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Biomedical subjects

L K Curtiss

Publications and source records attributed to L K Curtiss.

At least 91 records · Page 5Linked to original sources
PubMed1990

Platelet-mediated foam cell formation in atherosclerosis.

The early fatty streak lesions of atherosclerosis are characterized by the presence of cholesteryl ester-loaded macrophages or "foam cells." Platelets are also present in the early lesions of atherosclerosis and are often found in close association with foam cells. We have investigated the hypothesis that platelets contribute to foam cell formation by inducing macrophage cholesteryl ester accumulation. Using an in vitro culture system of human monocyte-derived macrophages and autologous platelets, we have demonstrated a platelet-dependent stimulation of macrophage cholesterol esterification and cholesteryl ester accumulation. The response is specific to platelets and is dependent upon activation of the platelets. An active fraction can be isolated from the releasates of thrombin-stimulated platelets that contain large cholesterol-rich platelet membrane vesicles. The results suggest that platelet-derived free cholesterol is required for platelet-induced macrophage foam cell formation.

Arteriosclerosis↗
PubMed1990

Glycosaminoglycans alter the capacity of low density lipoprotein to bind to monosodium urate crystals.

Low density lipoprotein (LDL) has previously been demonstrated to be a potent inhibitor of human inflammatory cell activation by monosodium urate (MSU) crystals in vitro. As suppression of cellular responses to crystals by LDL is known to be dependent on the binding of LDL to urate crystals we further evaluated the mechanism and regulation of LDL binding to urate crystals in vitro. Using nonlinear, least squares methodology to analyze binding, we found LDL to saturably and reversibly bind with high affinity (Kd 9.3 x 10(-9) M) to MSU crystals. LDL binding was competitively inhibited by LDL but not by a variety of other positively charged moieties. Glycosaminoglycans (GAG) (heparin, heparan sulfate, hyaluronate and chondroitin sulfate), in diminishing order of potency, inhibited the binding of LDL to crystals. This inhibitory activity was dose dependent, sensitive to digestion with glycosidases and appeared to be specific for polymerized GAG, as glucuronic acid, dextran, dextran sulfate, as well as isolated amino sugar constituents of GAG were either weakly inhibitory or inactive. GAG also promoted dissociation of bound LDL from the urate crystal surface. The results indicate that LDL binds saturably and reversibly to urate crystals and that polymerized, but not depolymerized, hyaluronate and other GAG inhibit LDL binding to urate crystals. This suggests that the amount of LDL coating intraarticular urate crystals could vary during the course of a gouty paroxysm.

Crystallization↗
PubMed1989

A method for localizing the early products of nonenzymatic glycosylation in fixed tissue.

The process of nonenzymatic glycosylation (NEG) may play a significant role in the development of chronic complications of diabetes. Early products of NEG can be measured by various biochemical methods. A method has been developed to localize these early products of glycosylation in vivo in fixed tissue sections of normal and diabetic skin using monoclonal antibodies specific for glucitollysine, which is formed when the early products of NEG are chemically reduced in vitro. Carnoy's-fixed, paraffin-embedded skin samples from six diabetic and 13 nondiabetic subjects were sectioned, mounted on glass slides, and reduced for one h in 100 mM NaBH4. Immunolocalization was by the avidin--biotin immunoperoxidase method. Diabetic skin consistently stained more intensely for glucitollysine than nondiabetic skin. Staining around vessels, in particular, and of the collagenous matrix in general, was markedly enhanced in diabetic skin compared with nondiabetic skin. Antigens present in both the epidermis and the eccrine structures reacted with the antibody in both diabetic and nondiabetic skin but with greater intensity in the diabetic skin. This study has shown that it is possible to localize the early products of NEG in tissue sections using monoclonal antibodies. The findings correlate with biochemical data that show increased NEG in diabetics compared with nondiabetics. This technique should prove valuable for further investigations of the role of NEG in the pathogenesis of diabetes.

Aged↗
PubMed1989

Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay as a candidate reference method for the measurement of apolipoprotein B-100.

A monoclonal antibody-based direct binding enzyme-linked immunosorbent assay (ELISA) for apoprotein (apo) B-100 has been developed for use as a reference method. The assay uses the two well-characterized monoclonal antibodies, MB24 and MB47. MB47, which recognizes an epitope at the low density lipoprotein (LDL) receptor-binding domain of apoB and is specific for apoB-100, is bound to the microtiter plate as the capture antibody. MB24, which binds an epitope in the amino terminal half of the apoB-100 and identifies both apoB-100 and apoB-48, is conjugated to horseradish peroxidase and is utilized as the indicating antibody. The assay was calibrated with LDL (d 1.030-1.050 g/ml) and the LDL protein was determined by a sodium dodecyl sulfate (SDS) Lowry procedure. The working range of the assay is 0.25-1.25 micrograms/ml. Optimal dilution of whole plasma was found to be 1:2000. In the assay, MB47 bound approximately 97% of the apoB in all low density lipoprotein, and greater than 90% of the apoB in the majority of very low density lipoprotein preparations. Small dense LDL from subjects with familial combined hyperlipidemia (FCHL) and large bouyant LDL from subjects with familial hypercholesterolemia (FH) exhibited binding properties similar to LDL from healthy normolipidemic subjects when tested in the reference ELISA. The intra- and interassay coefficients of variation averaged 2.5% and 6.0%, respectively. Plasma B-100 levels were not influenced by freezing and thawing or storage at 4 degrees C for up to 3 weeks or storage at -70 degrees C for up to 11 months. Excellent agreement was obtained between the reference ELISA and a polyclonal RIA which measures total apoB (r = 0.93, n = 105, mean ELISA B-100 value = 100 mg/dl, mean RIA value = 101 mg/dl, Sy = 9.6). Reference ELISA B-100 values of samples pretreated with bacterial lipase were not significantly increased in most samples with plasma triglyceride levels below 600 mg/dl. To help reduce the large among-laboratories variability of apoB measurements, we recommend that this candidate reference direct binding ELISA be used to assign apoB target values to apoB reference pools.

Antibodies, Monoclonal↗
PubMed1988

Localization of two epitopes of apolipoprotein A-I that are exposed on human high density lipoproteins using monoclonal antibodies and synthetic peptides.

To understand the structure of apolipoprotein A-I, we have used an immunochemical approach and identified specific regions of apoA-I that may be exposed on the apoprotein as it exists on high density lipoprotein (HDL). Twelve mouse monoclonal antibodies specific for human apoA-I were generated from six fusions. Thirteen synthetic peptides of between 5 and 16 amino acid residues in length, which span the amino-terminal two-thirds of apoA-I, were tested for their ability to react with each of the 12 antibodies. In a competitive solid-phase radioimmunoassay, a synthetic peptide, which represented residues 1-15 of mature apoA-I, inhibited the binding of antibody AI-16 to immobilized HDL. Similarly, a synthetic peptide, which represented residues 90-105 of apoA-I, inhibited the binding of antibody AI-18 to immobilized HDL. Using systematic changes in the size and sequence of the oligopeptides, the limits and essential amino acid residues of these epitopes were defined. Comparisons of the slopes of the competition curves obtained with immunoreactive peptides, isolated apoA-I, and HDL verified that these two regions of apoA-I are exposed on the surface of apoA-I as it exists on native HDL.

Animals↗
PubMed1988

Apoprotein E-rich high density lipoproteins inhibit ovarian androgen synthesis.

The influence of high density lipoproteins (HDL) on luteinizing hormone-stimulated rat ovarian theca/interstitial cell steroidogenesis was studied. Without HDL the cells produced primarily androgens from progestin precursors. In the presence of rat or human HDL steroid output increased 3-5-fold, but the type of steroid produced was dependent on the source of the HDL. Human HDL nonselectively amplified luteinizing hormone-stimulated steroid production, whereas rat HDL promoted progestin production without a concomitant increase in androgen output. Comparisons of the activities of apoprotein E-rich HDL (e.g. HDL from intact mature rats) with apoprotein E-poor HDL (e.g. human HDL or rat HDL from hypophysectomized immature rats) suggested that apoprotein E was responsible for the inhibition of androgen production. Furthermore, the inhibitory activity of rat HDL was abolished by depleting apoprotein E-containing lipoproteins with heparin affinity chromatography. Direct evidence that apoprotein E was the inhibitory constituent of rat HDL was obtained by showing that isolated lipid-free rat apoprotein E inhibited androgen production, whereas isolated rat apoproteins A-I and A-IV did not. The possible paracrine function of apoprotein E in ovarian follicular maturation of the ovary is discussed.

Androgens↗
PubMed1988

Quantitation of plasma apolipoprotein A-I using two monoclonal antibodies in an enzyme-linked immunosorbent assay.

A rapid sandwich enzyme-linked immunosorbent assay (ELISA) for the quantitation of human apolipoprotein (apo) A-I was developed. The assay uses a pair of noncompeting purified monoclonal antibodies to detect apoA-I in plasma. The antibodies used in this assay were selected because they bind greater than 90% of radioiodinated high density lipoprotein (HDL), they identify "fresh" nondeamidated epitopes on apoA-I, and they have comparable binding affinities for isolated HDL and HDL in plasma. The assay was standardized with a plasma secondary standard composed of lyophilized human serum. The assay was used to measure the apoA-I levels in normal subjects, patients with coronary artery disease, and patients with familial hypercholesterolemia. The results indicate that certain monoclonal antibodies can be used to reliably measure plasma levels of apoA-I in diverse groups of subjects.

Antibodies, Monoclonal↗
PubMed1988

Platelet-enhanced apolipoprotein E production by human macrophages: a possible role in atherosclerosis.

Cholesterol-loaded human monocyte-derived macrophages increase their production of apolipoprotein E (apoE). Although cholesterol loading is often achieved with modified plasma lipoproteins, macrophages can be loaded also by coculture with platelets. Therefore, the relationship between platelet-mediated cholesteryl ester accumulation and apoE secretion was examined. Macrophages were isolated by adherence and cultured for 6 days in serum-free medium. Secreted apoE was measured with a sensitive solid-phase radioimmunoassay. Maximum apoE secretion by the adherent macrophages from 5 x 10(6) peripheral blood mononuclear cells was obtained with 3 x 10(8) platelets and was ten-fold greater than control cells cultured in the absence of platelets. Platelet-mediated apoE secretion was consistently greater than that obtained by culture with either native or acetylated low density lipoproteins. Whereas the 1000 g supernatants of unstimulated platelets were poor inducers of apoE secretion, substances rich in cholesterol that were shed from thrombin-stimulated platelets and recovered in the 1000 g supernatants were almost as active as intact platelets. In all studies, platelet-induced secretion of apoE paralleled the capacity of platelets to induce macrophage cholesterol accumulation, indicating that macrophage apoE secretion was readily influenced by macrophage cholesterol metabolism.

Apolipoproteins E↗
PubMed1987

Lipoprotein B37, a naturally occurring lipoprotein containing the amino-terminal portion of apolipoprotein B100, does not bind to the apolipoprotein B,E (low density lipoprotein) receptor.

In 1979, Steinberg and colleagues described a unique kindred with familial hypobetalipoproteinemia (Steinberg, D., Grundy, S. M., Mok, H. Y. I., Turner, J. D., Weinstein, D. B., Brown, W. V., and Albers, J. J. (1979) J. Clin. Invest. 64, 292-301). Recently, we demonstrated the existence of an abnormal species of apolipoprotein (apo-) B, apo-B37 (Mr = 203,000) in nine members of that kindred (Young, S. G., Bertics, S. J., Curtiss, L. K., and Witztum, J. L. (1987) J. Clin. Invest. 79, 1831-1841; Young, S. G., Bertics, S. J., Curtiss, L. K., Dubois, B. W., and Witztum, J. L. (1987) J. Clin. Invest. 79, 1842-1851). Apolipoprotein B37 contains only the amino-terminal portion of apo-B100. In affected individuals most of the apo-B37 is contained in the high density lipoprotein (HDL) fraction (d = 1.063-1.21 g/ml), where it is the principal apolipoprotein in a unique lipoprotein (Lp) particle, Lp-B37, which contains little, if any, apo-A-I. However, the most abundant lipoprotein in the HDL density fraction is a smaller particle, which contains apo-A-I, but no apo-B. The Lp-B37 particles were isolated from the HDL of affected individuals by immunoabsorption of apo-B37. Selected affinity antibodies specific for apo-B37 were used to prepare an anti-apo-B37-Sepharose 4B column. Lipoproteins not bound by the column (unbound HDL fraction) contained apo-A-I, but no apo-B. The Lp-B37, which was eluted from the column with 3 M KI, contained apo-B37 and trace amounts of apo-A-I, but no apo-B100. Over a 4-h period, normal human fibroblasts degraded 10-fold more 125I-low density lipoprotein (LDL) than 125I-Lp-B37. Also, whereas addition of excess unlabeled LDL markedly reduced degradation of 125I-LDL, it did not significantly reduce the degradation of 125I-Lp-B37. Unlabeled Lp-B37 did not inhibit uptake and degradation of 125I-LDL by fibroblasts. These data suggest that the amino-terminal portion of apo-B100, when expressed on a naturally occurring lipoprotein particle, does not contain a functional apo-B,E(LDL) receptor binding domain.

Apolipoprotein B-100↗
PubMed1987

Kinetics of C1 activation by a monoclonal anti-C1q antibody and its (Fab)2 fragments.

Monoclonal antibody 1H11, which binds to the "head" portion of C1q, has been shown to be a strong, stoichiometric activator of C1, the first component of human complement, maximal activation being achieved at a ratio of one antibody-combining site per one C1q head; moreover, this activation occurs even in the presence of C1-inhibitor, as reported previously. In the present paper, the kinetics of activation are shown to be biphasic; that is, a portion of the C1 is activated very rapidly, and the remainder slowly. These two processes can be separated by the order of mixing of preincubated components; thus, only the rapid activation rate is observed if C1q and the monoclonal antibody are preincubated together and are added subsequently to a mixture of C1r2C1S2 and C1-inhibitor. Only the slow activation rate is observed when C1q, C1r2C1S2, and C1-inhibitor are preincubated and are added subsequently to monoclonal antibody 1H11. Similar results are obtained by using either the intact 1H11 antibody or else the (Fab)2 obtained from it by proteolytic digestion and purification. The rapid phase is independent of the concentration of 1H11 over the range employed; the slow phase depends on 1H11 concentration. Plausible activation schemes are presented to explain the two distinct activation rate processes, and kinetic models are developed which provide a reasonable simulation of the experimental data.

Antibodies, Monoclonal↗
PubMed1987

Detection of two apolipoprotein B species (apoBc and apoBg) by a monoclonal antibody.

A monoclonal antibody (MB-19) was used to investigate the polymorphism of apolipoprotein B in a large East Finnish family and in unrelated subjects. Apolipoprotein B was shown to exhibit high, intermediate or low affinity binding to this antibody. Thus, MB-19 bound strongly to the Ag(c) epitope, an Ag antigenic domain previously characterized by human antisera, while it bound only weakly to the allelic epitope Ag(g). It proved useful for the detection of the two corresponding allelic apoB species designated apoBc (= high affinity binding) and apoBg (= low affinity binding), and for confirming their co-dominant transmission. Intermediate binding resulted from the presence of a mixture of both apoB populations in heterozygous subjects.

Adolescent↗
PubMed1987

Characterization of an abnormal species of apolipoprotein B, apolipoprotein B-37, associated with familial hypobetalipoproteinemia.

Steinberg and colleagues have previously described a unique kindred with normotriglyceridemic hypobetalipoproteinemia (1979. J. Clin. Invest. 64:292-301). In a reexamination of this kindred, we found an abnormal apolipoprotein (apo) B species, apo B-37 (203,000 mol wt), in the plasma lipoproteins of multiple members of the kindred. In affected individuals apo B-37 was found in very low density lipoproteins, along with the normal apo B species, apo B-100 and apo B-48. High density lipoproteins (HDL) also contained apo B-37, but no other apo B species. The first 13 amino-terminal amino acids of apo B-37 were identical to those of normal apo B-100. We utilized a panel of 18 different apo B-specific monoclonal antibodies and polyclonal antisera specific for apo B-37 and the thrombin cleavage products of apo B-100 to map apo B-37 in relation to apo B-100, apo B-48, and the thrombin cleavage products of apo B-100. The results of those immunochemical studies indicated that apo B-37 contains only amino-terminal domains of apo B-100. In affected individuals, the majority of apo B-37 in plasma was contained in the HDL density fraction. Within that fraction apo B-37 was found on discrete lipoprotein particles, termed Lp-B37, that had properties distinct from normal HDL particles containing apo A-I. This report documents for the first time the existence of an abnormal apo B species in humans. Further study of apo B-37 and lipoprotein particles containing apo B-37 should lead to an improved understanding of apo B structure and function.

Antibodies, Monoclonal↗
PubMed1987

Genetic analysis of a kindred with familial hypobetalipoproteinemia. Evidence for two separate gene defects: one associated with an abnormal apolipoprotein B species, apolipoprotein B-37; and a second associated with low plasma concentrations of apolipoprotein B-100.

In 1979 Steinberg and colleagues recognized a unique kindred with normotriglyceridemic hypobetalipoproteinemia (1979. J. Clin. Invest. 64:292-301). We have undertaken an intensive reexamination of this kindred and have studied 41 family members in three generations. In this family we document the presence of two distinct apo B alleles associated with low plasma concentrations of apolipoprotein (apo) B and low density lipoprotein (LDL) cholesterol and we trace the inheritance of these two alleles over three generations. One of the alleles resulted in the production of an abnormal, truncated apo B species, apo B-37. The other apo B allele was associated with reduced plasma concentrations of the normal apo B species, apo B-100. H.J.B., the proband, and two of his siblings had both abnormal apo B alleles and were therefore compound heterozygotes for familial hypobetalipoproteinemia. Their average LDL-cholesterol level was 6 +/- 9 mg/dl. All of the offspring of the three compound heterozygotes had hypobetalipoproteinemia, and each had evidence of only one of the abnormal apo B alleles. In the entire kindred, we identified six heterozygotes for familial hypobetalipoproteinemia who had only the abnormal apo B-37 allele and their average LDL cholesterol was 31 +/- 12 mg/dl. We identified 10 heterozygotes who had only the allele for reduced plasma concentrations of apo B-100 and their LDL cholesterol level was 31 +/- 15 mg/dl. Unaffected family members (n = 22) had LDL cholesterol levels of 110 +/- 27 mg/dl. This report describes the first kindred in which two distinct abnormal apo B alleles have been identified, both of which are associated with familial hypobetalipoproteinemia.

Alleles↗
PubMed1987

New mechanism for foam cell generation in atherosclerotic lesions.

Because of a close association between platelets and macrophages in early fatty streak lesions, the hypothesis was tested that platelets contribute to lesion progression by directly enhancing macrophage cholesteryl ester (CE) accumulation. Both the rate of cholesterol esterification and the accumulation of CE were increased within 24 h of the co-culture of adherent macrophages with platelets. Maximum increases in esterification and CE accumulation were observed within 3 to 4 d of culture and were greater than 10-fold over controls. Optimum accumulation of CE by 5 X 10(5) was obtained with 5 X 10(8) autologous platelets. When similar amounts of free cholesterol were supplied with platelets, red blood cells (RBC), RBC ghosts, or sonicated RBC, only platelets enhanced macrophage CE accumulation, which indicates specificity for platelets. Products released from platelets 30 min after thrombin stimulation were active as well. The results suggest that platelets and/or substances shed by activated platelets are potent mediators of macrophage CE accumulation.

Apolipoproteins E↗
PubMed1986

Apolipoprotein B mediates the capacity of low density lipoprotein to suppress neutrophil stimulation by particulates.

Low density lipoprotein (LDL) inhibits phagocytosis of certain negatively charged particulates and also inhibits subsequent cellular secretory and oxidative responses to these particulates. In the present work, we have defined the structural features of LDL involved in this activity. Starch-heptane extraction depleted greater than 95% of neutral lipids but had little effect on the capacity of LDL to inhibit monosodium urate crystal- or polystyrene latex bead-induced neutrophil chemiluminescence (CL). Liposomes containing gamma-palmitoyl-beta-oleoylphosphatidylcholine (PC) with unesterified cholesterol (PC:cholesterol = 2:1), PC and sphingomyelin (PC:sphingomyelin = 2.3:1), or PC alone lacked the capacity to inhibit urate-induced CL. However, incorporation of apoB-100 into liposomes via cholate dialysis rendered them nearly as inhibitory for urate-induced neutrophil CL as LDL on a protein weight basis. Moreover, delipidated apoB-100, containing less than 3% residual phospholipid, inhibited neutrophil responses to urate crystals or latex beads (degranulation and superoxide anion release) in a stimulus-specific manner. Modifications of the lysine residues of apoB (e.g. acetylation) reduced both the capacity of LDL to inhibit urate crystal-induced CL and to bind to urate crystals. The effects of apoB lysine residue modification were reversible, proportional to the extent of modification, and were not attributable to alteration of the net charge of apoB. Thus, the apoB-100 of LDL both mediates and shares the capacity of native LDL to inhibit certain neutrophil responses to particulates.

Apolipoproteins B↗
PubMed1986

Activation of the first component of human complement, C1, by monoclonal antibodies directed against different domains of subcomponent C1q.

Two monoclonal antibodies directed against C1q, and their (Fab)2 and Fab fragments, were used to study the mechanism of C1 activation. Monoclonal antibody 2A10, an IgG2a, was digested by pepsin to yield fully immunoreactive (Fab')2. Monoclonal antibody 1H11, an IgG1, was digested by papain to yield fully immunoreactive, bivalent (Fab)2. Previously 1H11 had been shown to bind to the C1q "heads," whereas 2A10 bound to stalks. Activation of C1 was followed by the cleavage of 125I-C1s in the presence of C1 inhibitor (C1-Inh) at 37 degrees C. Spontaneous activation was minimal at inhibitor concentrations above 0.4 micron (1.3 X physiologic inhibitor concentration); all results were corrected for the spontaneous activation background. Heat-aggregated IgG activated completely in this system and was taken as 100% activation. Monoclonal antibody 2A10 caused precipitation of C1 and slow activation; neither the (Fab')2 nor the Fab' derived from 2A10-caused activation. Probably, aggregates of intact 2A10 and C1 were serving as immune complexes to activate other molecules of C1. In contrast, both 1H11 and its (Fab)2 activated completely and stoichiometrically; that is, maximal activation was achieved at a ratio of one C1q head to one antibody combining site. The monovalent Fab derived from 1H11 bound well to C1q, but no activation of C1 was observed. Thus, bivalent binding of this head-binding monoclonal is required for C1 activation, but not the presence of the antibody Fc portion. Neither 1H11 nor its (Fab)2 fragments caused C1 precipitation; however, the 1H11 did form complexes composed of two C1q cross-linked by multiple 1H11, which were visualized by electron microscopy. The presence of these dimeric complexes correlated well with activation. A model for C1 activation is proposed in which two C1q subcomponents are held together by multiple (Fab)2 bridging C1q heads. The model is roughly analogous to touching opposing pairs of fingers and thumb tips, the two hands representing the two C1q, forming a cage. C1-Inh, which probably binds to C1r through the open end of the C1 cone, is too long asymmetric to be included within the cage. Thus, according to this model, the dimers of C1 are released from the inhibitory action of C1-Inh, and activation proceeds spontaneously and rapidly at 37 degrees C.

Animals↗
PubMed1986

Monoclonal antibody detects Ag polymorphism of apolipoprotein B.

A monoclonal antibody (MB-19) was used to investigate the polymorphism of apolipoprotein B in a large family and in unrelated subjects. Apolipoprotein B was shown to exhibit high-, intermediate- or low-affinity binding to this antibody. Thus, MB-19 bound strongly to the Ag(c) epitope, An Ag antigenic domain previously characterized by human antisera, while it bound only weakly to the allelic epitope Ag(g). It proved therefore useful for the detection of the two corresponding allelic apoB species designated apoBc (high-affinity binding) and apoBg (low-affinity binding), and for confirming their co-dominant transmission. Intermediate binding resulted from the presence of a mixture of both apoB populations in heterozygous subjects.

Antibodies, Monoclonal↗