Performance of a semiautomated Papanicolaou smear screening system: results of a population-based study conducted in Guanacaste, Costa Rica

Cardiovascular disease is one of the most common complications of dialysis and renal transplant patients, and high levels of AGE are present in end-stage renal failure. To address the potential involvement of AGE and growth factors in the pathophysiology of cardiovascular complications, we performed immunostaining using cardiac tissues from autopsy cases of patients on maintenance dialysis (10 cases), long-term surviving renal transplant patients with functioning grafts (8 cases), control subjects with normal renal function (7 cases) and non diabetic subjects with mild renal insufficiency (8 cases). We used two types of AGE-antibodies, 6D12 [monoclonal anti-AGE antibody, recognizing N epsilon-(carboxymethyl) lysine(CML)-modified AGE] (oxidative AGE) and non-CML-PA [polyclonal, not recognizing CML], and antibodies against PDGFs, PDGF receptors and TGF beta. Positive 6D12 staining was observed in the coronary arterial walls and in macrophages. The accumulation of 6D12-reactive AGE in the coronary arterial walls of maintenance dialysis patients was significantly greater than that of control subjects (p < 0.05). Renal transplantation significantly reduced this accumulation (p < 0.05). On the other hand non-CML-PA mainly detected AGE in intracardiac arterioles and neural tissues. There was little difference in the accumulation of non-CML-AGE among the four groups. PDGFs and PDGF receptors were mainly detected in vascular endothelial cells and infiltrating cells of cardiac tissues of renal transplant patients, but not of maintenance dialysis patients. TGF beta was not detected in cardiovascular tissue of transplant patients. Our results indicated that the accumulation of oxidative AGE (CML-AGE) in the cardiac vascular tissue is one of the factors for cardiovascular complications of maintenance dialysis patients, and also that renal transplantation has a reducing effect on CML-AGE accumulation. PDGFs may be involved in the cardiovascular complications after renal transplantation.     

Nonenzymatic glycation of type IV collagen and matrix metalloproteinase susceptibility

The glomerular basement membrane (GBM) is damaged in diabetes through complex mechanisms that are not fully understood. Prominent among them is nonenzymatic protein glycation leading to the formation of so-called advanced glycation end products (AGEs). We examined the effects of in vitro glycation of intact collagen type IV in bovine lens capsule (LBM) and kidney glomerular (GBM) basement membranes on their susceptibility to matrix metalloproteinases, using stromelysin 1 (MMP-3) and gelatinase B (MMP-9). Sites of cleavage of unmodified LBM collagen were located in the triple helical region. In vitro glycation by glucose severely inhibited the release of soluble collagen cleavage peptides by MMP-3 and MMP-9. The distribution of AGEs within the three domains of collagen IV (7S, triple helical, and noncollagenous NC1) were compared for LBM glycation using AGE fluorescence, pentosidine quantitation, and immunoreactivity towards anti-AGE antibodies that recognize the AGE carboxymethyllysine (CML). Marked asymmetry was observed, with the flexible triple helical domain having the most pentosidine and fluorescent AGEs but the least CML. The in vivo relevance of these findings is supported by preliminary studies of AGE distribution in renal basement membrane (RBM) collagen IV domains from human kidneys of two insulin-dependent diabetics and one normal subject. Pentosidine and fluorescent AGE distributions of diabetic RBM were similar to LBM, but the CML AGE in diabetic kidney was less in the triple helical domain than in NC1. Our results support the hypothesis that nonenzymatic glycation of collagen IV contributes to the thickening of basement membranes, a hallmark of diabetic nephropathy.     

Implication of an increased oxidative stress in the formation of advanced glycation end products in patients with end-stage renal failure

Recent studies have demonstrated a marked increase in the level of advanced glycation end products (AGEs) in the plasma, skin and amyloid fibrils of hemodialysis (HD) patients. The presence of AGEs in (beta2m) forming amyloid fibrils has been established in a previous immunochemical study relying on a monoclonal anti-AGE antibody. In the present study, Western blot analysis and immunohistochemistry reveal that the epitope recognized by this antibody is N epsilon-(carboxymethyl)lysine (CML) and that CML is one of the AGE structures present in amyloid fibrils. Thus, two AGE structures, CML and pentosidine, are now recognized in dialysis-related amyloidosis. AGE accumulation in uremia is not accounted for by elevated glucose levels. Since CML and pentosidine formation are closely linked to oxidative processes, we tested the hypothesis that a high oxidative stress enhanced AGE formation in HD patients. We focused on ascorbic acid (AA) because AA is easily oxidized under oxidative stress and its oxidized form (oxiAA) is a source of CML and pentosidine. In vitro incubation of beta2m with AA under atmospheric oxygen resulted in: (1) the rapid appearance of characteristic physicochemical properties of AGEs (brown color, fluorescence, polymerization tendency); (2) the transformation of beta2m into AGE-modified beta2m recognized by a specific monoclonal antibody; and (3) the accelerated formation of CML in beta2m and beta2m-peptide, recognized by mass spectrometry. A similar in vitro incubation of human serum albumin disclosed a parallel production of pentosidine measured by high-performance liquid chromatographic assay. In HD patients, the degree of AA oxidation, assessed as the ratio of oxiAA to total ascorbate, was more than twice as high as that of normal subjects (0.87 +/- 0.16 vs. 0.35 +/- 0.11, P < 0.0001), suggesting the presence of an increased oxidative stress. Interestingly, plasma level of oxiAA was correlated with the plasma levels of protein linked (P < 0.01, r2 = 0.25) and free (P < 0.05, r2 = 0.22) pentosidine. Altogether these results demonstrate that AGE, that is, CML and pentosidine, production is accelerated under oxidative stress, even in the absence of glucose. They suggest that, in uremia, CML and pentosidine production is determined both by an increased oxidative stress and the availability of precursors such as oxiAA. Finally, both CML and pentosidine contribute to the AGEs present in dialysis-related amyloid fibrils.     

Advanced glycosylation end products in adrenal lipofuscin

The present study examined the presence of advanced glycosylation end products (AGEs) in lipofuscin present in the brain and adrenal gland of aging rats by immunohistochemistry using antibodies raised against AGEs. Lipofuscin identified as yellow to brown granules emitting bright yellow to orange autofluorescence with ultraviolet light were detected in cortical neurons, cerebellar Purkinje cells, and adrenal cells in the inner part of the zona reticularis. However, none of the antibodies visualized lipofuscin in these areas. The outer part of the zona reticularis contained yellow granules emitting a faint orange autofluorescence. These granules were immunostained by an antibody that reacted with AGEs structures unrelated to the carboxymethyllysine moiety. Newly formed adrenal cortical cells are thought to migrate from the outer layer to the inner layer of the zona reticularis. Therefore, our results suggest that glycosylation-related processes are involved in lipofuscinogenesis, at least in its early stage, in the adrenal zona reticularis.     

Ceroid/lipofuscin formation in cultured human fibroblasts: the role of oxidative stress and lysosomal proteolysis

The mechanisms involved in the accumulation of ceroid/lipofuscin within non-dividing cells are not totally understood. Oxidative stress, as well as diminished activity of lysosomal proteolytic enzymes, are known to induce ceroid/lipofuscin accumulation in a variety of cell types. In order to clarify the roles of oxidative stress and lysosomal proteolysis in ceroidogenesis/lipofuscinogenesis, and to study the fate of already formed ceroid/lipofuscin, confluent cultures of AG-1518 human fibroblasts were exposed to oxidative stress (40% ambient oxygen) and/or treated with the thiol protease inhibitor leupeptin for 2 weeks. Both oxidative stress and protease inhibition caused accumulation of ceroid/lipofuscin per se (estimated by fluorescent, confocal and electron microscopy). The combined effect of these factors was, however, almost three times as large as the sum of their isolated effects. The pigment accumulated progressively as long as the oxidative stress and/or protease inhibition acted; was not eliminated after re-establishment of normal conditions; and decreased in amount after subsequent passage. The results suggest that (i) ceroid/lipofuscin forms within secondary lysosomes due to peroxidative damage of autophagocytosed material, and (ii) it is not substantially eliminated from non-dividing cells by degradation or exocytosis.     

Yersinia enterocolitica impairs activation of transcription factor NF-kappaB: involvement in the induction of programmed cell death and in the suppression of the macrophage tumor necrosis factor alpha production

Advanced glycation endproducts (AGEs) have been implicated in the pathophysiology of coronary heart disease in ageing, diabetes and renal disease. Competitive enzyme-linked immunosorbent assays (ELISAs) have been developed to measure these compounds in serum, but as recognition of AGEs is both carrier protein- and antibody-dependent standardisation is problematic. We report here on another barrier to standardization, as yet unrecognised. During the development of an AGE ELISA, we found that serum samples did not dilute in parallel to AGE standards or each other. This finding was confirmed by recovery studies that showed over-recovery of AGEs at high serum concentrations, but under-recovery at high dilutions of serum in assay buffer. We developed an inhibition assay to detect factors in serum capable of interacting directly with AGEs immobilised on microtitre plates. Binding of these factors prevented recognition of AGEs by a CML monoclonal antibody and a polyclonal anti-AGE antibody, and was neither sugar- nor carrier protein-dependent. We detected the presence of this factor in all human sera tested and also in foetal calf serum. Pre-incubation of sera with AGEs or heat-treatment at 56 degrees C for 30 min. significantly reduced this binding. We are currently investigating the nature of this factor and the possibility that it may be complement. The effect of this factor on immunoassays for AGEs can only be detected by performing parallelism and recovery studies and we suggest the use of the method referred to in this paper to aid interpretation of parallelism data.     

Autoxidation products of both carbohydrates and lipids are increased in uremic plasma: is there oxidative stress in uremia?

BACKGROUND: Advanced glycation end products (AGEs), formed by non-enzymatic glycation and oxidation (glycoxidation) reactions, have been implicated in the pathogenesis of several diseases, including normoglycemic uremia. AGE research in uremia has focused on the accumulation of carbohydrate-derived adducts generated by the Maillard reaction. Recent studies, however, have demonstrated that one AGE, the glycoxidation product carboxymethyllysine (CML), could be derived not only from carbohydrates but also from oxidation of polyunsaturated fatty acids in vitro, raising the possibility that both carbohydrate and lipid autoxidation might be increased in uremia. METHODS: To address this hypothesis, we applied gas chromatography-mass spectrometry and high performance liquid chromatography to measure protein adducts formed in uremic plasma by reactions between carbonyl compounds and protein amino groups: pentosidine derived from carbohydrate-derived carbonyls, malondialdehyde (MDA)-lysine derived from lipid-derived carbonyls, and CML originating possibly from both sources. RESULTS: All three adducts were elevated in uremic plasma. Plasma CML levels were mainly (>95%) albumin bound. Their levels were not correlated with fructoselysine levels and were similar in diabetic and non-diabetic patients on hemodialysis, indicating that their increase was not driven by glucose. Pentosidine and MDA-lysine were also increased in plasma to the same extent in diabetic and non-diabetic hemodialysis patients. Statistical analysis indicated that plasma levels of CML correlated weakly (P < 0.05) with those of pentosidine and MDA-lysine, but that pentosidine and MDA-lysine varied independently (P > 0.5). CONCLUSIONS: These data suggest that the increased levels of AGEs in blood, and probably in tissues, reported in uremia implicate a broad derangement in non-enzymatic biochemistry involving alterations in autoxidation of both carbohydrates and lipids.     

Fluorescence properties of autofluorescent granules generated by cultured human RPE cells

PURPOSE: To compare the fluorescence properties of autofluorescent granules generated by retinal pigment epithelial (RPE) cells in vitro with those of the lipofuscin of RPE in vivo. METHODS: Cultured human RPE cells were maintained in basal medium for as long as 1 year, fed rod outer segments (ROS) daily for as long as 56 days, fed ROS in the presence and absence of leupeptin, or fed liposomes consisting of the major phospholipids in ROS. At different time points, cells were examined for overall fluorescence, and their fluorescence spectra were determined. In addition, chloroform-methanol extracts were examined by thin-layer chromatography and compared with those generated from RPE lipofuscin. RESULTS: Autofluorescent granules accumulated in cultured RPE cells, regardless of the presence of an exogenous substrate or the nature of the substrate. The rate of accumulation of autofluorescent granules was greatest in cells fed ROS. The autofluorescent material generated in cultured RPE cells had some spectral similarities with RPE lipofuscin but differed in solubility and chromatographic mobility of their constituent fluorophores. CONCLUSIONS. The autofluorescent granules generated by cultured RPE, even with different specific substrates, differ from lipofuscin granules in vivo, suggesting that additional properties of RPE cells or of the materials they phagocytose are required to produce autofluorescent materials with the characteristics of lipofuscin.     

Accumulation of advanced glycation endproducts in the rat nephron: link with circulating AGEs during aging

The accumulation of advanced glycosylation end products (AGEs) is believed to be a factor in the development of aging nephropathy. We have attempted to establish a link between the formation of AGEs and the onset of renal impairment with aging, indicated by albuminuria, using a fluorescence assay and immunohistochemical detection of AGEs in the renal extracellular matrix in rats. The fluorescence of collagenase-digested Type IV collagen from GBM increased with age, from 1.65 +/- 0.05 AU/mM OHPro (3 months) and 1.58 +/- 0.04 (10 months) to 2.16 +/- 0.06 (26 months) (p < 0.001) and 2.53 +/- 0.18 (30 months) (p < 0.001). In contrast, the extent of early glycation products significantly decreased from 5.35 +/- 0.25 nmol HCHO/nmol OHPro at 3 months to 3.14 +/- 0.19 at 10 months (p < 0.001), 3.42 +/- 0.38 at 26 months, and 0.74 +/- 0.08 at 30 months (p < 0.001). The urinary fluorescence of circulating AGE rose from 2.42 +/- 0.15 AU/mg protein (3 months), 1.69 +/- 0.07 (10 months), to 4.63 +/- 0.35 (26 months) (p < 0.01) and 4.73 +/- 0.72 (30 months), while the serum fluorescence increased from 0.39 +/- 0.02 AU/mg protein at 3 months and 0.43 +/- 0.02 at 10 months to 0.59 +/- 0.04 at 26 months (p < 0.001) and 0.54 +/- 0.03 at 30 months (p < 0.04). Polyclonal antibodies raised against AGE RNase showed faint areas of AGE immunoreactivity in mesangial areas in the nephrons of young rats. The immunolabeling of Bowman's capsule, the mesangial matrices, and the peripheral loops of glomerular and tubule basement membranes increased with rat age. The increase in circulating AGE peptides parallels the accumulation of AGEs in the nephron, and this parallels the pattern of extracellular matrix deposition, suggesting a close link between AGE accumulation and renal impairment in aging rats.     

Immunohistochemical localization of advanced glycosylation end products in coronary atheroma and cardiac tissue in diabetes mellitus

Advanced glycosylation end products (AGEs) accumulate on long-lived extracellular matrix proteins and have been implicated in the micro- and macrovascular complications of diabetes mellitus. Within the arterial wall, AGE-modified proteins increase vascular permeability, inactivate nitric oxide activity, and induce the release of growth-promoting cytokines. Recently developed anti-AGE antibodies were used in an immunohistochemical analysis of coronary arteries obtained from type II diabetic and nondiabetic patients. High levels of AGE reactivity were observed within the atherosclerotic plaque present in vessels from selected patients with diabetes. Considered together with the pathological effects of AGEs on vascular wall homeostasis, these data support the role of advanced glycosylation in the rapidly progressive atherosclerosis associated with diabetes mellitus.     

Advanced glycation end products in vitreous: Structural and functional implications for diabetic vitreopathy

PURPOSE: Advanced glycation end products (AGEs) form irreversible cross-links with many macromolecules and have been shown to accumulate in tissues at an accelerated rate in diabetes. In the present study, AGE formation in vitreous was examined in patients of various ages and in patients with diabetes. Ex vivo investigations were performed on bovine vitreous incubated in glucose to determine AGE formation and cross-linking of vitreous collagen. METHODS: By means of an AGE-specific enzyme-linked immunosorbent assay (ELISA), AGE formation was investigated in vitreous samples obtained after pars plana vitrectomy in patients with and without diabetes. In addition, vitreous AGEs were investigated in bovine vitreous collagen after incubation in high glucose, high glucose with aminoguanidine, or normal saline for as long as 8 weeks. AGEs and AGE cross-linking was subsequently determined by quantitative and qualitative assays. RESULTS: There was a significant correlation between AGEs and increasing age in patients without diabetes (r = 0.74). Furthermore, a comparison between age-matched diabetic and nondiabetic vitreous showed a significantly higher level of AGEs in the patients with diabetes (P < 0.005). Collagen purified from bovine vitreous incubated in 0.5 M glucose showed an increase in AGE formation when observed in dot blot analysis, immunogold labeling, and AGE ELISA. Furthermore, there was increased cross-linking of collagen in the glucose-incubated vitreous, when observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein separation. This cross-linking was effectively inhibited by coincubation with 10 mM aminoguanidine. CONCLUSIONS: This study suggests that AGEs may form in vitreous with increasing age. This process seems to be accelerated in the presence of diabetes and as a consequence of exposure to high glucose. Advanced glycation and AGE cross-linking of the vitreous collagen network may help to explain the vitreous abnormalities characteristic of diabetes.     

Structural characterization and immunochemical detection of a fluorophore derived from 4-hydroxy-2-nonenal and lysine

Aging and the progression of certain degenerative diseases are accompanied by increases in intracellular fluorescent material, termed "lipofuscin" and ceroid, respectively. These pigments are observed within granules composed, in part, of damaged protein and lipid. Modification of various biomolecules by aldehyde products of lipid peroxidation is believed to contribute to lipofuscin and ceroid formation. However, little direct evidence currently exists because the structures responsible for the fluorescent, cross-linked nature of this material are not well characterized. In this study, we have identified a fluorescent product formed in the reaction of Nalpha-acetyllysine and 4-hydroxy-2-nonenal (HNE), a major product of lipid peroxidation and the most reactive of these compounds under physiological conditions [Esterbauer, H., Shaur, R. J. & Zollner, H. (1991) Free Radical Biol. Med. 11, 81-128]. This fluorescent compound, characterized as a 2-hydroxy-3-imino-1,2-dihydropyrrol derivative, appears to form upon oxidative cyclization of the nonfluorescent 2:1 lysine-HNE Michael adduct-Schiff base cross-link. Polyclonal antibody was raised to the Nalpha-acetyllysine-HNE fluorophore and found to be highly specific to the chromophore structure of the compound. This antibody has been used to conclusively demonstrate that the lysine-HNE derivative of this fluorophore forms on protein upon exposure to HNE. The results of this study therefore provide the basis for future investigations on the contribution(s) of HNE-derived fluorophore formation to lipofuscin and ceroid accumulation.     

Kinetics of nonenzymatic glycation of ribonuclease A leading to advanced glycation end products. Paradoxical inhibition by ribose leads to facile isolation of protein intermediate for rapid post-Amadori studies

Nonenzymatic glycation (Maillard reaction) of long-lived proteins is a major contributor to the pathology of diabetes and possibly aging and Alzheimer's disease. We report here kinetic studies of the glycation of the model protein ribonuclease A by glucose and ribose leading to the formation of antigenic advanced glycation end products ("AGEs"), detectable by AGE-specific polyclonal antibodies, and pentosidine, an acid-stable fluorescent AGE. As anticipated, the kinetics of glycation by ribose were considerably faster than by glucose, and the rate of AGE formation initially increased with increasing sugar concentrations. However, ribose above 0.15 M appeared to paradoxically slow the kinetics of AGE formation, suggesting ribose inhibits the conversion of "early" Amadori rearrangement products to "late" AGEs and thus favors the accumulation of reactive Amadori intermediates. The facile isolation of such protein intermediates was achieved by an "interrupted glycation" protocol which free and reversibly bound (Schiff base) ribose was removed following a short (24h) initial incubation of 0.5 M ribose at 37 degrees C. The kinetics of buildup of the Amadori intermediates and the kinetics of their post-Amadori conversion to antigenic AGEs were independently studied. A rapid and reversible inhibition of the post-Amadori kinetics by free ribose was verified by direct re-addition of ribose to the isolated, sugar-free intermediate. The pH dependence of the kinetics of antigenic AGE formation from such intermediates was measured and exhibited an unusual bell-shaped profile over the pH range of 5.0-9.5 with a maximum near pH 8.0. Aminoguanidine, a pharmacological AGE inhibitor, was found to moderately or weakly inhibit antigenic AGE formation in such post- Amadori steps. The isolation of the glycated ribonuclease intermediate thus simplifies kinetic and mechanistic studies of AGE formation, permits AGE studies in the absence of complications arising from free or Schiff base bound sugar, and provides a novel methodology for evaluating the mechanism and efficacy of therapeutic agents that may inhibit AGE formation.     

Increased levels of advanced glycation endproducts in the lenses and blood vessels of cigarette smokers

BACKGROUND: Advanced glycation endproducts (AGEs) arise from the spontaneous reaction of reducing sugars with the amino groups of macromolecules. AGEs accumulate in tissue as a consequence of diabetes and aging and have been causally implicated in the pathogenesis of several of the end-organ complications of diabetes and aging, including cataract, atherosclerosis, and renal insufficiency. It has been recently proposed that components in mainstream cigarette smoke can react with plasma and extracellular matrix proteins to form covalent adducts with many of the properties of AGEs. We wished to ascertain whether AGEs or immunochemically related molecules are present at higher levels in the tissues of smokers. MATERIALS AND METHODS: Lens and coronary artery specimens from nondiabetic smokers and nondiabetic nonsmokers were examined by immunohistochemistry, immunoelectron microscopy, and ELISA employing several distinct anti-AGE antibodies. In addition, lenticular extracts were tested for AGE-associated fluorescence by fluorescence spectroscopy. RESULTS: Immunoreactive AGEs were present at significantly higher levels in the lenses and lenticular extracts of nondiabetic smokers (p < 0.003). Anti-AGE immunogold staining was diffusely distributed throughout lens fiber cells. AGE-associated fluorescence was significantly increased in the lenticular extracts of nondiabetic smokers (p = 0.005). AGE-immunoreactivity was significantly elevated in coronary arteries from nondiabetic smokers compared with nondiabetic nonsmokers (p = 0.015). CONCLUSIONS: AGEs or immunochemically related molecules are present at higher levels in the tissues of smokers than in nonsmokers, irrespective of diabetes. In view of previous reports implicating AGEs in a causal association with numerous pathologies, these findings have significant ramifications for understanding the etiopathology of diseases associated with smoking, the single greatest preventable cause of morbidity and mortality in the United States.     

Reduction of advanced glycation end-product (AGE) levels in nervous tissue proteins of diabetic Lewis rats following islet transplants is related to different durations of poor metabolic control

Advanced glycation end-products (AGEs) are irreversible compounds which, by abnormally accumulating over proteins as a consequence of diabetic hyperglycaemia, can damage tissues and thus contribute to the pathogenesis of diabetic complications. This study was performed to evaluate whether restoration of euglycaemia by islet transplantation modifies AGE accumulation in central and peripheral nervous tissue proteins and, as a comparison, in proteins from a non-nervous tissue. Two groups of streptozotocin diabetic inbred Lewis rats with 4 (T1) or 8 (T2) months disease duration were grafted into the liver via the portal vein with 1200-1500 islets freshly isolated from normal Lewis rats. Transplanted rats, age-matched control and diabetic rats studied in parallel, were followed for a further 4-month period. At study conclusion, glycaemia, glycated haemoglobin and body weight were measured in all animals, and an oral glucose tolerance test (OGTT) performed in transplanted rats. AGE levels in cerebral cortex, spinal cord, sciatic nerve proteins and tail tendon collagen were measured by enzyme-linked immunosorbent assay (ELISA). Transplanted animal OGTTs were within normal limits, as were glycaemia and glycated haemoglobin. Diabetic animal AGEs were significantly higher than those of control animals. Protein AGE values were reduced in many transplanted animals compared to diabetic animals, reaching statistical significance in spinal cord (P < 0.05), sciatic nerve (P < 0.02) and tail tendon collagen (P < 0.05) of T1 animals. Thus, return to euglycaemia following islet transplantation after 4 months of diabetes with poor metabolic control reduces AGE accumulation rate in the protein fractions of the mixed and purely peripheral nervous tissues (spinal cord and sciatic nerve, respectively). However, after a double duration of bad metabolic control, a statistically significant AGE reduction has not been achieved in any of the tissues, suggesting the importance of an early therapeutic intervention to prevent the possibly pathological accumulation of AGEs in nervous and other proteins.     

Advanced glycation end products in Alzheimer's disease and other neurodegenerative diseases

Advanced glycation end products (AGEs) have been implicated in the chronic complications of diabetes mellitus and have been reported to play an important role in the pathogenesis of Alzheimer's disease. In this study, we examined the immunohistochemical localization of AGEs, amyloid beta protein (A beta), apolipoprotein E (ApoE), and tau protein in senile plaques, neurofibrillary tangles (NFTs), and cerebral amyloid angiopathy (CAA) in Alzheimer's disease and other neurodegenerative diseases (progressive supranuclear palsy, Pick's disease, and Guamanian amyotrophic lateral sclerosis/Parkinsonism-dementia complex). In most senile plaques (including diffuse plaques) and CAA from Alzheimer's brains, AGE and ApoE were observed together. However, approximately 5% of plaques were AGE positive but A beta negative, and the vessels without CAA often showed AGE immunoreactivity. In Alzheimer's disease, AGEs were mainly present in intracellular NFTs, whereas ApoE was mainly present in extracellular NFTs. Pick's bodies in Pick's disease and granulovacuolar degeneration in various neurodegenerative diseases were also AGE positive. In non-Alzheimer neurodegenerative diseases, senile plaques and NFTs showed similar findings to those in Alzheimer's disease. These results suggest that AGE may contribute to eventual neuronal dysfunction and death as an important factor in the progression of various neurodegenerative diseases, including Alzheimer's disease.     

Immunohistochemistry of matrix metalloproteinases (MMP), their substrates, and their inhibitors (TIMP) during trophoblast invasion in the human placenta

The invasion of extravillous trophoblast cells into the maternal endometrium is one of the key events in human placentation. The ability of these cells to infiltrate the uterine wall and to anchor the placenta to it as well as their ability to infiltrate and to adjust utero-placental vessels to pregnancy depends, among other things, on their ability to secrete enzymes that degrade the extracellular matrix. Most of the latter enzymes belong to the family of matrix metalloproteinases. Their activity is regulated by the tissue inhibitors of matrix metalloproteinases. We have studied the distribution patterns of matrix metalloproteinases-1, -2, -3, and -9 and their inhibitors TIMP-1 and TIMP-2 as compared to the distribution of their substrates along the invasive pathway of extravillous trophoblast of 1st, 2nd, and 3rd trimester placentas by means of light microscopy on paraffin and cryostat sections as well as at the ultrastructural level (only 3rd trimester placenta). The comparison of different methods proved to be necessary, since the immunohistochemical distribution patterns of these soluble enzymes are considerably influenced by the pretreatment of tissues. All three methods revealed immunoreactivities of both, proteinases and their inhibitors, not only intracellularly in the extravillous trophoblast but also extracellularly in its surrounding matrix, the distribution patterns depending on the stage of pregnancy and on the degree of differentiation of trophoblast cells along their invasive pathway. Within the extracellular matrix, immunolocalization of matrix metalloproteinases as well as their inhibitors showed a specific relation to certain extracellular matrix molecules.     

An IgG monoclonal antibody against Dictyostelium discoideum glycoproteins specifically recognizes Fucalpha1,6GlcNAcbeta in the core of N-linked glycans. Localized expression of core-fucosylated glycoconjugates in human tissues

Core fucosylation of N-linked oligosaccharides (GlcNAcbeta1, 4(Fucalpha1,6)GlcNAcbeta1-Asn) is a common modification in animal glycans, but little is known about the distribution of core-fucosylated glycoproteins in mammalian tissues. Two monoclonal antibodies, CAB2 and CAB4, previously raised against carbohydrate epitopes of Dictyostelium discoideum glycoproteins (Crandall, I. E. and Newell, P. C. (1989) Development 107, 87-94), specifically recognize fucose residues in alpha1,6-linkage to the asparagine-bound GlcNAc of N-linked oligosaccharides. These IgG3 antibodies do not cross-react with glycoproteins containing alpha-fucoses in other linkages commonly seen in N- or O-linked sugar chains. CAB4 recognizes core alpha1,6 fucose regardless of terminal sugars, branching pattern, sialic acid linkage, or polylactosamine substitution. This contrasts to lentil and pea lectins that recognize a similar epitope in only a subset of these structures. Additional GlcNAc residues found in the core of N-glycans from dominant Chinese hamster ovary cell mutants LEC14 and LEC18 progressively decrease binding. These antibodies show that many proteins in human tissues are core-fucosylated, but their expression is localized to skin keratinocytes, vascular and visceral smooth muscle cells, epithelia, and some extracellular matrix-like material surrounding subpopulations of lymphocytes. The availability of these antibodies now allows for an extended investigation of core fucose epitope expression in development and malignancy and in genetically manipulated mice.     

Distribution and localization of galectin purified from Rana catesbeiana oocytes

Galectins are a family of lectins that recognize beta-D-galactosides independently of calcium ions, and are widely distributed in animals. To characterize a galectin previously purified from oocytes of Rana catesbeiana (American bullfrog), we studied its distribution and localization in several tissues from this frog. Hemagglutination assay and western blotting showed that this lectin is present in many tissues including the liver, skin, kidney, skeletal muscle, and sciatic nerve, but is particularly concentrated in the ovary. Light microscopic immunohistochemistry showed that this lectin is localized in such places as cell-cell junctions, basement membranes, extracellular matrix, or secretory substances in several organs, indicating that this galectin is mainly distributed extracellularly. However, in the ovary, light microscopy showed that this lectin is present in or associated with the yolk platelet. Electron microscopy further revealed that it is localized in the periphery of the yolk platelet (the yolk plasm), but not in the cortical granule. These results indicate that Rana oocytes contain abundant galectin in their yolk platelets in contrast to Xenopus laevis oocytes, which have been found not to contain galectins but other classes of lectins in their yolk platelets and cortical granules.     

The kinetics and extent of engraftment of chronic myelogenous leukemia cells in non-obese diabetic/severe combined immunodeficiency mice reflect the phase of the donor's disease: an in vivo model of chronic myelogenous leukemia biology

In vitro studies have provided little consensus on the kinetic abnormality underlying the myeloid expansion of chronic myelogenous leukemia (CML). Transplantation of human CML cells into non-obese diabetic mice with severe immunodeficiency disease (NOD/SCID mice) may therefore be a useful model. A CML cell line (BV173) and peripheral blood cells collected from CML patients in chronic phase (CP), accelerated phase (AP), or blastic phase (BP) were injected into preirradiated NOD/SCID mice. Animals were killed at serial intervals; cell suspensions and/or tissue sections from different organs were studied by immunohistochemistry and/or flow cytometry using antihuman CD45 monoclonal antibodies (MoAbs), and by fluorescence in situ hybridization (FISH) for the BCR-ABL fusion gene. One hour after injection, cells were sequestered in the lungs and liver, but 2 weeks later they were no longer detectable in either site. Similar short-term kinetics were observed using 51Cr-labeled cells. The first signs of engraftment for BV173, AP, and BP cells were detected in the bone marrow (BM) at 4 weeks. At 8 weeks the median percentages of human cells in murine marrow were 4% (range, 1 to 9) for CP, 11% (range, 5 to 36) for AP, 38.5% (range, 18 to 79) for BP, and 54% (range, 31 to 69) for BV173. CP cells progressively infiltrated BM (21%) and spleen (6%) by 18 to 20 weeks; no animals injected with the cell line or BP cells survived beyond 12 weeks. The rate of increase in human cell numbers was higher for BP (7.3%/week) as compared with CP (0.9%/week) and AP (0. 5%/week). FISH analysis with BCR and ABL probes showed that some of the human cells engrafting after injection of CP cells lacked a BCR-ABL gene and were presumably normal. We conclude that CML cells proliferate in NOD/SCID mice with kinetics that recapitulate the phase of the donor's disease, thus providing an in vivo model of CML biology.