antigens↗
PubMed1986

Distribution of charged sites on lymphatic endothelium.

The charge distribution on the luminal and abluminal aspects of fixed and living lymphatic endothelium was examined with particular emphasis on the endocytotic vesicular system and interendothelial junctions. Native ferritin (NF; pl = 4.5), when administered abluminally to perfused lymphatics, entered endocytotic vesicles and abluminal and luminal caveolae; NF was also found in intercellular channels, in contrast, NF when applied luminally was largely excluded from both luminal caveolae and intercellular channels. Cationic ferritin (CF; pl = 8.4) bound to the discontinuous basal lamina and to the abluminal plasma membrane, clustering preferentially around the stomata of abluminal caveolae. CF did not, however, bind to the plasma membrane of, or enter, either the vesicular system or intercellular channels, when administered abluminally. When added to the perfusion fluid CF bound to the luminal membrane and to the infundibula of intercellular channels. Ruthenium red (RR) and alcian blue (AB), both cationic stains, bound intensely to the luminal membrane and much less so to the abluminal surface, thus simulating the binding pattern of CF. Unlike CF, however, RR and AB bound to the membranes of abluminal and luminal caveolae with the same level of staining as to the plasma membrane to which they were attached. These results reflect a marked asymmetry in the membrane charge characteristics of endothelial cells.

Alcian Blue↗
PubMed1987

Anti-histone antibodies in idiopathic and drug-induced lupus recognize distinct intrahistone regions.

We have identified regions within core histones that are antigenic for autoantibodies in systemic lupus erythematosus (SLE) and drug-induced lupus. An immunoblotting technique was used to determine the reactivity of lupus antibodies for intact histones and for trypsin-resistant histone fragments that lack the amino- and carboxyl-terminal amino acids that are normally exposed in native nucleosomes. In SLE, the predominant anti-histone response was restricted to epitopes in the trypsin-sensitive regions. Of 20 SLE sera that had strong antibody activity for multiple intact histones, 17 showed minimal activity with any of the corresponding trypsin-resistant fragments. A markedly different pattern of reactivity was present in sera of patients with procainamide (Pr)-induced lupus in which antibodies to H2A, H2B, and the H2A-H2B complex had strong fragment activity. Interestingly, recognition of trypsin-resistant fragments was also noted in a small number of SLE sera that contained antibodies to the H2A-H2B complex. In contrast to both SLE and Pr-induced lupus, antibodies induced by hydralazine (Hy) reacted primarily with H3 and H4. Furthermore, these antibodies bound equally well to the corresponding trypsin-resistant regions that are thought to be relatively unexposed in native nucleosomes. Thus, the specificities of anti-histone antibodies in SLE, Pr-induced lupus, and Hy-induced lupus are markedly different, but in each disease reactivity appears to be restricted to a limited number of histone determinants. The data raise the possibility that autoantigen in the form of native nucleosomes may be recognized in SLE and possibly in Pr-induced lupus. In contrast, the propensity of Hy to induce autoantibodies to determinants usually not recognized in SLE or Pr-induced lupus may suggest a different immunogenic stimulus in this disease.

Animals↗
PubMed1979

Permselectivity of cat blood-lymph barrier to endogenous macromolecules.

The restrictive properties of liver blood-lymph barrier to endogenous plasma protein fractions of varying molecular size were studied at different hepatic venous pressures (and lymph flows) using steady-state lymph to plasma protein concentration ratios. At control hepatic venous pressures (0-2 mmHg) the lymph to plasma venous concentration ratios of the various protein fractions indicate significant selectivity by the liver blood-lymph barrier to macromolecules on the basis of molecular size. The sieving observed was consistent with equivalent pore radii of 180-250 A. The transsinusoidal oncotic pressure gradient ranged between 4.0 and 8.0 mmHg at control venous pressures. As venous pressure was increased, liver lymph flow and lymph to plasma protein concentration ratio increased while the blood-lymph oncotic pressure gradient decreased. Liver lymph flow was linearly related to hepatic venous pressure. At lymph flow less than or equal to 10 x control, there is no longer a sieving effect on plasma protein; findings consistent with por radii in excess of 1000 A. The results of this study suggest that maximal sieving by the liver blood-lymph barrier occur at normal capillary filtration rates and support the possibility that the interstitium is the rate limiting barrier for blood to lymph transport of macromolecules in the liver.

Animals↗
PubMedDate not supplied

[Changes in lipid composition of mucosa secretion from chick glandular stomach intermediate zone in A-vitaminosis].

The lipid composition of secretion and the vitamin A content in it were studied. Secretion of normal chickens is shown to contain 96 +/- 3.1 mg of lipids, 82.5 +/- 3.4 of substances extracted by the solvent hexane-diethyl ester, 30 +/- 2.1 of unsaponifiable lipids and 2.43 +/- 0.285 micrograms of vitamin A per 1 g of dry residue. With A-avitaminosis the secretion amount is twice as large with vitamin A absent; the lipid content is almost twice as low, the total weight of substances extracted by the solvent hexane-diethyl ester is four times as low and that of the lipid unsaponifiable fraction--2.5 times as low. Quantitative and qualitative changes in lipid zones were detected by means of thin-layer chromatography and UV-spectroscopy, and structural changes in the secretion--by electron microscopy. Vitamin A is suggested to participate in formation of macromolecular lipid systems of the secretion.

Animals↗
PubMed1981

An abnormality of immune complex kinetics in murine lupus.

In order to understand better the role of immune complex metabolism in the pathogenesis of autoimmune diseases, we have investigated the early stages of immune complex uptake by the liver, the major organ responsible for clearance of soluble complexes in the mouse. Livers were perfused in situ via the portal vein over 3 to 5 min with trace amounts of radiolabeled soluble model immune complexes. In 4 nonautoimmune strains (BALB/c, DBA/2, CAF1, NZW) 60 to 72% of the model complexes perfused were taken up and remained in the liver after 20 min of continuous perfusion with oxygenated Krebs-Henseleit buffer. In NZB and NZB/W F1 female mice at ages 0.5 to 11 mo, 66 to 78% of the model complexes remined in the liver. However, when a dose of heat-aggregated human gamma-globulin sufficient to saturate the reticuloendothelial system was perfused 7 min after radiolabeled complexes, 15.2 +/- 7.2% (mean +/- SD) of the complexes were displaced in the nonautoimmune strains. In contrast, 32.6 +/- 10.5% of the complexes were displaced from the liver in NZB and NZB/W F1 female mice (p < 0.001). Thus, although hepatic uptake of immune complexes in autoimmune mice appears to be normal or even enhanced, there may be impaired phagocytosis by the hepatic RES or weaker binding of complexes to the surface of the Kupffer cells. Such surface-bound immune complexes remaining accessible to the circulation may contribute to the autoimmune process.

Aging↗