Low ALT Levels Independently Associated with 22-Year All-Cause Mortality Among Coronary Heart Disease Patients.

BACKGROUND: Low alanine aminotransferase (ALT) blood levels are known to be associated with frailty and increased risk of long-term mortality in certain populations. However, the contribution of this marker to long-term outcome has not been assessed in patients with chronic coronary heart disease. OBJECTIVE: The aim of the current study was to assess the association between low ALT values and long-term, 22.8-year, all-cause mortality in this population. PARTICIPANTS: We examined the association of low ALT (<17 IU/l) with long-term all-cause mortality in the Bezafibrate Infarction Prevention (BIP) Registry population. KEY RESULTS: Appropriate laboratory and survival data were available for 6,575 patients, without known liver pathology, included in the BIP registry, with a median follow-up period of 22.8 years. The cumulative probability of all-cause mortality was significantly higher in the low ALT group compared with patients with higher ALT levels (65.6 % vs. 58.4 %; log-rank p < 0.001). Consistently, multivariate analysis, adjusted for multiple established predictors of mortality in this population, demonstrated that low ALT is independently associated with 11 % greater long-term (22.8 years) mortality risk [HR 1.11 (95 % confidence interval: 1.03-1.19; adjusted p < 0.01)]. CONCLUSIONS: Low ALT levels are associated with increased long-term mortality among middle-aged patients with stable coronary heart disease. This association remained statistically significant after adjustment for other well-established risk factors for mortality in this population.

Isolation by cell-column chromatography of immunoglobulins specific for cell surface carbohydrates.

A new method of affinity chromatography using glutaraldehyde-fixed cells immobilized on Sephadex beads has been used to isolate immunoglobulins (Ig's) specific for cell surface glycoproteins. Ig's that specifically bound and agglutinated the same cells as those originally fixed on the columns were isolated from nonimmune sera of various species. Periodate treatment of the cell-columns and the free cells destroyed their ability to bind the Ig's, and the binding of the Ig's to untreated cells was inhibited by monosaccharides such as D-galactose and sialic acid. The binding of antibodies directed against cell surfaces obtained by immunizing animals with the same mouse tumor cell lines used on the columns (P388 and EL4) was not inhibited by various saccharides. Surface glycoproteins obtained from the mouse tumor cells by immunoprecipitation with the column-isolated Ig's yielded specific electrophoretic patterns that differed from those obtained using Ig's from the sera of rabbits immunized with the tumor cells. The data suggest that the Ig's isolated by cell-column chromatography were directed against carbohydrates, probably those in terminal positions of the polysaccharide portions of the tumor cell surface glycoproteins. Column-isolated Ig's specific for carbohydrates were also useful in studies of cell interactions in nonmammalian systems including Dictyostelium discoideum and Saccharomyces cerevisiae. The cell-column method appears to be adaptable to the isolation of a variety of molecules in addition to antibodies.

Lymphocyte activation by monovalent fragments of antibodies reactive with cell surface carbohydrates.

Antibodies reactive with cell surface carbohydrates were isolated from normal chicken serum and were found to be mitogenic for mouse splenic lymphocytes as assayed by both blast transformation and [3H]thymidine incorporation. The Fab' fragments of these carbohydrate-binding immunoglobulins were just as mitogenic as the divalent native antibody. Moreover, succinylated Fab' fragments, which probably would not form self-associating aggregates, showed similar mitogenic properties. All of these results indicate that, at least for saccharide-specific ligands, multipoint attachment and receptor cross-linkage on the cell to which the ligand is attached may not be a stringent requirement for activation.

Homocysteine levels in adolescent schizophrenia patients.

Homocysteine is a sulfur containing amino acid that has been widely investigated for its putative role in cardiovascular and neuropsychiatric disorders. It has been suggested that homocysteine has implications especially in young, male schizophrenia patients. In this prospective case-control study, we compared plasma homocysteine levels in a group of adolescent schizophrenia inpatients (aged 14-21 years; n=23) to normal healthy controls (n=51). Mean plasma homocysteine levels were significantly higher in the patient group than in the control group (15.40+/-2.00 and 9.78+/-0.33 micromol/L, respectively, p<0.032). The difference was almost entirely attributable to the male schizophrenia subgroup (18.18+/-5.65 in male patients vs. 10.31+/-5.33 micromol/L in female patients). The group x sex interaction was statistically significant (p=0.0035). These data indicate that a subgroup of male adolescent schizophrenia patients has high homocysteine blood levels. The role of homocysteine in the pathophysiology of adolescent-onset schizophrenia merits further investigation.

Levels of disialogangliosides in sera of melanoma patients monitored by sensitive thin-layer chromatography and immunostaining.

Levels of GD2, GD3, and 9-O-acetyl GD3 were monitored in sera of patients with melanoma and healthy adults with two monoclonal antibodies that specifically detect these gangliosides. By direct measurement of radioactivity in the immunolabeled chromatogram, GD2 could be detected in normal sera at 2 ng/mL. Serum levels of GD2 and GD3 were increased approximately sixfold and fivefold, respectively, in patients with disseminated melanoma, compared with those of healthy adults. The acetylated derivative of GD3, which is highly specific for melanoma cells, was not detected in serum. This sensitive assay allows the quantitation of tumor-associated gangliosides that are circulating in sera of melanoma patients.

Characterization of tenascin secreted by human melanoma cells.

Tenascin is a large glycoprotein of the extracellular matrix. It shows a site-restricted expression during embryogenesis and can be found in adult tissues during wound healing and tumorigenesis. Because of the potential involvement of tenascin in adhesion and invasion during metastasis, the study of the interactions of tumor cells with tenascin is of considerable interest. Using five anti-melanoma monoclonal antibodies to four different epitopes of human tenascin, we found that most melanoma cells secrete tenascin in vitro constitutively. Transforming growth factor beta 1 in the medium increased secretion in tenascin-producing cells. Tenascin was present in sera of melanoma patients, with significantly elevated levels in patients with advanced melanomas as compared to patients with low tumor burden or to normal donors. Normal and malignant melanocytes did not attach to tenascin as substrate within 1 to 2 h and tenascin could also inhibit fibronectin-dependent adhesion. These results indicate that tenascin may play a critical role in cell-substrate interactions of melanoma cells.

A differential scanning calorimetry study of the interaction of gangliosides with peanut lectin, serotonin and daunomycin.

Thermotropic behaviour of human Tay-Sachs ganglioside and of mixed bovine brain gangliosides, before and after interaction with peanut lectin, serotonin and daunomycin, was investigated. Interaction of mixed brain gangliosides with peanut lectin or serotonin causes a decrease in the enthalpy of melting, whereas interaction of this lectin with Tay-Sachs ganglioside does not influence the enthalpy of melting. Serotonin causes a small increase in the enthalpy of melting of the Tay-Sachs ganglioside.

Calorimetric studies on the interaction of gangliosides with phospholipids and myelin basic protein.

Differential scanning calorimetry was employed to investigate the interaction of GM1 gangliosides with phospholipids (phosphatidylethanolamine, phosphatidylserine or phosphatidylcholine). It was found that GM1 is completely miscible with phosphatidylethanolamine; however, the interaction with phosphatidylserine is minimal. Addition of excess Ca2+ to the interaction products of GM1 with phosphatidylcholine or phosphatidylethanolamine did not induce phase separation. The influence of myelin basic protein on the thermotropic behaviour of GM1 was also studied. It was found that basic protein has a very strong perturbing effect on GM1 micelles.

Calorimetric studies on various gangliosides and ganglioside-lipid interactions.

Differential scanning calorimetry was used to investigate the thermotropic behaviour of various gangliosides differing in size and in the net negative charge. It was found that the number and the position of the negative charges in the headgroup region influence strongly the phase transition profiles. Interaction of GM1 ganglioside with egg phosphatidylcholine or cholesterol was also investigated. GM1 is completely miscible with egg phosphatidylcholine, giving only one transition peak at all ratios of the two components, implying that when gangliosides are in a more fluid lipid environment in biological membranes they will be randomly distributed. Interaction with cholesterol decreases the enthalpy of melting of the ganglioside. The decrease in enthalpy reaches a plateau at about 30 mol% cholesterol, suggesting a lower affinity of cholesterol for gangliosides than for sphingomyelin.

Microalbuminuria immunoassay based on antibodies covalently conjugated to Eupergit C-coated beads.

OBJECTIVE: To develop a reliable, simple, and sensitive assay for microalbuminuria, based on covalent attachment of anti-HSA to oxirane-bearing polymethylmethacrylate beads (Eupergit CB6200). RESEARCH DESIGN AND METHODS: Anti-HSA antibodies were coupled to CB6200 beads by reaction of their amino groups with the oxirane groups of the matrix. The capability of the beads to bind HSA from standard solutions or urine was evaluated and compared with the state of the art ELISA test. RESULTS: The new bead immunoassay is sensitive and linear in the range of 1-25 mg/L, which is considered the low microalbuminuria range. When HSA levels in urine were tested, the intra- and interassay CV values ranged between 2.7 and 3.9% and between 5.6 and 6.6%, respectively. The long-term storage stability of the antibodies covalently bound on the beads was higher than of the same antibodies adsorbed on ELISA plates. After 16 wk of storage, the CV was about 7.3% with the bead assay, compared with 14% obtained for the ELISA test under the same experimental conditions. CONCLUSIONS: A new procedure for microalbuminuria assay was developed, with Eupergit CB6200 beads as a solid support for covalent binding of the first antibody. Accuracy, sensitivity, reproducibility, and precision of the bead immunoassay were similar to those of commonly used immunoassays, as exemplified by the analysis of HSA in 53 clinical urine samples. The bead assay retains a low degree of variability over long storage periods, and the beads may be reapplied after a simple acid-washing procedure.

[ADMA (asymmetric dimethylarginine)--the inhibitor of nitric oxide (NO) synthesis: a new marker for vascular pathology].

Ample evidence is accumulating to suggest that asymmetric dimethylarginine (ADMA), an endogenous competitive inhibitor of nitric oxide synthase, is significantly elevated during phases of endothelial dysfunction. ADMA inhibits NO synthesis, hence its arterial infusion induces local arterial constriction. ADMA is generated ubiquitously in numerous tissues, by proteolysis of methylated proteins, while its degeneration is carried out mainly by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). Administration of L-arginine can override partially NO synthesis by ADMA, yet it cannot eliminate the primary factors involved in the endothelial dysfunction. ADMA measurements might add valuable information about this new risk factor or at least a marker for adverse endothelial events.

Homocystinuria in the Arab population of Israel: identification of two novel mutations using DGGE analysis.

This study describes, for the first time, a thorough genetic investigation in Israeli Arab homocystinuric patients. By using a DGGE methodology and sequencing we were able to identify the disease causing mutation in all. Of the mutations that were detected, two are novel: a 785C>G transversion in exon 7 (T262R) and a 5-bp deletion in the 5' of IVS17 including the T in the +2 position that is crucial for correct splicing (g18327-18331del5). In spite of the highly consanguineous nature of this population several different mutations were found. This may suggest that the mutations arose only recently in the population. The results of our study would enable early prenatal diagnosis, genetic counseling and screening for the mutations in population at risk. Hum Mutat 16:372, 2000.

Cerebrovascular events in patients with significant stenosis of the carotid artery are associated with hyperhomocysteinemia and platelet antigen-1 (Leu33Pro) polymorphism.

BACKGROUND AND PURPOSE: Although risk factors for carotid artery stenosis caused by atherosclerosis are known, it is unclear what triggers "activation" of the atherosclerotic plaques and the ensuing thromboembolic cerebral events. The aim of this study was to evaluate whether thrombophilic factors, platelet glycoprotein (GP) polymorphisms, and homocysteine are associated with a risk of ischemic events in patients with significant carotid stenosis. METHODS: Consecutive patients with >/=50% carotid stenosis, whether symptomatic (with ipsilateral ischemic events) or asymptomatic, who were evaluated and followed in a neurovascular clinic were tested for plasma levels of homocysteine, C677T mutation in methylenetetrahydrofolate reductase, G20210A mutation of factor II, factor V Leiden, antiphospholipid antibodies, and polymorphisms of platelet membrane GP: human platelet antigen (HPA)-1, GP Ia (C807T), and GP Ib (variable number of tandem repeats, Kozak, and HPA-2). RESULTS: Eighty-six asymptomatic and 67 symptomatic patients were evaluated. The former group was older (73.7+/-6.9 versus 69.5+/-9.1 years, P=0.02). Major risk factors for stroke were similar in both groups. In symptomatic patients versus asymptomatic patients, hyperhomocysteinemia was 3-fold more frequent (34.3% versus 12.8%, respectively; P=0.002) and HPA-1a/b was almost 2-fold more common (38.8% versus 20.9%, respectively; P=0.01). All other thrombophilic factors and platelet polymorphisms studied did not differ significantly between the 2 groups. Multivariate analysis revealed that hyperhomocysteinemia and the HPA-1a/b genotype conferred a significant risk of cerebral ischemic events, with odds ratios (95% CI) of 4.07 (1.7 to 9.7) and 3.4 (1.5 to 7.8), respectively. CONCLUSIONS: Hyperhomocysteinemia and HPA-1a/b are independent risk factors for ischemic events in patients with significant carotid stenosis.

Cross genotype sex hormone treatment in two cases of hypogonadal osteoporosis.

BACKGROUND: Sex hormone deficiency is the most common cause of bone loss. Reduced bone mass and an increased risk for osteoporotic fractures have been described in hypogonadal subjects of both sexes. We present here the results of treating two patients showing abnormal sexual differentiation (an XX male and an XY female), who suffered from bone loss related to sex hormone deficiency, with cross genotype sex hormones. SUBJECTS AND METHODS: Patient 1 was an asymptomatic 39-yr-old XY female with complete androgen insensitivity. Her testes had been removed, and she later discontinued estrogen treatment. Patient 2, a 37-yr-old XX male, had congenital adrenal hyperplasia, which led to a masculine phenotype. He was ovariectomized and reared as a male. He was treated with glucocorticoids but refused androgen treatment for many years. We treated both patients with phenotypically matched sex hormones (patient 1 received conjugated estrogens 1.25 mg/day, and patient 2 received 250 mg testosterone every 4 weeks) and followed their bone mineral density (BMD) using dual-energy X-ray absorptiometry, urine calcium, and hydroxyproline excretion. RESULTS: Before treatment both patients had low sex hormones and highly elevated gonadotropins. As a result of treatment urine hydroxyproline excretion decreased from 45 and 26.7 mg/g creatinine to 15 and 15.9 mg/g creatinine in patients 1 and 2 respectively. In patient 1, lumbar BMD rose from 0.912gr/cm2 to 0.976gr/cm2 and femoral neck BMD rose from 0.716gr/cm2 to 0.836gr/cm2 after 4 years of treatment. In patient 2, lumbar BMD rose from 0.717gr/cm2 to 0.815gr/cm2 and the femoral neck BMD rose from 0.509gr/cm2 to 0.635gr/cm2 after 27 months of treatment. CONCLUSIONS: Phenotypically-matched sex hormone therapy in patients with abnormal sexual differentiation is essential not only to maintain external appearance but also for the preservation of bone mass.

Regenerative capacity of the goldfish visual system is affected by antibodies specific to gangliosides injected intraocularly.

An association between gangliosides and neuronal regeneration in goldfish is demonstrated in the present study. A single intraocular injection of affinity purified anti-GM1 antibodies administered simultaneously with crush injury of the optic nerve, inhibits the regenerating process as expressed by two parameters: protein synthesis in the retina and in vitro sprouting ability from the retina. The retinal level of several gangliosides (such as GD3, GD1a, GD1b and GT1b) is enhanced during regeneration. Although GM1 appears to be a minor retinal ganglioside, antibodies to GM1 exert a marked effect on retinal regenerative process. It is assumed that such antibodies could interact with more abundant retinal gangliosides such as GD1b which shows enhanced biosynthesis during regeneration and which shares a similar disaccharide terminal residue with GM1.

Analysis of cell-free human alpha1 integrin with a monoclonal antibody to the I-domain: detection in ocular fluid and function as an adhesion substrate.

The alpha1 beta1 integrin, an inserted (1) domain containing collagen receptor, is expressed in the cell surface membrane of normal and malignant cells, and may play a role in their migration through tissues or in metastatic spread. Here we report that a functional anti-human alpha1beta1 integrin monoclonal antibody (mAb) (1B3.1) directly and specifically binds plastic bound recombinant human alpha1 I-domain protein containing the collagen binding site. Detection was diminished by acidification of the I-domain protein but was enhanced by increasing concentrations of Mg2+ cation. Furthermore, we detected binding of the mAb to proteins from the ocular fluids of 6 patients, with the highest concentration, corresponding to 22.1 ng/ml of I-domain, found in a sample from the eye of a patient with metastatic lung adenocarcinoma. Interestingly, we found that both SKNSH neuroblastoma cells and virally transformed human T cells adhered specifically to plastic wells coated with either immobilized collagen IV or alpha1 I-domain. MAb I B3.1 inhibited adhesion to collagen IV but not to immobilized I-domain. These results suggest a novel function for cell free alpha1 I-domain as a substrate for cellular adhesion, which may have relevance in tumor spread in vivo.

Interaction of the chlorinated hydrocarbon insecticide lindane or DDT with lipids--a differential scanning calorimetry study.

The interaction of DDT and lindane with glycosphingolipids and phospholipids was investigated by employing differential scanning calorimetry. The degree of perturbation produced by lindane is stronger than that of DDT and depends also on the lipid.

Multiple sclerosis: cell-mediated immunity to human brain gangliosides.

Cell-mediated immunity (CMI) to myelin components has been implicated in Multiple Sclerosis (MS) pathogenesis: two targets were suggested, Myelin Basic Protein with controversial results and, more recently, gangliosides. In order to investigate their possible involvement, we have performed Leukocyte Migration inhibition (LMI) tests in the presence of human brain gangliosides. Thirty nine MS patients (twenty four being "definite", according to McDonald and Halliday's classification), twenty nine patients with Other Neurological Diseases (OND), thirty six patients with Inflammatory diseases (ID) and forty healthy controls were tested. MS patients were divided into two groups, depending on the clinical stage of the disease. The mean migration inhibition percentage of the MS-attack group was found to be significantly different from the four others (p less than 0.01) (24.4 +/- 16.2 versus 10.9 +/- 8.5 in MS without attack, 4.4 +/- 12.9 in OND, 3.9 +/- 13.9 in ID and 11.1 +/- 12.1 in healthy subjects). LMI to gangliosides is therefore significantly increased during the attack stage in MS. These results support the notion of a Delayed Type Hypersensitivity to these glycolipids during the active stage of the disease.

Assessment of the coagulation profile in hemato-oncological patients receiving ATG-based conditioning treatment for allogeneic stem cell transplantation.

Antithymocyte globulin (ATG) is increasingly used in pre-allogeneic stem cell transplantation (allo-SCT) conditioning regimens to prevent graft rejection and graft-versus-host disease. However, ATG was also found to be associated with increased incidence of thrombosis during organ transplantation. In the present study, we tested the coagulation status of 21 patients with hematologic malignancies undergoing allo-SCT who received ATG-based (11 patients) or non-ATG-based (10) conditioning treatment. We assessed several thrombophilia markers as well as circulating total and endothelial microparticles (TMP/EMP) and soluble CD40 ligand (CD40L). No significant difference in the mean values of prothrombin time, partial thromboplastin time, fibrinogen, antithrombin, protein C, protein S, thrombin-antithrombin III complex, homocysteine levels, prevalence of genetic thrombophilia markers and levels of EMP, TMP or CD40L was observed between the ATG-treated and ATG-untreated patients, as well as before and after conditioning in each group separately. Platelet counts decreased significantly in ATG-treated patients; however, this decrease was not associated with clinical or laboratory evidence of disseminated intravascular coagulation. No patient developed thromboembolic event or veno-occlusive liver disease. Our results suggest that allo-SCT is not associated with increased hypercoagulability and addition of ATG to conditioning regimen has no significant procoagulant effect.

Alterations in gangliosides of rat liver cells in hyperlipidemic state.

The possibility that liver cell membrane is modified in hyperlipidemic state was studied using nephrotic hyperlipidemic rats. Liver cells of normal and nephrotic rats were isolated and subjected to labeling of cell surface components using lactoperoxidase catalyzed radioiodination. The labeling of total surface lipids of hepatocytes of nephrotic rats was about five times higher than that of normal ones and was particularly higher in glycosphingolipids. Cultivation of the isolated hepatocytes as primary cultures reduced drastically labeling of surface lipids in liver cells of both nephrotic and normal rats and abolished the differences observed in liver cells of the two types. Determination of cell associated gangliosides, showed that the level in nephrotic rat hepatocytes was only 35% higher than that of normal rats. Yet, in both types of liver cells 24 h cultivation decreased markedly the ganglioside content. However, similar to the effect observed in hyperlipidemic rats, supplementing the culture medium with very low density lipoproteins (VLDL) increased considerably the ganglioside level of cultured hepatocytes. These treatments did not affect the activity of enzymes involved in the synthesis of gangliosides. It is suggested that ganglioside content in liver cell membrane is modulated in the hyperlipidemic state.