N-cadherin distinguishes pleural mesotheliomas from lung adenocarcinomas: a ThinPrep immunocytochemical study.

BACKGROUND: Cadherins are a family of cell-cell adhesion proteins. The homotypic binding of cadherins is critical for cell sorting and tissue formation during organogenesis. Different cadherin subtypes show lineage specific tissue expression, which has been exploited to differentiate histologically similar tumors of varying ontogeny. By applying immunohistochemistry to tissue sections, the authors have previously documented the utility of N-cadherin in distinguishing between pleural mesotheliomas and lung adenocarcinomas, based on the observation that N-cadherin is expressed in the former disease but not in the latter. Because the diagnosis of these diseases is frequently rendered on cytologic material rather than tissue biopsies, the authors wanted to assess the utility of N-cadherin immunocytochemistry in evaluating material prepared with ThinPrep. METHODS: The authors examined the cytologic material from 12 patients for the expression of N-cadherin using immunocytochemistry. Four patients had mesotheliomas and eight had adenocarcinomas. ThinPrep slides of the patients' pleural fluid were prepared and stained with the monoclonal antibody 13A9, which is specific for N-cadherin. RESULTS: Of the 12 cases studied, all 4 cases of pleural mesothelioma expressed N-cadherin in a specific cell-cell membrane location, and all 8 cases of lung adenocarcinoma were negative for N-cadherin. CONCLUSIONS: These results show that the N-cadherin specific antibody 13A9 is a suitable marker in material prepared with ThinPrep for the differentiation of pleural mesotheliomas from lung adenocarcinomas. This antibody should be included in the diagnostic immunocytochemical panel for evaluation of these malignancies.

Glucose-modified low density lipoprotein enhances human monocyte chemotaxis.

In diabetes mellitus the progression of atherosclerosis is accelerated. The interaction of glucose with atherogenic lipoproteins may be relevant to the mechanisms responsible for this vascular damage. The aim of this study was to examine the effect of glucose-modified low density lipoprotein (LDL) on human monocyte chemotaxis and to investigate the roles of oxidation and glycation in the generation of chemotactic LDL. Cu(II)-mediated LDL oxidation was potentiated by glucose in a dose-dependent manner and increased its chemotactic activity. Incubation with glucose alone, under conditions where very little oxidation was observed, also increased the chemotactic property of LDL. Neither diethylenetriamine pentaacetic acid (DETAPAC) nor aminoguanidine, which both inhibited LDL oxidation, completely inhibited the chemotactic activity of glycated oxidised LDL. The results suggest that both oxidation and glycation contribute to increased chemotactic activity.

Plasma oxygen during cardiopulmonary bypass: a comparison of blood oxygen levels with oxygen present in plasma lipid.

1. Although not often appreciated, it is a fact that molecular oxygen is more soluble in lipids than in aqueous solution. We have recently developed a method to monitor oxygen within the lipid content of plasma. Monitoring plasma oxygen is one essential element during open heart surgery using a cardiopulmonary bypass pump and oxygenator. Currently oxygen is monitored electrochemically and is based upon monitoring the partial pressure of oxygen in a gas equilibrated with whole blood. 2. To determine the relative importance of lipid-associated oxygen in blood and assess the potential use of such a measurement we present comparisons of changes in oxygen associated with whole blood and lipid content of plasma before, during and after cardiac surgery. 3. In a limited number of patients studied (n = 28), aged between 34 and 86 years, oxygen in lipid increased with decreased extracorporeal blood temperature during cardiopulmonary bypass, increased in proportion to oxygen supplied and appeared to be a better monitor of oxygen than conventional electrochemical systems currently in use. Oxygen associated with whole blood and plasma lipid was markedly below normal on aortic declamping after cardiopulmonary bypass, suggesting an hypoxic episode at this point. Levels of oxygen in the lipid phase of plasma returned to normal presurgical values 6-8 h after surgery. 4. Calculation of the concentration of lipid-associated oxygen present in plasma suggests that plasma lipids contain up to 25% of that typically ascribed to haemoglobin. Thus, we suggest that monitoring lipid-associated oxygen may prove a better alternative to current methods of measuring oxygen status. Furthermore, we suggest that plasma lipid is a hitherto unsuspected pool of circulating oxygen which may play a significant role in tissue oxygen supply.

Micellar acceleration of oxygen-dependent reactions and its potential use in the study of human low density lipoprotein.

The reaction rate between superoxide and nitro-blue tetrazolium (NBT) is known to be accelerated/catalysed by micellar systems. Previous reports suggest that an accelerated rate of NBT reduction by micellar systems may be the result of either the binding of organic substrates such as NBT to the micellar phase giving a more favourable environment for superoxide reduction (an orientation effect), or the electrostatic interaction between micelles and superoxide. Here we show, using three different superoxide generating systems, that micelles composed of a number of different lipids or human low density lipoprotein (LDL) accelerates the apparent reaction between superoxide and NBT. Evidence in favour of an accelerated production of superoxide as opposed to the accelerated reduction of NBT is provided and we propose that the accelerated production of superoxide is a consequence of increased oxygen solubility in the lipid, rather than aqueous, phase. This is supported by: 1. The absence of any spectrophotometric changes due to interaction between lipid or LDL and reagents used. 2. The ability of micelles composed of a number of different fatty substances, including LDL, to accelerate superoxide generation, assessed by NBT reduction. 3. The behaviour of micelles, which appears to be one of substrate rather than catalyst, during the acceleration of NBT reduction. This is confirmed by the use of a known micellar catalyst, Triton-X100. This suggests that lipids contribute to the reaction as a substrate rather than a catalyst. 4. The inability of LDL to accelerate NBT reduction by potassium superoxide, a reaction which is independent of bimolecular oxygen. 5. The inability of LDL to accelerate NBT reduction when added after superoxide generation. 6. Studies that show LDL can enhance an NBT-independent monitor of oxidation, namely the transition metal-catalysed oxidation of vitamin C. 7. Estimations of the solubility of oxygen in LDL which appear to be consistent with reported physical measurements. Furthermore, we show that LDL modification can alter LDL-mediated micellar acceleration of superoxide generation. Extensive oxidation of LDL decreases micellar acceleration and minimal oxidation enhances it. We suggest that LDL micellar acceleration might serve as a novel approach to studying human LDL.

Effect of glucose-mediated LDL oxidation on the P388D1 macrophage-like cell line.

Oxidised human low density lipoprotein (LDL) is thought to play a role in the development of atherosclerosis. Recent reports suggest that glucose-derived oxidants are capable of oxidising LDL. In this report, the effect of glucose-mediated oxidation of LDL upon the macrophage like cell line, P388D(1), was examined. Glucose-mediated oxidation of LDL was assessed by changes in the electrophoretic mobility of LDL and by analysis of lipid content using gas chromatography. The presence of Cu(II) (0.5 microM) was essential for the oxidation of LDL. The oxidation was potentiated by glucose in a dose- and time-dependent manner. At the concentration of LDL used (1 mg/ml), high concentrations of glucose (up to 500 mM) were required to oxidise LDL. The electrophoretic mobility of LDL correlated with the degree of lipid oxidation; both correlated with an inhibitory effect of oxidised LDL upon P388D(1) DNA synthesis. Diethylenetriaminepentaacetic acid (DETAPAC), a transition metal chelator, and aminoguanidine (AMG), an anti-glycation agent, inhibited the oxidation of LDL and attenuated the effects on DNA synthesis. Thus, glucose can mediate transition metal-dependent oxidation of LDL to a level that can affect P388D(1) cells, a mechanism which might have relevance to accelerated atherosclerosis in diabetic patients.

Glucose modification of human serum albumin: a structural study.

Structural changes associated with the exposure of human serum albumin (HSA) to glucose with or without the presence of Cu (II) have been characterized using a bank of methods for structural analysis including circular dichroism (CD), amino acid analysis (AAA), fluorescence measurements, SDS-PAGE, and boronate binding (which is a measure of Amadori product formation). We show that in the short-term (10 d) incubation mixtures, HSA is resistant to Cu (II)-mediated oxidative damage and that the early products of glycation of HSA had minimal effects on the folded structure. Amino acid analysis showed that there was no formation of advanced glycation endproducts (AGE), which can be measured by loss of lysine. This remained the case in longer term incubation of HSA (56 d) in the hyperglycemic concentration range (5-25 mM glucose) despite increased levels of Amadori product (60% boronate binding) and the formation of glycophore (Excitation 350, Emission 425). At high, nonphysiological concentrations (100 mM and 500 mM) of glucose, glycophore formation increased and 3 and 11 mol Lysine-glucose adduct/mol HSA were converted to AGE, respectively. This was accompanied by increased damage to tryptophan and protein-protein crosslinking but only minor tertiary structural change. In the presence of Cu (II), however, AGE formation was accompanied by extensive damage to histidine and tryptophan side chains, main chain fragmentation, and loss of both secondary and tertiary structure. Thus, changes in structure appear to be the result of oxidation as opposed to glycation, per se.

Aminoguanidine and its pro-oxidant effects on an experimental model of protein glycation.

Recent reports show a pro-oxidant activity of aminoguanidine. Aminoguanidine is able to generate hydrogen peroxide in the presence of Cu (II). These observations have been confirmed by the present studies in that aminoguanidine is, indeed, able to generate oxidants similar in reactivity to the hydroxyl radical and is also able to fragment BSA in a Cu (II)-dependent manner. Studies on glycated bovine serum albumin show that aminoguanidine can affect a number of parameters associated with the nonenzymatic glycation of protein. This includes an ability to decrease glucose attachment and levels of protein fluorescence termed glycophore, resulting from protein glycation. Aminoguanidine also increases the generation of dicarbonyl compounds by glycated protein. All of these effects on parameters of glycation appear to be Cu (II) dependent. Further studies show that one effect of protein glycation is to decrease its susceptibility to proteolysis. The reverse is true of protein oxidation, which has previously been shown to increase the susceptibility of proteins to proteolytic digestion. Evidence is presented suggesting that aminoguanidine is able to enhance the proteolytic digestion of glycated BSA, a protein shown to be protease resistant. Our observations are discussed within the context of current concepts of protein glycation in the development of diabetic complications and aminoguanidine's potential use as a prophylactic agent in diabetes mellitus.

Apparent superoxide dismutase-like activity of immunoglobulin.

Using various superoxide generating systems and nitroblue tetrazolium or cytochrome c as superoxide detector molecules it is possible to assess the superoxide dismutase activity of proteins. Intact antibodies raised to different antigens, the Fab' fragment of anti-TNF [M632] and well-characterized recombinant Fv fragments of the murine antibody NQ11.7.22 appear to possess superoxide dismutase (SOD)-like activity. Kinetic characteristics of the SOD-like activity of NQ11.7.22-Fv fragments suggest an enzymatic property and these fragments behave in an analogous manner to human erythrocyte Cu-Zn SOD. Furthermore, the SOD-like activity of the NQ11.7.22-Fv fragment is affected by certain single-point mutations in the amino acid composition and has a pH optimum of 6.2-6.6 which is unlike Cu-Zn SOD (pH 7.8-8.2). A change in tyrosine at the 32 position in the heavy chain and histidine at position 27 of the light chain of the NQ11.7.22-Fv fragment results in a profound reduction in SOD-like activity. Tyrosine at the 32 position in the heavy chain is known to play a significant role in antigen binding suggesting that the SOD-like activity occurs at the antigen-binding site itself. Single-point mutations at the periphery of the antigen combining site on the NQ11.7.22-Fv fragment had little or no effect on SOD-like activity. Further studies show that immunoglobin (lgG-1), a commercially available murine monoclonal antibody, can also enhance the generation of hydrogen peroxide, the product of superoxide dismutation, when present in superoxide producing systems. The generation of hydrogen peroxide was increased by low pH (pH 6.25) with lgG-1 but reduced with Cu-Zn SOD.

The assessment of protein glycation in human atherosclerotic plaques by affinity chromatography.

Human atherosclerotic plaques are characterized by a massive deposition of lipid within arterial walls. The lipids accumulated are partly oxidized, as assessed by gas chromatography of lipids and their oxidation products. Both advancing age and diabetes mellitus are associated with an increased prevalence and severity of atherosclerosis. In diabetes mellitus the development of secondary complications appear to be increased by poor glucose control. Indeed, the post-translational modification of protein by non-enzymatic glycation may provide the link between abnormal glucose control and diabetic complications. For atherosclerosis however, the relationship between glucose control and disease is unclear, with evidence available to support and discount such a link. To study protein glycation in a condition associated with a significant level of lipid oxidation products poses several methodological problems, most of which are associated with interference by lipid-derived aldehydes. Many chemical assays of protein glycation monitor aldehydic products common to the chemistry of both protein glycation and lipid oxidation. Studies of protein glycation in human atheroma, obtained at necropsy, are presented which make use of a commercially available boronic acid affinity-based chromatographic assay of glycated protein. The commercially available affinity-based chromatographic assay of glycated protein appears to be free from such interference and may well prove useful in the study of other conditions in which the non-enzymatic glycation of protein is suspected.

The inhibition of foam cell formation by sodium diethyldithiocarbamate.

A prominent feature of human atherosclerosis is the lipid-laden foamy macrophage, which often also contains the insoluble pigment, ceroid. The culture of macrophage-like cells, P388D1s, with artificial lipoproteins composed of cholesteryl linoleate (CL) and bovine serum albumin (BSA) results in foam cell formation with lipoprotein uptake and the intracellular accumulation of ceroid. Ceroid accumulation is accompanied by the oxidation of the cholesterol ester as monitored by gas chromatography. The sodium salt of diethyldithiocarbamic acid (DDC) at 1-5 microM effectively inhibited lipoprotein uptake, cholesteryl linoleate oxidation and ceroid accumulation in cultures of P388D1. Further studies showed that intracellular ceroid accumulation appeared to require the presence of cystine in the medium. Lipoprotein oxidation by this macrophage-like cell therefore appears to involve a mechanism dependent on cystine metabolism which is consistent with previous reports of macrophage-mediated lipoprotein oxidation. Studies on CL/BSA-induced ceroid accumulation in human monocytes also showed that DDC behaved in much the same manner. This inhibitory effect of DDC on foam cell formation, often considered a primary event of atherosclerosis, at concentrations as low as 1 microM, suggests the need for further, more comprehensive, studies on this compound's activities.

Lipid oxidation, lipoprotein cell-association and ceroid accumulation in P388D1 macrophage-like cells.

Flow cytometry can be used to quantify the accumulation of ceroid in macrophages, the result of cellular handling of certain lipoproteins. Using P388D1 cells, a murine-derived macrophage-like cell line, the effect of the lipophilic antioxidant, DL-alpha-tocopherol, upon the uptake and accumulation of ceroid by the cells was monitored on culture with artificial lipoproteins containing a single lipid species. Ceroid accumulation was greater for artificial lipoprotein composed of BSA complexed with cholesteryl arachidonate, than with cholesteryl linoleate. alpha-Tocopherol inhibited the ceroid accumulation, which was also dependent upon cell density. Thus, since these findings are similar to recent observations in primary cultures of murine peritoneal macrophages, it would appear that macrophage-like cell lines such as P388D1 cells are appropriate for the study of potential agonists and antagonists of lipid oxidation. Culture of P388D1 cells with oxidised human low-density lipoprotein (LDL) also resulted in ceroid formation, shown to be dependent upon the level of LDL oxidation as assessed by thiobarbituric acid-reactivity, the xylenol orange assay of peroxides and gas chromatographic analysis of cholesterol and fatty acid content. Ceroid accumulation reflected changes in the level of LDL oxidation better than did the cell association of oxidised radiolabelled LDL, monitored as that bound and retained by the cell.

Protein glycation and fluorescent material in human atheroma.

Samples of normal human aorta, atherosclerotic lesions and atheroma (necrotic 'gruel' from the interior of advanced lesions) were obtained at necropsy from subjects with no history of diabetes mellitus. Components of each were extracted by ethanol:diethylether (3:1) and, subsequently, by 10% sodium dodecyl sulphate (SDS). Both fractions, organic and aqueous (SDS), were assessed for their relative fluorescence (excitation: 350 nm/emission: 430 nm). The amount of early products of glycated protein was assessed by affinity chromatography in the SDS-soluble fraction. Age-matched plasma samples, obtained from non-diabetic individuals, were also examined. Material from atherosclerotic lesions appeared to exhibit an inverse correlation between protein glycation and fluorescent material which was best reflected within the organic extract. This was not the case for normal aorta. A linear correlation between fluorescent material in the organic extract and SDS extract existed in the normal aorta alone. The only age-dependent change was found in normal aorta in which there was an increase in SDS-soluble fluorescence with increasing age. In plasma samples alone, protein glycation and protein fluorescence appeared to be linearly correlated. The observations are discussed in the context of the possible contribution of protein glycation to atherogenesis.

Apolipoprotein oxidation in the absence of lipid peroxidation enhances LDL uptake by macrophages.

A characteristic of the antioxidant, probucol, is its inability to inhibit apolipoprotein B fragmentation in low density lipoprotein (LDL), despite a pronounced ability to inhibit lipid oxidation on relatively lengthy exposure to Cu(II). Here we show that a short exposure of LDL to hydrogen peroxide and Cu(II) leads to 125I-labelled apolipoprotein B fragmentation, the production of malondialdehyde and hydroperoxides and leads to increased uptake by macrophages on subsequent culture. However, pre-loading LDL with probucol protects LDL from lipid oxidation but not protein fragmentation or macrophage uptake. The use of probucol to conduct studies on apolipoprotein B oxidation without extensive lipid oxidation may prove useful when studying LDL apolipoprotein damage on exposure to an aqueous free radical insult.

Glucose oxidation and low-density lipoprotein-induced macrophage ceroid accumulation: possible implications for diabetic atherosclerosis.

The exposure of proteins to high concentrations of glucose in vitro is widely considered a relevant model of the functional degeneration of tissue occurring in diabetes mellitus. In particular, the enhanced atherosclerosis in diabetes is often discussed in terms of glycation of low-density lipoprotein (LDL), the non-enzymic attachment of glucose to apolipoprotein amino groups. However, glucose can undergo transition-metal-catalysed oxidation under near-physiological conditions in vitro, producing oxidants that possess a reactivity similar to the hydroxyl radical. These oxidants can fragment protein, hydroxylate benzoic acid and induce lipid peroxidation in human LDL. In this study, glycation of LDL in vitro is accompanied by such oxidative processes. However, the oxidation of LDL varies with glucose concentration in a manner which does not parallel changes in protein glycation. Glycation increases in proportion to glucose concentration, whereas in our studies maximal oxidation occurs at a glucose concentration of approx. 25 mM. The modification of LDL resulting from exposure to glucose alters macrophage ceroid accumulation, a process which occurs in the human atherosclerotic plaque. The accumulation of ceroid in macrophages is shown to be related to LDL oxidation rather than LDL glycation, per se, as it too occurs at a maximum of approx. 25 mM. Oxidative sequelae of protein glycation appear to be a major factor in LDL-macrophage interactions, at least with respect to ceroid accumulation. Our observations are discussed in the context of the observed increase in the severity of atherosclerosis in diabetes.

Measurement of ceroid accumulation in macrophages by flow cytometry.

The accumulation in macrophages of ceroid, an autofluorescent polymer composed of oxidised protein and lipid, can be monitored semiquantitatively by staining techniques. However, such methods are crude and give little information about the amount of ceroid within cells. Flow cytometry, however, can give a quantitative assessment of cellular ceroid accumulation in vitro. Recently, flow cytometry was explored as a method for measurement of the accumulation in macrophages of ceroid. The accumulation appeared to be diminished in the presence of the antioxidant, alpha-tocopherol. This is consistent with the role of lipoprotein oxidation in ceroid accumulation. Here the optimum wavelengths of emission and excitation, using both conventional fluorescence spectroscopy of cellular ceroid and flow cytometric measurements with a number of optical filters, are determined. The use of optimal wavelengths determined in these studies enhances overall sensitivity. The findings are discussed in the context of future investigation of cell-mediated lipid oxidation and its potential antagonists.

Differing effects of probucol and vitamin E on the oxidation of lipoproteins, ceroid accumulation and protein uptake by macrophages.

Studies using 125I-low density lipoprotein (125I-LDL) show that probucol (10 microM) and alpha-tocopherol (100 microM) inhibit protein degradation in LDL exposed to Cu (II) in vitro. The inhibitory effect of alpha-tocopherol on protein fragmentation exceeded that of probucol. On the other hand, probucol was more able to inhibit lipid peroxidation. The subsequent uptake of Cu (II)-oxidised 125I-LDL by murine peritoneal macrophages (MPM) was virtually unaffected by the presence of probucol during LDL oxidation. The same was not true for alpha-tocopherol which led to lower levels of 125I-LDL uptake by MPM. Thus, it appears that although the antioxidant activity of probucol exceeds that of alpha-tocopherol for lipid oxidation, the reverse is true for protein degradation and, perhaps more significantly, for subsequent macrophage uptake. Further studies used artificial lipoproteins composed of cholesteryl linoleate or cholesteryl arachidonate complexed with bovine serum albumin. Culture of these artificial lipoproteins with MPM resulted in protein uptake, protein degradation, cholesterol oxidation to cholest-5-en-3 beta,7 beta-diol and the intracellular accumulation of ceroid in MPM. The presence of alpha-tocopherol (0-100 microM) inhibited all of these processes. Probucol (0-10 microM) inhibited ceroid accumulation and cholesterol oxidation to the same degree as alpha-tocopherol (0-100 microM) but had no effect upon protein degradation and protein uptake. Control studies of lipoproteins incubated without cells showed that protein degradation by cell-independent processes was also inhibited by alpha-tocopherol, but not by probucol. These observations are discussed in the context of the role of lipoprotein oxidation in atherogenesis.