[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stromal cells (MSCs) modulate the immune response and represent a potential treatment for inflammatory and autoimmune diseases. We hypothesized that this feature could be potentiated by co-administering anti-inflammatory cytokines. In this article, we asked whether engineering of Wharton Jelly–derived human MSCs (WJ-hMSCs) to express an anti-inflammatory cytokine increases cell immunomodulatory properties without altering their native features.
[Show abstract][Hide abstract] ABSTRACT: Despite several pathological conditions are associated with free light chains (FLC) deposition in human tissues, only few cases of human diseases caused by the specific binding activity of monoclonal FLC are described. A 65-year old male patient, with highly abnormal functional coagulation tests and undetectable functional fibrinogen was admitted to the Hematological Clinic of the University Hospital of Pisa. The same tests were within the reference intervals one year before. After excluding a number of causes for abnormal coagulation tests, we focused on potential causes of acquired dysfibrinogenemia. Due to the presence of abnormal values of FLC, we performed an immunofixation: while serum did not show any detectable monoclonal band, the immunofixation of a plasma sample revealed the presence of monoclonal FLC of kappa type co-migrating with fibrinogen. The serum kappa FLC concentrations were much lower than plasma levels, suggesting that the majority of these FLC were bound to fibrinogen, remaining associated to fibrin after clotting. Bone marrow biopsy showed 4% monoclonal plasma cells producing kappa light chains. The patient was diagnosed as affected by a FLC MGUS. After two courses of dexamethasone, the plasma concentration of kappa FLC decreased substantially and most of the coagulation tests normalized. The nature of the interaction between fibrinogen and kappa FLC is currently under investigation to elucidate the mechanism able to inhibit fibrinogen polymerization.
No preview · Article · Jan 2015 · Biochimica clinica
[Show abstract][Hide abstract] ABSTRACT: Objective
The atherosclerotic plaque that is vulnerable to rupture and to superimposed thrombosis is mainly represented by a thin-cap fibroatheroma with or without ulceration/thrombosis and inflammatory infiltrates. Total serum gamma-glutamyltransferase (GGT) activity is an independent predictor for cardiovascular events. Four GGT fractions have been identified in plasma and only one of them (b-GGT) in atherosclerotic plaques, but the possible role of GGT in plaque pathophysiology has not been assessed yet. We investigated the relationships between plaque b-GGT activity and the histological features of plaque vulnerability.
Methods and Results
Plaque GGT activity was investigated in 65 patients undergoing carotid endarterectomy; plaques were histologically characterized and immunostained for GGT. Intra-plaque total and fractional GGT activity was determined by a cost-effective test of molecular size exclusion chromatography, and compared with histological markers of plaque vulnerability. Plaque cholesterol content was also measured by chromatography.
b-GGT was the only fraction detected within the atherosclerotic plaques and intra-plaque b-GGT activity correlated to plaque cholesterol content (r = 0.667, P < 0.0001), plasma b-GGT and f-GGT fractions (r = 0.249; r = 0.298, both P < 0.05). Higher b-GGT activity was found in thin-cap fibroatheromas and it was associated to histological markers of vulnerable plaques, i.e. larger necrotic areas, greater macrophage infiltration and higher cholesterol content (P<0.05).
intra-plaque b-GGT activity correlates with the histological markers of vulnerable plaque and with plasma b-GGT in human carotid atherosclerosis; these data support the possible role of b-GGT in clinically significant atherosclerotic disease.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Four 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 behavior of fractional GGT in cirrhotic patients evaluated for liver transplantation.
This was a single-center, cross-sectional study; GGT fractions were determined by gel-filtration chromatography.
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 (25(th) -75(th) 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 AST, ALT, LDH, ALP and bilirubin, and negative with albumin. The b-GGT fraction showed a positive correlation with albumin, fibrinogen, and platelet counts, and negative with INR, bilirubin and LDH.
The ratio performs as a sensitive biomarker of the liver parenchymal rearrangement, irrespective of etiology of cirrhosis and presence of hepatocellular carcinoma, even in patients with total GGT values within the reference range. This article is protected by copyright. All rights reserved.
No preview · Article · Jan 2014 · Liver international: official journal of the International Association for the Study of the Liver
[Show abstract][Hide abstract] ABSTRACT: Despite the increasing number of papers on decellularised scaffolds, there is little consensus on the optimum method of decellularising biological tissue such that the micro-architecture and protein content of the matrix are conserved as far as possible. Focusing on the liver, the aim was therefore to develop a method for the production of well-characterised and reproducible matrices which best preserves the structure and composition of the native extra cellular matrix (ECM). Given the importance of matrix stiffness in regulating cell response, the mechanical properties of the decellularised tissue were also considered. The testing and analysis framework is based on the characterisation of decellularised and untreated samples in the same reproducible initial state (i.e. the equilibrium swollen state). Decellularised ECMs (dECMs) were characterised using biochemical, histological, mechanical and structural analyses to identify the best procedure which ensures complete cell removal while preserving most of the native ECM structure and composition. Using this method, sterile decellularised porcine ECMs with highly conserved intra-lobular micro-structure and protein content were obtained in a consistent and reproducible manner using the equilibrium swollen state of tissue or matrix as a reference. A significant reduction in the compressive elastic modulus was observed for liver dECM with respect to native tissue, suggesting a re-examination of design parameters for ECM-mimicking scaffolds for engineering tissues in-vitro.
No preview · Article · Oct 2013 · Acta biomaterialia
[Show abstract][Hide abstract] ABSTRACT: Methods for the determination of warfarin and warfarin alcohols in plasma and oral fluid were developed as a first step towards a minimally invasive monitoring of warfarin therapy and to clarify the role of warfarin alcohols in the anticoagulation process.
Warfarin is the most common anticoagulant drug prescribed for the treatment of many diseases such as atrial fibrillation and pulmonary embolism. It is metabolized by the cytochrome P450 to inactive hydroxylated metabolites (major pathway) and by ketone reductases to warfarin alcohols, which show a little anticoagulant activity. The large number of factors that may interact with this therapy (diet, comorbidities, other drugs, etc.) makes it relatively easy to go out of the optimal range, so that patients need to be monitored over long periods of time. The standardized evaluation of the coagulation time (international normalized ratio, INR), which of course requires blood collection, is the primary assay used in monitoring warfarin therapy. This test is performed on a daily basis at the onset of therapy, then once every 2-3 weeks when stable coagulation levels are achieved. However, there is a subset of patients who almost never reach stability and need more frequent controls, with high social and economic costs.
The determination of warfarin and warfarin alcohols in oral fluid samples could offer an alternative approach to INR assay, because the oral fluid concentration of warfarin is expected to mirror the concentration of the unbound warfarin in plasma (i.e. the pharmacologically active fraction, about 1%) and could anticipate the INR variations, thus allowing a more effective prevention of adverse events.
In this study oral fluid and plasma samples were acidified with H2SO4 (0.5 M) and then extracted with a 1:5 dichloromethane/hexane mixture for the determination of the total content. The unbound plasmatic fraction was obtained by ultrafiltration of the sample at a molecular weight cut-off of 3 KDa. HPLC separation was carried out in isocratic conditions at 25 °C on a C-18 reversed-phase column with a mobile phase consisting of a 70% buffer phosphate 25 mM at pH = 7 and 30% methanol at a flow rate of 0.7 mL/min. Fluorescence detection was performed at 390 nm (excitation wavelength 310 nm). Neither method showed any detectable interference or matrix effect. LODs for warfarin and warfarin alcohols were 0.2 and 0.1 ng/mL respectively, whereas recoveries were 85 and 70% for the extracted samples, 70 and 90% for the ultracentrifuged samples. The intra- and inter-day precisions were <10% (RSD) for all methods.
The correlations between oral fluid and plasmatic (total and unbound) levels of warfarin and warfarin alcohols, and between these concentrations and INR were evaluated in a longitudinal study involving 10 patients.
[Show abstract][Hide abstract] ABSTRACT: Methods for the determination of warfarin and warfarin alcohols in oral fluid and plasma were developed in order to establish the best oral fluid sampling that provide the highest correlation between the oral fluid and unbound plasmatic concentration of warfarin and warfarin alcohols.
Warfarin is essential and not replaceable in a large number of clinical conditions. It is metabolized by the cytochrome P450 to inactive hydroxylated metabolites (major pathway) and by ketone reductases to warfarin alcohols, which show a little anticoagulant activity. The large number of factors (diet, comorbidities, other drugs, etc.) that may interact with the therapy due to the narrow therapeutic range of the drug, entails constantly monitoring the patient by means of continuous and frequent blood analysis, even for long periods of time, to evaluate the coagulation time expressed as International Normalized Ratio (INR).
Clearly new alternative methods to blood tests are needed that would be less invasive, simple to use, implementable in low cost devices, and, if possible, allowing self-monitoring. However, if oral fluid is to be used for anticoagulant monitoring, the oral fluid/plasma ratio should be constant over a wide plasma concentration range and not be influenced by oral fluid sampling method (stimulated and non-stimulated). For acidic drugs (such as warfarin, pKa = 5.15 ± 0.04 at T = 25°C) the oral fluid/plasma ratio is largely dependent of variations of the oral fluid pH, and an optimization of stimulated sampling is required. In this case the pH values remains within a relatively narrow range and the oral fluid/plasma ratio should be expected to be more constant than in non-stimulated oral fluid.
In this work, oral fluid samples were acidified with H2SO4 (0.5 M) and then extracted with a 1:5 dichloromethane/hexane mixture; whereas, the unbound plasmatic fraction was obtained by ultrafiltration of the plasma sample at a molecular weight cut-off of 3 KDa. The chromatographic separation was carried out in isocratic conditions at 25 °C on a C-18 reversed-phase column with a 70% buffer phosphate 25 mM at pH = 7 and 30% methanol mobile phase at a flow rate of 0.7 mL/min. Fluorescence detection was performed at 390 nm (excitation wavelength 310 nm). Methods were not affected by interferences or matrix effect. LODs for warfarin and warfarin alcohols were 0.2 and 0.1 ng/mL respectively, whereas recoveries were 85 and 70% for the extracted samples, 70 and 90% for the ultracentrifuged samples. The intra- and inter-day precisions were <10% (RSD) for all methods.
A cross-sectional study was performed involving 10 patients undergoing warfarin therapy in order to identify the correlations between salivary flow rate, pH and concentration of warfarin and warfarin alcohols. These results confirmed the importance of pH in regulating the drug transfer from plasma and indicate that oral fluid may be a clinical tool for therapy monitoring.
[Show abstract][Hide abstract] ABSTRACT: An HPLC method with spectrofluorimetric detection is presented for the determination of warfarin (WAR) and some of its metabolites (warfarin alcohols, WAROHs) in saliva and human plasma (total content and fraction not bound to albumin). The aim of research is to develop a minimally invasive monitoring technique for this oral anticoagulant and clarify the role of WAROHs in the anticoagulation process. Saliva and total plasma samples were acidified with H2SO4 (0.5 M) and then extracted with a 1:5 dichloromethane/hexane mixture. The unbound plasmatic fraction was ultrafiltrated at a molecular weight cut-off of 3 KDa. The chromatographic separation was carried out in isocratic conditions at 25 °C on a C-18 reversed-phase column Poroshell 120 EC-C18 (Agilent, 100 × 4.6 mm, 2.7 µm) with a 70% buffer phosphate 25 mM at pH = 7 and 30% methanol mobile phase at a flow rate of 0.7 mL/min. The excitation and emission wavelengths for the spectrofluorimetric measurements were 310 and 390 nm respectively. Methods were not affected by interferences or matrix effect. In standard working solutions, the areas of WAR and WAROH peaks linearly increased with concentration in the range 1 – 2000 ng/mL. LODs for WAR and WAROHs were 0.2 and 0.1 ng/mL respectively, whereas recoveries were 85 and 70% for the extracted samples, 70 and 90% for the ultracentrifuged samples. The intra- and inter-day precisions were <10% (RSD) for all methods. A cross-sectional study was performed in patients undergoing warfarin therapy to identify possible correlations between salivary flow rate, pH and concentration of WAR and WAROH. A later longitudinal study investigated the correlations between salivary and plasmatic (total and unbound) levels of WAR and WAROH and between these concentrations and INR. Results highlighted the key role played by salivary pH.
[Show abstract][Hide abstract] ABSTRACT: Intestinal acute GVHD (I-aGVHD) is a life-threatening complication after allografting. Non-invasive bed-side procedures to evaluate extension and treatment response are still lacking. We hypothesized that, during I-aGVHD, contrast-enhanced ultrasound sonography (CEUS) could detect microcirculation changes (MVC) of the bowel wall (BW) and help to monitor treatment response. We prospectively employed CEUS in 83 consecutive patients. Of these, 14 patients with biopsy-proven intestinal GVHD (I-GVHD) were defined as the study group, whereas 16 patients with biopsy-proven stomach GVHD (U-GVHD) without intestinal symptoms, 6 normal volunteers and 4 patients with neutropenic enterocolitis were defined as the control group. All patients were evaluated with both standard ultrasonography (US) and CEUS at the onset of intestinal symptoms, during clinical follow-up and at flare of symptoms. Standard US revealed BW thickening of multiple intestinal segments, useful to determine the extension of GVHD. CEUS showed MVC, which correlated with GVHD activity, treatment response, and predicted flare of intestinal symptoms. US and CEUS findings were superimposable at diagnosis and in remission. CEUS was, however, more sensitive and specific to identify subclinical activity in patients with clinical relevant improvement. These findings were not observed in the control groups. CEUS is a non-invasive, easily reproducible bed-side tool useful to monitor I-aGVHD.Bone Marrow Transplantation advance online publication, 13 May 2013; doi:10.1038/bmt.2013.65.
Full-text · Article · May 2013 · Bone marrow transplantation
[Show abstract][Hide abstract] ABSTRACT: The GGT enzyme, considered for years only as a marker of liver disease and alcohol abuse, has now revealed a risk of death for many causes. Through a molecular exclusion chromatography on FPLC system (Fast Protein Liquid Chromatography), it is possible to discriminate four fractions of GGT, defined according to the molecular weight: big-GGT, medium-GGT, small-GGT and free-GGT. The objective was to study the preventing meaning of GGT fractions for asbestos-related diseases. This study was conducted on 129 workers previously exposed to asbestos, 22 patients affected by Malignant Pleural Mesothelioma and 107 healthy workers. Our data demonstrated a statistical significant correlation between the fraction free-GGT with the presence of MPM, suggesting a possible role for this molecule as a biomarker for MPM diagnosis. However, being a preliminary study, further studies are warranted to confirm our results.
No preview · Article · Feb 2013 · Giornale italiano di medicina del lavoro ed ergonomia
[Show abstract][Hide abstract] ABSTRACT: Background:
We assessed GGT fractions correlates and their reference values in the Offspring Cohort of the Framingham Heart Study.
Correlates of GGT fractions were assessed by multivariable regression analysis in 3203 individuals [47% men, mean age (SD): 59 (10) years]. GGT fractions reference values were established by empirical quantile analysis in a reference group of 432 healthy subjects [45% men, 57 (10) years].
Fractional GGT levels were higher in men than in women (P<0.0001). In both sexes, fractions were associated with: triglycerides were associated with b-GGT, alcohol consumption with m-, s- and f-GGT. C-reactive protein with m- and s-GGT, while plasminogen activator inhibitor-1 with b- and f-GGT. Body mass index, blood pressure, glucose and triglycerides correlated with b- and f-GGT. In comparison with the reference group [b-GGT/s-GGT median (Q1-Q3): 0.51 (0.35-0.79)U/L], subjects affected by cardiovascular disease or diabetes showed no change of b/s ratio [0.52 (0.34-0.79)U/L, 0.57 (0.40-0.83)U/L, respectively]. The b/s ratio was higher in presence of metabolic syndrome [0.61 (0.42-0.87)U/L, P<0.0001], while lower in heavy alcohol consumers [0.41 (0.28-0.64)U/L, P<0.0001].
Metabolic and cardiovascular risk markers are important correlates of GGT fractions, in particular of b-GGT.
No preview · Article · Dec 2012 · Clinica chimica acta; international journal of clinical chemistry
[Show abstract][Hide abstract] ABSTRACT: Background
Warfarin is an essential and not replaceable drug in a large number of clinical conditions. Therapeutic Drug Monitoring (TDM) is necessary because several factors may interact with the therapy due to the narrow therapeutic range of the drug. Saliva is suitable for TDM because the salivary concentration only mirrors the free plasma concentration.
Materials and methods
A method was developed to determine warfarin in saliva by HPLC and fluorimetric detection. The chromatographic separation was performed at 25°C on a C-18 reversed-phase column, mobile phase 65% phosphate buffer (pH = 7.0) and 35% methanol, flow rate 0.7 mL/min, injection volume 25 μL, excitation wavelength 310 nm, emission wavelength 400 nm.
The method was free from interference and matrix effect, linear in the range 0.2 – 100 ng/mL, detection limit of 0.2 ng/mL, and coefficient of variation of <3% and <5% for intra-day and inter-day measurements. The average salivary concentration of 50 patients was 2.5 ± 1.6 ng/mL (range 0.8-7.6 ng/mL). Dosage was not correlated to INR (r = -0.03, p = 0.85) but positively correlated to warfarin concentration (r = 0.39, p = 0.006). A correlation between warfarin concentration and INR was found in samples with pH ≥ 7.2 (r = 0.84, p = 0.004) confirming the key-role of the salivary pH.
The method allows to measure warfarin concentration in saliva and to analyze correlations with INR and other parameters.
[Show abstract][Hide abstract] ABSTRACT: Warfarin is the most used anticoagulant for the prevention of thrombosis
and thromboembolism. It is a weakly acid drug (pKa = 5.19, at 25 °C),
highly bound to plasma albumin (> 99%). Only the unbound fraction can
carry out a therapeutic action and cross the biological membranes (e.g.
salivary glands). The drug is metabolized by the CYP450 system to inactive
hydroxylated metabolites (major pathway), and by ketone reductases to
warfarin alcohols with a little anticoagulant activity (1, 2).
In this paper, two analytical procedures are presented for the determination
of bound and unbound fraction of both warfarin and its metabolites in
human plasma. HPLC separation was carried out in isocratic conditions at
25 °C on a C-18 reversed-phase column with a 85% buffer phosphate 25
mM at pH = 7 and 15% acetonitrile mobile phase at a flow rate of 1.2
mL/min. Spectrophotometric detection was performed at 310 nm, and
spectrofluorimetric one at 400 nm (excitation wavelength of 310 nm). In
both methods, no interference and effect matrix were observed. Analytes
recoveries were between 95 and 105% for both total and unbound fractions.
The intraday and interday precision were <10% (RSD). The methods were
successfully applied to pooled plasma samples obtained from patients
undergoing warfarin therapy
[Show abstract][Hide abstract] ABSTRACT: Background:
Four fractions of gamma-glutamyltransferase (GGT) with different molecular weight (b-, m-, s-, and f-GGT) are present in human plasma. Differential GGT fraction pattern is found in non-alcoholic liver disease (NAFLD) and chronic viral hepatitis, characterized by normal or decreased b-GGT/s-GGT (b/s) ratio, respectively.
Chromatographic fractional GGT analysis was performed on plasma obtained from 51 subjects: 27 alcoholics (mean (SD), age 45 (9) years; 23 males; 14 positive for viral infection), 24 abstinents from at least 1 month (43 (12) years; 20 males; 6 positive for viral infection). Twenty-seven blood donors matched for age and gender (44 (9) years; 23 males) were selected as controls.
All fractions were significantly increased in alcoholics (P<0.001), s-GGT showing the largest increase, while only m-GGT and s-GGT were elevated in abstainers (P<0.01), in comparison with controls. The b/s ratio was significantly lower in both alcoholics and abstainers than in controls (median (25th-75th perc.): 0.10 (0.07-0.15), 0.16 (0.10-0.24), 0.35 (0.29-0.53), respectively, P<0.001). Viral infection did not significantly changes absolute values of individual GGT fractions in alcoholics, but the b/s ratio was significantly lower in virus positive than in virus negative subjects (0.08 (0.05-0.12), 0.14 (0.09-0.20), respectively, P<0.01).
The fraction pattern analysis might increase the specificity of GGT as biomarker of alcohol abuse, especially concerning the differential diagnosis between alcoholism and NAFLD, a common cause of elevated GGT level in the general population.
No preview · Article · Jun 2012 · Drug and alcohol dependence
[Show abstract][Hide abstract] ABSTRACT: Cystic fibrosis (CF) is an autosomal recessive disorder characterized by a chronic neutrophilic airways inflammation, increasing levels of oxidative stress and reduced levels of antioxidants such as glutathione (GSH). Gamma-glutamyltransferase (GGT), an enzyme induced by oxidative stress and involved in the catabolism of GSH and its derivatives, is increased in the airways of CF patients with inflammation, but the possible implications of its increase have not yet been investigated in detail.
The present study was aimed to evaluate the origin and the biochemical characteristics of the GGT detectable in CF sputum. We found GGT activity both in neutrophils and in the fluid, the latter significantly correlating with myeloperoxidase expression. In neutrophils, GGT was associated with intracellular granules. In the fluid, gel-filtration chromatography showed the presence of two distinct GGT fractions, the first corresponding to the human plasma b-GGT fraction, the other to the free enzyme. The same fractions were also observed in the supernatant of ionomycin and fMLP-activated neutrophils. Western blot analysis confirmed the presence of a single band of GGT immunoreactive peptide in the CF sputum samples and in isolated neutrophils.
In conclusion, our data indicate that neutrophils are able to transport and release GGT, thus increasing GGT activity in CF sputum. The prompt release of GGT may have consequences on all GGT substrates, including major inflammatory mediators such as S-nitrosoglutathione and leukotrienes, and could participate in early modulation of inflammatory response.