Enrico Di Stasio

Thrombotic risk factors in patients with liver cirrhosis: correlation with MELD scoring system and portal vein thrombosis development.

BACKGROUND/AIMS Prognostic scores currently used in cirrhotic patients do not include thrombotic risk factors (TRFs). Predicting factors of portal vein thrombosis (PVT) development are still unknown. We wanted to describe TRFs as a function of liver disease severity using the MELD score and assess the role of local and systemic TRFs as predictors of PVT development in cirrhotic patients. METHODS One hundred consecutive patients with liver cirrhosis were included in the study. TRFs, D-dimers, MELD score, portal vein patency and flow velocity were evaluated in all subjects at baseline and every 6 months thereafter. Variables able to predict PVT development within 1 year were identified by means of multiple logistic regression. RESULTS The plasma levels of protein C and antithrombin were lower and the concentration of D-dimers was higher in patients with advanced disease. Plasma levels of antithrombin, protein C and protein S resulted significantly lower in PVT group at univariate analysis, but reduced portal vein flow velocity was the only variable independently associated with PVT development. CONCLUSIONS Lower concentrations of natural coagulation inhibitors are frequently detected in patients with liver cirrhosis. A reduced portal flow velocity seems to be the most important predictive variable for PVT development in patients with cirrhosis.

Structure-Function Analysis of Hepatitis C Virus Envelope-CD81 Binding

Hepatitis C virus (HCV) is a major human pathogen causing chronic liver disease. We have recently found that the large extracellular loop (LEL) of human CD81 binds HCV. This finding prompted us to assess the structure-function features of HCV-CD81 interaction by using recombinant E2 protein and a recombinant soluble form of CD81 LEL. We have found that HCV-E2 binds CD81 LEL with a K(d) of 1.8 nM; CD81 can mediate attachment of E2 on hepatocytes; engagement of CD81 mediates internalization of only 30% of CD81 molecules even after 12 h; and the four cysteines of CD81 LEL form two disulfide bridges, the integrity of which is necessary for CD81-HCV interaction. Altogether our data suggest that neutralizing antibodies aimed at interfering with HCV binding to human cells should have an affinity higher than 10(-9) M, that HCV binding to hepatocytes may not entirely depend on CD81, that CD81 is an attachment receptor with poor capacity to mediate virus entry, and that reducing environments do not favor CD81-HCV interaction. These studies provide a better understanding of the CD81-HCV interaction and should thus help to elucidate the viral life cycle and to develop new strategies aimed at interfering with HCV binding to human cells.

No protective effect of calcitriol on β-cell function in recent-onset type 1 diabetes the IMDIAB XIII trial.

OBJECTIVE We investigated whether supplementation of the active form of vitamin D (calcitriol) in recent-onset type 1 diabetes can protect β-cell function evaluated by C-peptide and improve glycemic control assessed by A1C and insulin requirement. RESEARCH DESIGN AND METHODS Thirty-four subjects (aged 11–35 years, median 18 years) with recent-onset type 1 diabetes and high basal C-peptide >0.25 nmol/l were randomized in a double-blind trial to 0.25 μg/day calcitriol or placebo and followed-up for 2 years. RESULTS At 6, 12, and 24 months follow-up, A1C and insulin requirement in the calcitriol group did not differ from the placebo group. C-peptide dropped significantly (P < 0.001) but similarly in both groups, with no significant differences at each time point. CONCLUSIONS At the doses used, calcitriol is ineffective in protecting β-cell function in subjects (including children) with recent-onset type 1 diabetes and high C-peptide at diagnosis.

Characterization of the S-denitrosylating activity of bilirubin.

Bilirubin‐IX‐α (BR) is an endogenous molecule with a strong antioxidant feature due to its ability to scavenge free radicals. In this paper, we demonstrated that BR, at concentrations close to those found within the cell (0.1–2.5 μM), acted as a denitrosylating agent and increased the release of nitric oxide from S‐nitrosoglutathione (GSNO) and S‐nitrosocysteine (SNOC) (2.5 μM). The complexation of BR with saturating concentrations of human serum albumin (HSA, 2.5 μM) did not further increase nitric oxide release from GSNO and SNOC. At concentrations similar to those reached in plasma (5–20 μM), BR denitrosylated S‐nitroso‐HSA (2.5 μM), the main circulating S‐nitrosothiol, and this effect was potentiated by the complexation of BR with saturating HSA (20 μM). Furthermore, the product(s) of the reaction between nitric oxide and BR were identified. Ultraviolet and mass spectrometry analysis revealed that nitric oxide binds to BR forming a N‐nitroso derivative (BR–nitric oxide) with extinction coefficients of 1.393 mM−1cm−1 and 2.254 mM−1cm−1 in methanol and NaOH, respectively. The formation of BR–nitric oxide did not occur only in a reconstituted system, but was confirmed in rat fibroblasts exposed to pro‐oxidant stimuli. These results provided novel insights on the antioxidant characteristic of BR through its interaction with nitric oxide, a gaseous neurotransmitter with a well‐known dual effect, namely neuroprotective under physiological conditions or neurotoxic if produced in excess, and proposed BR–nitric oxide as a new biomarker of oxidative/nitrosative stress.

The effect of shear stress on protein conformation: Physical forces operating on biochemical systems: The case of von Willebrand factor

Macromolecules and cells exposed to blood flow in the circulatory tree experience hydrodynamic forces that affect their structure and function. After introducing the general theory of the effects of shear forces on protein conformation, selected examples are presented in this review for biological macromolecules sensitive to shear stress. In particular, the biochemical effects of shear stress in controlling the von Willebrand Factor (VWF) conformation are extensively described. This protein, together with blood platelets, is the main actor of the early steps of primary haemostasis. Under the effect of shear forces >30 dyn/cm², VWF unfolding occurs and the protein exhibits an extended chain conformation oriented in the general direction of the shear stress field. The stretched VWF conformation favors also a process of self aggregation, responsible for the formation of a spider web network, particularly efficient in the trapping process of flowing platelets. Thus, the effect of shear stress on conformational changes in VWF shows a close structure-function relationship in VWF for platelet adhesion and thrombus formation in arterial circulation, where high shear stress is present. The investigation of biophysical effects of shear forces on VWF conformation contributes to unraveling the molecular interaction mechanisms involved in arterial thrombosis.

Early prediction of response to sorafenib in patients with advanced hepatocellular carcinoma: The role of dynamic contrast enhanced ultrasound

BACKGROUND & AIMS Sorafenib has become the standard first-line treatment for patients with advanced HCC and acts by inducing alterations in tumor vascularity. We wanted to evaluate the feasibility of dynamic CEUS (D-CEUS) as a predictor of early tumor response to sorafenib and to correlate functional parameters with clinical efficacy end points. METHODS Twenty-eight HCC patients treated with sorafenib 400mg bid were prospectively enrolled. CEUS was performed at baseline (T0) and after 15 (T1) and 30 (T2) days of treatment. Tumor vasculature was assessed in a specific harmonic mode associated with a perfusion and quantification software (Q-Lab, Philips). Variations between T1/T2 and T0 were calculated for five D-CEUS functional parameters (peak intensity, PI; time to PI, TP; area under the curve, AUC; slope of wash in, Pw; mean transit time, MTT) and were compared for responders and non-responders. The correlation between D-CEUS parameters, overall survival (OS), and progression-free survival (PFS) was also assessed. A p value <0.05 was considered statistically significant. RESULTS The percentage variation at T1 significantly correlated with response in three D-CEUS parameters (AUC, PI and Pw; p=0.002, <0.001, and 0.003, respectively). A decrease of AUC (p=0.045) and an increased/unchanged value of TP (p=0.029) and MTT (p=0.010) were associated with longer survival. Three D-CEUS parameters (AUC, TP, Pw) were significantly associated with PFS. CONCLUSIONS D-CEUS provides a reliable and early measure of efficacy for anti-angiogenic therapies and could be an excellent tool for selecting patients who will benefit from treatment.

Association Between Osteoprotegerin G1181C and T245G Polymorphisms and Diabetic Charcot Neuroarthropathy: A case-control study

OBJECTIVE Charcot neuroarthropathy is a disabling complication of diabetes. Although its pathogenesis remains unknown, we suppose that genetics may play a relevant role. RESEARCH DESIGN AND METHODS We performed a case-control study with 59 subjects with diabetic Charcot neuroarthropathy (Ch group), 41 with diabetic neuropathy without Charcot neuroarthropathy (ND group), and 103 healthy control subjects (H group) to evaluate the impact of two single nucleotide polymorphisms (SNPs) of the osteoprotegerin gene (G1181C and T245G) on the risk of Charcot neuroarthropathy. RESULTS Regarding the SNPs of G1181C, we found a significant linkage between the G allele and Charcot neuroarthropathy (Ch vs. ND, odds ratio [OR] 2.32 [95% CI 1.3–4.1], P = 0.006; Ch vs. H, 2.10 [1.3–3.3], P = 0.002; and ND vs. H, 0.90 [0.7–1.9], P = 0.452); similarly, we found a linkage with the G allele of T245G (Ch vs. ND, 6.25 [2.2–19.7], P < 0.001; Ch vs. H, 3.56 [1.9–6.7], P = 0.001; and ND vs. H, 0.54 [0.6–5.7], P = 0.304), supporting a protective role for the allele C and T, respectively. For this reason we investigated the frequency of the protective double homozygosis CC + TT (7% in Ch) that was significantly lower in Ch compared with H (0.18 [0.06–0.5], P = 0.002) and with ND (0.17 [0.05–0.58], P = 0.006), whereas there was no difference between H and ND (1.05 [0.43–2.0], P = 0.468). In a multivariate logistic backward regression model, only weight and the lack of CC and TT genotypes were independently associated with the presence of Charcot neuroarthropathy. CONCLUSIONS This is the first study that shows an association between genetic regulation of bone remodeling and Charcot neuroarthropathy.

Role of asymmetric-dimethyl-L-arginine (ADMA) and nitrite/nitrate (NOx) in the pathogenesis of oxidative stress in female subjects with uncomplicated type 1 diabetes mellitus.

AIMS To explore the role of asymmetric-dimethyl-L-arginine (ADMA), an endogenous nitric oxide synthetases (NOS) inhibitor, and nitrite/nitrate (NOx) in the pathogenesis of oxidative stress in early stages of type 1 diabetes mellitus. METHODS We measured in 99 female subjects with uncomplicated type 1 diabetes (duration disease <10 years) and in 44 sex-matched controls (comparable for age, smoking habit, diet and physical activity) plasma levels of NOx, glycosylated haemoglobin (HbA1c), glucose, uric acid, total cholesterol, high density lipoprotein (HDL) cholesterol, triglycerides and serum ADMA. RESULTS Type 1 diabetic subjects have higher levels of glycemia, HbA1c, LDL cholesterol and NOx, but lower ADMA and serum uric acid (UA), compared with the control group; no further differences were found. A significant linear and inverse correlation was found between NOx and ADMA levels (R(2)=0.237, p<0.001). CONCLUSIONS This study suggests a reduced ADMA inhibition of NOS as possible mechanism involved in the pathogenesis of oxidative stress in female subjects with a short duration and uncomplicated type 1 diabetes.

Association Between Osteoprotegerin G1181C and T245G Polymorphisms And Charcot Diabetic Neuroarthropathy: a case – control study

OBJECTIVE Charcot neuroarthropathy is a disabling complication of diabetes. Although its pathogenesis remains unknown, we suppose that genetics may play a relevant role. RESEARCH DESIGN AND METHODS We performed a case-control study with 59 subjects with diabetic Charcot neuroarthropathy (Ch group), 41 with diabetic neuropathy without Charcot neuroarthropathy (ND group), and 103 healthy control subjects (H group) to evaluate the impact of two single nucleotide polymorphisms (SNPs) of the osteoprotegerin gene (G1181C and T245G) on the risk of Charcot neuroarthropathy. RESULTS Regarding the SNPs of G1181C, we found a significant linkage between the G allele and Charcot neuroarthropathy (Ch vs. ND, odds ratio [OR] 2.32 [95% CI 1.3–4.1], P = 0.006; Ch vs. H, 2.10 [1.3–3.3], P = 0.002; and ND vs. H, 0.90 [0.7–1.9], P = 0.452); similarly, we found a linkage with the G allele of T245G (Ch vs. ND, 6.25 [2.2–19.7], P < 0.001; Ch vs. H, 3.56 [1.9–6.7], P = 0.001; and ND vs. H, 0.54 [0.6–5.7], P = 0.304), supporting a protective role for the allele C and T, respectively. For this reason we investigated the frequency of the protective double homozygosis CC + TT (7% in Ch) that was significantly lower in Ch compared with H (0.18 [0.06–0.5], P = 0.002) and with ND (0.17 [0.05–0.58], P = 0.006), whereas there was no difference between H and ND (1.05 [0.43–2.0], P = 0.468). In a multivariate logistic backward regression model, only weight and the lack of CC and TT genotypes were independently associated with the presence of Charcot neuroarthropathy. CONCLUSIONS This is the first study that shows an association between genetic regulation of bone remodeling and Charcot neuroarthropathy.

Fatigue Is Associated With Serum Interleukin-6 Levels and Symptoms of Depression in Patients on Chronic Hemodialysis

CONTEXT Little is known about activated immune-inflammatory pathways and interleukin-6 (IL-6) in the development of fatigue and/or depression in patients with end-stage renal disease on chronic hemodialysis (HD). OBJECTIVES To evaluate the possible correlation between fatigue and serum levels of IL-6 in patients on chronic HD. METHODS One hundred HD patients were assessed for the presence of fatigue using the SF-36 Vitality subscale and were administered the Beck Depression Inventory (BDI), the Hamilton Anxiety Rating Scale (HARS), the Mini-Mental State Examination (MMSE), the activities of daily living (ADL), and the instrumental activities of daily living (IADL). We also calculated the time of recovery after hemodialysis (TIRD) and the number/severity of comorbidities using the Charlson Comorbidity Index (CCI). Laboratory parameters were measured as well as serum IL-6. RESULTS Forty-three patients constituted the fatigued group and 57 the nonfatigued group. Age, CCI, BDI, HARS, and TIRD were significantly higher in fatigued patients than in the nonfatigued patients. Conversely, the scores of ADL, IADL, and MMSE were significantly lower in fatigued than in nonfatigued patients. Serum IL-6 levels (pg/mL) were higher in the fatigued group (5.1 ± 3.4) than in the nonfatigued group (1.6 ± 1.5; P < 0.001); serum albumin and creatinine levels were significantly lower. Twenty-six patients (26%) had no symptoms of depression (BDI score <10), and 74 patients (74%) had symptoms of depression (BDI score >9). Patients with a BDI score >9 were older; had a higher CCI; a lower MMSE; a higher TIRD; lower serum albumin, creatinine, and urea levels; and higher serum IL-6 levels. The correlation analyses showed that the score of the SF-36 Vitality subscale was associated with age, dialytic age, TIRD, ADL, IADL, CCI, BDI, HARS, MMSE, serum urea, creatinine, albumin, and IL-6 levels. On multivariate general linear model analyses, with fatigue as the dependent variable and gender as a second factor, BDI and serum IL-6 levels were independently associated with the score of the SF-36 Vitality subscale. A canonical correlation analysis was performed including in the model fatigue, BDI, and biomarkers; the correlation was 0.679 (R(2) = 0.462). Fatigue, BDI, and IL-6 among biomarkers showed the strongest association with the underlying construct (standardized canonical coefficients = -0.989, 0.015, and 0.852, respectively), thus explaining a correlation of IL-6 with both depression and fatigue. CONCLUSION Fatigue was significantly associated with symptoms of depression and serum IL-6 levels in patients receiving chronic HD.