Orientation of capture antibodies on gold nanoparticles to improve the sensitivity of ELISA-based medical devices.

The sensitivity of ELISA-based devices strongly depends on the right orientation of antibodies on the sensor surface. The aim of this work was to increase the analytical performance of a commercial ELISA-based medical device (VIDAS®), thanks to the specific orientation of antibodies on gold nanostructured disposables. For this purpose, fPSA VIDAS® assay was used as model and the disposable providing the antigen binding surface (SPR®) was functionalized with gold nanostructures coated with monovalent half-fragment antibodies (reduced IgG, rIgG). The functionalization of polystyrene SPRs® with gold nanostructures was achieved through a one-step incubation of gold dispersions in a mixture of non-toxic solvents. Five different concentrations of gold nanoparticles (NPs) were tested with a maximum fluorescence enhancement for NPs density around 3-8 *103 NPs/µm2 (752 ± 11 RFV vs 316 ± 5 RFV of bare SPRs®). The comparison of the dose-response curve obtained with commercial and gold coated-SPRs® revealed a significant improvement (p < 0.0001) of the analytical sensitivity of the VIDAS® system using nanostructured disposables. This improved version of SPRs® allows to distinguish small variations of fPSA concentrations opening the way to the application of this biomarker to other kinds of cancer as recently described in the literature.

Signal Enhancement in Oriented Immunosorbent Assays: A Balance between Accessibility of Antigen Binding Sites and Avidity.

The sensitivity of immunoassays was reported to be increased by the orientation of antibodies. We investigated how the size and valence of antigens and orientation and valence of antibodies contribute to the analytical sensitivity of ELISA. Antigens differing in size and number of epitopes were compared using oriented and non-oriented ELISAs: the orientation of antibodies was obtained coating half-fragment antibodies on maleimide microplates, while, in non-oriented ELISA, whole antibodies were randomly physisorbed. The oriented assay performed better than the non-oriented one at each concentration (0.4-3.3 ng/mL) of a small monomeric antigen (cardiac Troponin I, 24 kDa, Rh 3 nm). No significant differences were observed with a large monovalent antigen (prostate-specific antigen-alpha(1) antichymotrypsin, 90 kDa, Rh > 3 nm), since its steric hindrance overcame the increased availability of antigen binding sites given by orientation. Large multivalent antigens (ferritin, 280 kDa, Rh 6 nm; α-fetoprotein, >70 kDa, Rh > 3.3 nm) performed better in non-oriented assays. In this case, the repeated epitopes on the surface of the antigens favored the engagement of both antigen binding sites of the whole IgG, thus suggesting that avidity represented the leading force in this experimental setting. In conclusion, the design of high-sensitivity ELISAs should consider the dimension and valency of antigens in addition to the affinity and avidity of antibodies.

Induction of Gamma-Glutamyltransferase Activity and Consequent Pro-oxidant Reactions in Human Macrophages Exposed to Crocidolite Asbestos.

Asbestos is the main causative agent of malignant pleural mesothelioma. The variety known as crocidolite (blue asbestos) owns the highest pathogenic potential, due to the dimensions of its fibers as well as to its content of iron. The latter can in fact react with macrophage-derived hydrogen peroxide in the so called Fenton reaction, giving rise to highly reactive and mutagenic hydroxyl radical. On the other hand, hydroxyl radical can as well originate after thiol-dependent reduction of iron, a process capable of starting its redox cycling. Previous studies showed that glutathione (GSH) is one such thiol, and that cellular gamma-glutamyltransferase (GGT) can efficiently potentiate GSH-dependent iron redox cycling and consequent oxidative stress. As GGT is expressed in macrophages and is released upon their activation, the present study was aimed at verifying the hypothesis that GSH/GGT-dependent redox reactions may participate in the oxidative stress following the activation of macrophages induced by crocidolite asbestos. Experiments in acellular systems confirmed that GGT-mediated metabolism of GSH can potentiate crocidolite-dependent production of superoxide anion, through the production of highly reactive dipeptide thiol cysteinyl-glycine. Cultured THP-1 macrophagic cells, as well as isolated monocytes obtained from healthy donors and differentiated to macrophages in vitro, were investigated as to their expression of GGT and the effects of exposure to crocidolite. The results show that crocidolite asbestos at subtoxic concentrations (50-250 ng/1000 cells) can upregulate GGT expression, which raises the possibility that macrophage-initiated, GSH/GGT-dependent pro-oxidant reactions may participate in the pathogenesis of tissue damage and inflammation consequent to crocidolite intoxication.

Pilot, Open, Randomized, Prospective Trial for Normothermic Machine Perfusion Evaluation in Liver Transplantation From Older Donors.

Ex situ normothermic machine perfusion (NMP) might minimize ischemia/reperfusion injury (IRI) of liver grafts. In this study, 20 primary liver transplantation recipients of older grafts (≥70 years) were randomized 1:1 to NMP or cold storage (CS) groups. The primary study endpoint was to evaluate graft and patient survival at 6 months posttransplantation. The secondary endpoint was to evaluate liver and bile duct biopsies; IRI by means of peak transaminases within 7 days after surgery; and incidence of biliary complications at month 6. Liver and bile duct biopsies were collected at bench surgery, end of ex situ NMP, and end of transplant surgery. Interleukin (IL) 6, IL10, and tumor necrosis factor α (TNF-α) perfusate concentrations were tested during NMP. All grafts were successfully transplanted. Median (interquartile range) posttransplant aspartate aminotransferase peak was 709 (371-1575) IU/L for NMP and 574 (377-1162) IU/L for CS (P = 0.597). There was 1 hepatic artery thrombosis in the NMP group and 1 death in the CS group. In NMP, we observed high TNF-α perfusate levels, and these were inversely correlated with lactate (P < 0.001). Electron microscopy showed decreased mitochondrial volume density and steatosis and an increased volume density of autophagic vacuoles at the end of transplantation in NMP versus CS patients (P < 0.001). Use of NMP with older liver grafts is associated with histological evidence of reduced IRI, although the clinical benefit remains to be demonstrated.

Association between plasma gamma-glutamyltransferase fractions and metabolic syndrome among hypertensive patients.

Among the risk factors associated to metabolic syndrome (MetS), hypertension shows the highest prevalence in Italy. We investigated the relationship between the newly identified serum γ-glutamyltransferase (GGT) fractions, b- s- m- f-GGT, and risk factors associated to MetS in hypertensive patients. A total of ninety-five consecutive hypertensive patients were enrolled. GGT fractions were analysed by gel-filtration chromatography, and hepatic steatosis was evaluated by ultrasound. MetS was diagnosed in 36% of patients. Considering the whole group, b- and f-GGT showed the highest positive correlation with BMI, glucose, triglycerides and insulin, and the highest negative correlation with HDL cholesterol. While both serum triglycerides and insulin were independently associated with b-GGT levels, only triglycerides were independently associated with f-GGT. The values of b-GGT activity increased with steatosis grade (g0 = 1.19; g2 = 3.29; ratio g2/g0 = 2.75, p < 0.0001 linear trend). Patients with MetS showed higher levels of b-GGT, m-GGT and f-GGT [median (25th-75th) U/L: 3.19 (1.50-6.59); 0.55 (0.26-0.81); 10.3 (9.1-13.6); respectively] as compared to subjects presenting with one or two MetS criteria [1.75 (0.95-2.85), p < 0.001; 0.33 (0.19-0.60), p < 0.05; 8.8 (7.0-10.6), p < 0.001]. Our data point to a potential role for b- and f-GGT fractions in identifying MetS patients among hypertensive subjects, thus providing a minimally invasive blood-based tool for MetS diagnosis.

Effect of the three-dimensional organization of liver cells on the biogenesis of the γ-glutamyltransferase fraction pattern.

Context Four gamma-glutamyltransferase (GGT) fractions with different molecular weights (big-, medium-, small- and free-GGT) are detectable in human plasma. Objective Verify if liver cells can release all four GGT fractions and if the spatial cell organization influences their release. Methods Hepatoma (HepG2) and melanoma (Me665/2/60) cells were cultured as monolayers or spheroids. GGT released in culture media was analysed by gel-filtration chromatography. Results HepG2 and Me665/2/60 monolayers released the b-GGT fraction, while significative levels of s-GGT and f-GGT were detectable only in media of HepG2-spheroids. Bile acids alone or in combination with papain promoted the conversion of b-GGT in s-GGT or f-GGT, respectively. Conclusions GGT is usually released as b-GGT, while s-GGT and f-GGT are likely to be produced in the liver extracellular environment by the combined action of bile acids and proteases.

Monocytes/macrophages activation contributes to b-gamma-glutamyltransferase accumulation inside atherosclerotic plaques.

BACKGROUND: Gamma-glutamyltransferase (GGT) is a well-established independent risk factor for cardiovascular mortality related to atherosclerotic disease. Four GGT fractions have been identified in plasma, but only b-GGT fraction accumulates in atherosclerotic plaques, and correlates with other histological markers of vulnerability. The present study was aimed to evaluate whether macrophagic lineage cells may provide a source of b-GGT within the atherosclerotic plaque. METHODS: GGT expression and release were studied in human monocytes isolated from peripheral blood of healthy donors. The growth factors GM-CSF and M-CSF were used to induce differentiation into M1-like and M2-like macrophages, respectively. Plaque GGT was investigated in tissue samples obtained from patients undergoing carotid endoarterectomy. RESULTS: We found that M1-like macrophages express higher levels of GGT as compared to M2-like, and that both monocytes and M1-like macrophages-but not M2-like-are able to release the b-GGT fraction upon activation with pro-inflammatory stimuli. Western blot analysis of b-GGT extracted from plaques confirmed the presence of a GGT immunoreactive peptide coincident with that of macrophages. CONCLUSIONS: Our data indicate that macrophages characterized by a pro-inflammatory phenotype may contribute to intra-plaque accumulation of b-GGT, which in turn may play a role in the progression of atherosclerosis by modulating inflammatory processes and favouring plaque instability.

Helicobacter, gamma-glutamyltransferase and cancer: further intriguing connections.

Virulence of Helicobacter pylori, Helicobacter suis and other bacteria appears to be partly mediated through a release of gamma-glutamyltransferase (GGT), an enzyme activity capable of promoting biochemical reactions ultimately resulting in damage to gastric epithelium and suppression of immune response. Recently published studies show that secretion of bacterial GGT occurs in the form of exosome-like vesicles. Very similar GGT-rich exosomes have been described to originate from human cancer cells, and the hypothesis is thus forwarded that in the resistant and invasive phenotype of malignant cells such vesicular/exosomal GGT may play roles akin to those described for Helicobacter infection, thus providing a significant contribution to the establishment of cancer metastases.

Gamma-glutamyltransferase fractions in human plasma and bile: characteristic and biogenesis.

Total plasma gamma-glutamyltransferase (GGT) activity is a sensitive, non-specific marker of liver dysfunction. Four GGT fractions (b-, m-, s-, f-GGT) were described in plasma and their differential specificity in the diagnosis of liver diseases was suggested. Nevertheless fractional GGT properties have not been investigated yet. The aim of this study was to characterize the molecular nature of fractional GGT in both human plasma and bile. Plasma was obtained from healthy volunteers; whereas bile was collected from patients undergoing liver transplantation. Molecular weight (MW), density, distribution by centrifugal sedimentation and sensitivity to both detergent (deoxycholic acid) and protease (papain) were evaluated. A partial purification of b-GGT was obtained by ultracentrifugation. Plasma b-GGT fraction showed a MW of 2000 kDa and a density between 1.063-1.210 g/ml. Detergent converted b-GGT into s-GGT, whereas papain alone did not produce any effect. Plasma m-GGT and s-GGT showed a MW of 1,000 and 200 kDa, and densities between 1.006-1.063 g/ml and 1.063-1.210 g/ml respectively. Both fractions were unaffected by deoxycholic acid, while GGT activity was recovered into f-GGT peak after papain treatment. Plasma f-GGT showed a MW of 70 kDa and a density higher than 1.21 g/ml. We identified only two chromatographic peaks, in bile, showing similar characteristics as plasma b- and f-GGT fractions. These evidences, together with centrifugal sedimentation properties and immunogold electronic microscopy data, indicate that b-GGT is constituted of membrane microvesicles in both bile and plasma, m-GGT and s-GGT might be constituted of bile-acid micelles, while f-GGT represents the free-soluble form of the enzyme.

Circulating gamma-glutamyltransferase fractions in cirrhosis.

BACKGROUND & AIMS: Four gamma-gultamyltransferases (GGT) fractions (b-, m-, s-, and f-GGT) have been identified in human plasma and their concentrations and ratios vary in different pathological conditions. To assess the behaviour of fractional GGT in cirrhotic patients evaluated for liver transplantation. METHODS: This was a single-centre, cross-sectional study; GGT fractions were determined by gel-filtration chromatography. RESULTS: 264 cirrhotic patients (215 males; median age 54.5 years) were included and compared against a group of 200 healthy individuals (100 males; median age 41.5). Median (25th-75th percentile) total and fractional GGT were higher in cirrhotics, with s-GGT showing the greatest increase [36.6 U/L (21.0-81.4) vs. 5.6 U/L (3.2-10.2), P<0.0001], while the median b-GGT/s-GGT ratio was lower in cirrhotics than in healthy controls [0.06 (0.04-0.10)] vs. 0.28 (0.20-0.40), P<0.0001]. The ratio showed higher diagnostic accuracy (ROC-AUC, 95% CI: 0.951, 0.927-0.969) then either s-GGT (0.924, 0.897-0.947; P<0.05) or total GGT (0.900, 0.869-0.925; P<0.001). The diagnostic accuracy of the ratio was maintained (0.940, 0.907-0.963) in cirrhotic patients (n=113) with total GGT values within the reference range. The s-GGT fraction consisted of two components, with one (s2-GGT) showing a significant positive correlation with serum aspartate aminotransferases, alanine aminotransferase, lactate dehydrogenases (LDH), alkaline phosphatases and bilirubin, and negative with albumin. The b-GGT fraction showed a positive correlation with albumin, fibrinogen, and platelet counts, and negative with international normalized ratio, bilirubin and LDH. CONCLUSIONS: The ratio performs as a sensitive biomarker of the liver parenchymal rearrangement, irrespective of aetiology of cirrhosis and presence of hepatocellular carcinoma, even in patients with total GGT values within the reference range.

Developmental variations of plasma gamma-glutamyltransferase fractions in humans and in laboratory mammalians.

Plasma samples from human cord blood, and fetuses, newborns, and adults of different mammalians species were analyzed by gel-filtration chromatography, to ascertain whether gamma-glutamyltransferase (GGT) fractions reflect liver maturation. Human cord blood plasma showed higher b-, m-, and s-GGT fraction as compared to adult women. In rat and mouse fetuses and in newborns, b-GGT was the most abundant fraction. As in adult humans, in adult rats, mice, rabbits, sheep, and mini pigs, f-GGT was the most abundant fraction. GGT fractions are a common feature of all mammalian species tested. Their pattern changes seem to reflect liver postnatal maturation, function.

Accuracy of b-GGT fraction for the diagnosis of non-alcoholic fatty liver disease.

BACKGROUND: Serum gamma-glutamyltransferase (GGT) activity is a sensitive but non-specific marker of non-alcoholic fatty liver disease (NAFLD). Recently, four GGT fractions (big-, medium-, small-, free-GGT) were described in humans. AIM: We aimed to investigate whether a specific GGT fraction pattern is associated with NAFLD. METHODS: Gamma-glutamyltransferase fractions were determined in patients with NAFLD (n = 90), and compared with those in control subjects (n = 70), and chronic hepatitis C (CHC, n = 45) age and gender matched. RESULTS: Total GGT was elevated in NAFLD as compared to controls (median, 25°-75° percentile: 39.4, 20.0-82.0 U/L vs. 18.4, 13.2-24.9 U/L respectively, P < 0.001). All fractions were higher in NAFLD than in controls (P < 0.001). The b-GGT showed the highest diagnostic accuracy for NAFLD diagnosis [area under ROC curve (ROC-AUC): 0.85; cut-off 2.6 U/L, sensitivity 74%, specificity 81%]. Also subjects with CHC showed increased GGT (41.5, 21.9-84.5 U/L, P < 0.001 vs. controls, P = n.s. vs. NAFLD), as well as m-, s-, and f-GGT, while b-GGT did not show any significant increase (P = n.s. vs. HS, P < 0.001 vs. NAFLD). In subjects with CHC, s-GGT showed the best diagnostic value (ROC-AUC: 0.853; cut-off 14.1 U/L, sensitivity 73%, specificity 90%). Serum GGT did not show any value in the differential diagnosis between NAFLD and CHC (ROC-AUC 0.507, P = n.s.), while b-GGT/s-GGT ratio showed the highest diagnostic accuracy for distinguishing NAFLD and CHC (ROC-AUC: 0.93; cut-off value 0.16, sensitivity 82%, specificity 90%). CONCLUSIONS: b-GGT increases in NAFLD, but not in CHC. GGT fraction analysis might help in improving the sensitivity and specificity of the diagnosis of NAFLD and other liver dysfunctions.

Serum gamma-glutamyltransferase fractions in myotonic dystrophy type I: differences with healthy subjects and patients with liver disease.

OBJECTIVES: Elevation of serum gamma-glutamyltransferase (GGT), in absence of a clinically significant liver damage, is often found in Myotonic Dystrophy type-1 (DM1). In this study we investigated if a specific GGT fraction pattern is present in DM1. DESIGNS AND METHODS: We compared total and fractional GGT values (b-, m-, s-, f-GGT) among patients with DM1 or liver disease (LD) and healthy subjects (HS). RESULTS: The increase of GGT in DM1 and LD, vs HS, was mainly due to s-GGT (median: 32.7; 66.7; and 7.9 U/L, respectively), and b-GGT (8.5; 18.9; and 2.1 U/L). The subset of DM1 patients matched with HS with corresponding serum GGT showed higher b-GGT (6.0 vs 4.2 U/L). CONCLUSIONS: DM1 patients with normal total GGT values showed an alteration of the production and release in the blood of GGT fractions. Since increased s-GGT is also found in LD, a sub-clinical liver damage likely occurs in DM1 subjects apparently free of liver disease.

Cardiovascular risk factors and gamma-glutamyltransferase fractions in healthy individuals.

BACKGROUND: Serum gamma-glutamyltransferase activity (GGT), even when within its normal reference range, catalyzes low density lipoprotein oxidation in vitro and predicts cardiovascular events. Of the four GGT fractions (b-GGT, m-GGT, s-GGT, and f-GGT) recently identified in blood, only b-GGT is found within atherosclerotic plaques. Our goal was to identify the determinants of the GGT fractions (b-, m-, s-, and f-GGT) and their association with established cardiovascular risk factors in healthy subjects. METHODS: Multiple linear regression analysis was applied to estimate the association of fractional GGT with gender, age, body mass index, arterial pressure (AP), plasma glucose, alanine aminotransferase (ALT), high and low density lipoproteins (LDL-C) cholesterol (HDL-C), triglycerides (TG) and C-reactive protein (CRP) in 200 healthy subjects. RESULTS: All GGT fractions were associated with ALT; b-GGT with AP, TG, and CRP; m-GGT with LDL-C, TG and CRP; s-GGT with TG and CRP, and f-GGT only with LDL-C, whereas gender was associated with s-GGT and f-GGT only. CONCLUSIONS: In healthy individuals, cardiovascular risk factors are associated with high molecular weight GGT fractions, namely with b-GGT, the only form present within the plaque. This finding adds to the present knowledge concerning the relevance of GGT, within its reference range, for atherosclerosis-related events.

Nuclear translocation of glutathione transferase omega is a progression marker in Barrett's esophagus.

Barrett's esophagus (BE) represents a major risk factor for esophageal adenocarcinoma (AC). For this reason, patients with BE are subjected to a systematic endoscopic surveillance to detect initial evolution towards non-invasive neoplasia (NiN) and cancer, that eventually occurs only in a small fraction of BE patients. This study was aimed to investigate the possible role of glutathione-S-transferase-omega 1 (GSTO1), a recently discovered member of the glutathione-S-transferase family, as a progression marker in the Barrett's disease in order to improve the diagnosis of NiN in BE and to understand the mechanisms of the progression from BE to AC. We investigated the expression and subcellular localization of GSTO1 in biopsies from patients with BE and in human cancer cell lines subjected to heath shock treatment. A selective nuclear localisation of GSTO1 was found in 16/16 biopsies with low- or high-grade NiN, while it appeared in only 4/22 BE biopsies without signs of NiN (P<0.0001). Among biopsies of BE without NiN, diffuse (nuclear and cytoplasmic) staining was found in 5/22 cases, while selective cytoplasmic localisation was found in 13/22. The 6 cases with indefinite grade of NiN were equally divided between nuclear, cytoplasmic and diffuse staining (2 each, respectively). Experiments in vitro showed that in human HeLa cancer cells, GSTO1 translocates into the nucleus as a consequence of heath shock. These findings suggested that the nuclear translocation of glutathione-S-transferase-omega 1 could be involved in the stress response of human cells playing a role in the cancer progression of Barrett's esophagus. Its immunohistochemical detection could represent a useful tool in the grading of Barrett's disease.