Advancing Thrombosis Research: A Novel Device for Measuring Clot Permeability.

Thromboembolism, a global leading cause of mortality, needs accurate risk assessment for effective prophylaxis and treatment. Current stratification methods fall short in predicting thrombotic events, emphasizing the need for a deeper understanding of clot properties. Fibrin clot permeability, a crucial parameter in hypercoagulable states, impacts clot structure and resistance to lysis. Current clot permeability measurement limitations propel the need for standardized methods. Prior findings underscore the importance of clot permeability in various thrombotic conditions but call for improvements and more precise, repeatable, and standardized methods. Addressing these challenges, our study presents an upgraded, portable, and cost-effective system for measuring blood clot permeability, which utilizes a pressure-based approach that adheres to Darcy's law. By enhancing precision and sensitivity in discerning clot characteristics, this innovation provides a valuable tool for assessing thrombotic risk and associated pathological conditions. In this paper, the authors present a device that is able to automatically perform the permeability measurements on plasma or fibrinogen in vitro-induced clots on specific holders (filters). The proposed device has been tailored to distinguish clot permeability, with high precision and sensitivity, between healthy subjects and high cardiovascular-risk patients. The precise measure of clot permeability represents an excellent indicator of thrombotic risk, thus allowing the clinician, also on the basis of other anamnestic and laboratory data, to attribute a risk score to the subject. The proposed instrument was characterized by performing permeability measurements in plasma and purified fibrinogen clots derived from 17 Behcet patients and 15 sex- and age-matched controls. As expected, our results clearly indicate a significant difference in plasma clot permeability in Behcet patients with respect to controls (0.0533 ± 0.0199 d vs. 0.0976 ± 0.0160 d, p < 0.001). This difference was confirmed in the patient's vs. control fibrin clots (0.0487 ± 0.0170 d vs. 0.1167 ± 0.0487 d, p < 0.001). In conclusion, our study demonstrates the feasibility, efficacy, portability, and cost-effectiveness of a novel device for measuring clot permeability, allowing healthcare providers to better stratify thrombotic risk and tailor interventions, thereby improving patient outcomes and reducing healthcare costs, which could significantly improve the management of thromboembolic diseases.

Mediterranean Diet and Oxidative Stress: A Relationship with Pain Perception in Endometriosis.

BACKGROUND: Endometriosis is a chronic and inflammatory disease associated with pelvic pain. Dietary changes may be therapeutic for chronic inflammatory processes, reducing visceral input. The aim was to evaluate the role of dietary changes according to the Mediterranean Diet (MD) on pain perception in endometriosis and their relationship with oxidative stress. METHODS: in this prospective study, we included 35 endometriosis women. At baseline (T0) and after 3 (T1) and 6 (T2) months from the start of the diet, we investigated pain intensity with VAS (Visual Analogue Scale, from 0 to 10), vitamin profile, and oxidative stress. RESULTS: we found a significant increase in the diet score (p < 0.001). At T1, patients reduced pain in terms of dyspareunia (p = 0.04), non-menstrual pelvic pain (p = 0.06), dysuria (p = 0.04), and dyschezia (p < 0.001). Dyspareunia (p = 0.002) and dyschezia (p < 0.001) were further significantly reduced also at T2. We observed a significant positive correlation between lipid peroxidation and VAS non-menstrual pelvic pain and dysuria and a significant negative correlation between Oxygen radical absorbance capacity and VAS non-menstrual pain and dyschezia. CONCLUSIONS: our findings show a clear tendency toward a relationship between pain relief in endometriosis and MD. This appears promising to treat endometriosis-related symptoms and could be considered a new effective strategy for chronic pain management in the long term.

Serum Interleukin-36 α as a Candidate Biomarker to Distinguish Behçet's Syndrome and Psoriatic Arthritis.

Behçet's syndrome (BS) is a rare systemic vasculitis characterized by different clinical manifestations. As no specific laboratory tests exist, the diagnosis relies on clinical criteria, and the differential diagnosis with other inflammatory diseases can be challenging. Indeed, in a relatively small proportion of patients, BS symptoms include only mucocutaneous, articular, gastrointestinal, and non-typical ocular manifestations, which are frequently found also in psoriatic arthritis (PsA). We investigate the ability of serum interleukin (IL)-36α-a pro-inflammatory cytokine involved in cutaneous and articular inflammatory diseases-to differentiate BS from PsA. A cross-sectional study was performed on 90 patients with BS, 80 with PsA and 80 healthy controls. Significantly lower IL-36α concentrations were found in patients with BS as compared to PsA, although in both groups IL-36α was significantly increased compared to healthy controls. An empirical cut-off of 420.6 pg/mL displayed a specificity of 0.93, with a sensitivity of 0.70 (AUC 0.82) in discriminating PsA from BS. This cut-off displayed a good diagnostic performance also in BS patients lacking highly specific BS manifestations. Our results indicate that IL-36α might be involved in the pathogenesis of both BS and PsA, and might be a candidate biomarker to support the differential diagnosis of BS.

Chemical Modification of Auranofin Yields a New Family of Anticancer Drug Candidates: The Gold(I) Phosphite Analogues.

A panel of four novel gold(I) complexes, inspired by the clinically established gold drug auranofin (1-Thio-ß-D-glucopyranosatotriethylphosphine gold-2,3,4,6-tetraacetate), was prepared and characterized. All these compounds feature the replacement of the triethylphosphine ligand of the parent compound auranofin with a trimethylphosphite ligand. The linear coordination around the gold(I) center is completed by Cl-, Br-, I- or by the thioglucose tetraacetate ligand (SAtg). The in-solution behavior of these gold compounds as well as their interactions with some representative model proteins were comparatively analyzed through 31PNMR and ESI-MS measurements. Notably, all panel compounds turned out to be stable in aqueous media, but significant differences with respect to auranofin were disclosed in their interactions with a few leading proteins. In addition, the cytotoxic effects produced by the panel compounds toward A2780, A2780R and SKOV-3 ovarian cancer cells were quantitated and found to be in the low micromolar range, since the IC50 of all compounds was found to be between 1 µM and 10 µM. Notably, these novel gold complexes showed large and similar inhibition capabilities towards the key enzyme thioredoxin reductase, again comparable to those of auranofin. The implications of these results for the discovery of new and effective gold-based anticancer agents are discussed.

Erythrocyte oxidative stress and thrombosis.

Thrombosis is a common disorder with a relevant burden of morbidity and mortality worldwide, particularly among elderly patients. Growing evidence demonstrated a direct role of oxidative stress in thrombosis, with various cell types contributing to this process. Among them, erythrocytes produce high quantities of intracellular reactive oxygen species (ROS) by NADPH oxidase activation and haemoglobin autoxidation. Concomitantly, extracellular ROS released by other cells in the blood flow can be uptaken and accumulate within erythrocytes. This oxidative milieu can alter erythrocyte membrane structure, leading to an impaired erythrocyte function, and promoting erythrocytes lysis, binding to endothelial cells, activation of platelet and of coagulation factors, phosphatidylserine exposure and release of microvesicles. Moreover, these abnormal erythrocytes are able to adhere to the vessel wall, contributing to thrombin generation within the thrombus. This process results in accelerated haemolysis and in a hypercoagulable state, in which structurally impaired erythrocytes contribute to increase thrombus size, to reduce its permeability and susceptibility to lysis. However, the wide plethora of mechanisms by which oxidised erythrocytes contribute to thrombosis is not completely elucidated. This review discusses the main biochemical aspects linking erythrocytes, oxidative stress and thrombosis, addressing their potential implication for clinical and therapeutic management.

A unique circulating miRNA profile highlights thrombo-inflammation in Behçet's syndrome.

OBJECTIVES: Behçet's syndrome (BS) is a rare systemic vasculitis often complicated by thrombotic events. Given the lack of validated biomarkers, BS diagnosis relies on clinical criteria.In search of novel biomarkers for BS diagnosis, we determined the profile of plasmatic circulating microRNAs (ci-miRNAs) in patients with BS compared with healthy controls (HCs). METHODS: ci-miRNA profile was evaluated by microarray in a screening cohort (16 patients with BS and 18 HCs) and then validated by poly(T) adaptor PCR (PTA-PCR) in a validation cohort (30 patients with BS and 30 HCs). Two disease control groups (30 patients with systemic lupus erythematosus (SLE) and 30 patients with giant cell arteritis (GCA) were also analysed. RESULTS: From the microarray screening, 29 deregulated (differentially expressed (DE)) human ci-miRNAs emerged. A hierarchical cluster analysis indicated that DE ci-miRNAs clearly segregated patients from controls, independently of clinical features. PTA-PCR analysis on the validation cohort confirmed the deregulation of miR-224-5p, miR-206 and miR-653-5p. The combined receiver operating characteristic (ROC) curve analyses showed that such ci-miRNAs discriminate BS from HCs (and BS with active vs inactive disease), as well as BS from patients with SLE and GCA.The functional annotation analyses (FAAs) showed that the most enriched pathways affected by DE ci-miRNAs (ie, cell-matrix interaction, oxidative stress and blood coagulation) are related to thrombo-inflammatory mechanisms. Accordingly, the expression of the three ci-miRNAs from the validation cohort significantly correlated with leucocyte reactive oxygen species production and plasma lipid peroxidation. CONCLUSIONS: The ci-miRNA profile identified in this study may represent a novel, poorly invasive BS biomarker, while suggesting an epigenetic control of BS-related thrombo-inflammation.

Neutrophil-mediated mechanisms of damage and in-vitro protective effect of colchicine in non-vascular Behçet's syndrome.

Behçet's syndrome (BS) is a systemic vasculitis with several clinical manifestations. Neutrophil hyperactivation mediates vascular BS pathogenesis, via both a massive reactive oxygen species (ROS) production and neutrophil extracellular traps (NETs) release. Here, we investigated neutrophil-mediated mechanisms of damage in non-vascular BS manifestations and explored the in-vitro effects of colchicine in counteracting these mechanisms. NETs and intracellular ROS production was assessed in blood samples from 80 BS patients (46 with active non-vascular BS, 34 with inactive disease) and 80 healthy controls. Moreover, isolated neutrophils were incubated for 1 h with an oxidating agent [2,2'-azobis (2-amidinopropane) dihydrochloride; 250 nM] and the ability of pure colchicine pretreatment (100 ng/ml) to counteract oxidation-induced damage was assessed. Patients with active non-vascular BS showed remarkably increased NET levels [21.2, interquartile range (IQR) = 18.3-25.9 mU/ml] compared to patients with inactive disease (16.8, IQR = 13.3-20.2 mU/ml) and to controls (7.1, IQR = 5.1-8.7 mU/ml, p < 0.001]. Also, intracellular ROS tended to increase in active BS, although not significantly. In active non-vascular BS, NETs correlated with neutrophil ROS production (p < 0.001) and were particularly increased in patients with active mucosal (p < 0.001), articular (p = 0.004) and gastrointestinal symptoms (p = 0.006). In isolated neutrophils, colchicine significantly reduced oxidation-induced NET production and cell apoptosis, although not via an anti-oxidant activity. Neutrophil-mediated mechanisms might be directly involved in non-vascular BS, and NETs, more than ROS, might drive the pathogenesis of mucosal, articular and intestinal manifestations. Colchicine might be effective in counteracting neutrophils-mediated damage in BS, although further studies are needed.

Co-expression of the SARS-CoV-2 entry receptors ACE2 and TMPRSS2 in healthy human conjunctiva.

The purpose of this study was to evaluate the expression of the SARS-CoV-2 receptors ACE2 and TMPRSS2 in an immortalized human conjunctival epithelial cell line and in healthy human conjunctiva excised during ocular surgery, using Western blot, confocal microscopy and immunohistochemistry. The Western blot showed that ACE2 and TMPRSS2 proteins were expressed in human immortalized conjunctival cells, and this was confirmed by confocal microscopy images, that demonstrated a marked cellular expression of the viral receptors and their co-localization on the cell membranes. Healthy conjunctival samples from 11 adult patients were excised during retinal detachment surgery. We found the expression of ACE2 and TMPRSS2 in all the conjunctival surgical specimens analyzed and their co-localization in the superficial conjunctival epithelium. The ACE2 Western blot levels and immunofluorescence staining for ACE2 were variable among specimens. These results suggest the susceptibility of the conjunctival epithelium to SARS-CoV-2 infection, even though with a possible interindividual variability.

Low-Calorie Vegetarian Versus Mediterranean Diets for Reducing Body Weight and Improving Cardiovascular Risk Profile: CARDIVEG Study (Cardiovascular Prevention With Vegetarian Diet).

BACKGROUND: Only a few randomized dietary intervention studies that investigated the effects of lacto-ovo vegetarian diet (Vd) in clinically healthy omnivorous subjects are available. METHODS: We randomly assigned to overweight omnivores with a low-to-moderate cardiovascular risk profile a low-calorie Vd compared with a low-calorie Mediterranean diet (MD), each lasting 3 months, with a crossover design. The primary outcome was the difference in body weight, body mass index, and fat mass changes between the 2 groups. Secondary outcomes were differences in circulating cardiovascular disease risk parameters changes between the 2 groups. RESULTS: One hundred eighteen subjects (mean age: 51.1 years, females: 78%) were enrolled. The total participation rate at the end of the study was 84.7%. No differences between the 2 diets in body weight were observed, as reported by similar and significant reductions obtained by both Vd (-1.88 kg) and MD (-1.77 kg). Similar results were observed for body mass index and fat mass. In contrast, significant differences between the 2 interventions were obtained for low-density lipoprotein cholesterol, triglycerides, and vitamin B12 levels. The difference between the Vd and MD groups, in terms of end-of-diet values, was recorded at 9.10 mg/dL for low-density lipoprotein cholesterol (P=0.01), 12.70 mg/dL for triglycerides (P<0.01), and 32.32 pg/mL for vitamin B12 (P<0.01). Finally, no significant difference was found between Vd and MD interventions in oxidative stress markers and inflammatory cytokines, except for interleukin-17, which improved only in the MD group. Forty-six participants during the Vd period and 35 during the MD period reached the target values for ≥1 cardiovascular risk factor. CONCLUSIONS: Both Vd and MD were effective in reducing body weight, body mass index, and fat mass, with no significant differences between them. However, Vd was more effective in reducing low-density lipoprotein cholesterol levels, whereas MD led to a greater reduction in triglyceride levels. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02641834.

ROS-challenged keratinocytes as a new model for oxidative stress-mediated skin diseases.

In the current study, the effects of the reactive oxygen species (ROS) generator 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) on extracellular and intracellular ROS production in human keratinocytes (HACAT) were studied. AAPH is a water-soluble compound able to generate ROS at known and constant rates at 37°C. The short treatment (2 h) with AAPH brought a significant dose-dependent increase in NADPH oxidase activity in intact keratinocytes. The long-term treatment (24 h) with AAPH led to a persistent increase in NADPH oxidase activity for up to 48 hour following the AAPH removal from cell incubation medium. ROS and nitric oxide levels, lipoperoxidation, intracellular calcium, mitochondrial superoxide production, and membrane potential were significantly modified in AAPH-treated HACAT. Superoxide dismutase (SOD) and/or catalase addition to HACAT revealed that untreated keratinocytes produce mostly superoxide anion (O 2- ), while AAPH-treated keratinocytes overproduce hydrogen peroxide (H 2 O 2 ) in extracellular medium. H 2 O 2 is particularly stable and plays important roles in several cell signaling pathways. Taken together, our findings suggest a cost-effective and easily reproducible in vitro model of stressed human keratinocytes releasing significantly elevated ROS amounts in extracellular medium with respect to control keratinocytes. The possible application of the proposed model for keratinocytes-melanocytes cross-talk studies is also suggested. The model of AAPH-stressed human keratinocytes described here can represent a useful tool for redox cross-talk studies between keratinocytes and other skin cell types, and applied for researches regarding skin pathologies associated with oxidative stress.

The right place of interleukin-1 inhibitors in the treatment of Behçet's syndrome: a systematic review.

Behçet's syndrome (BS) is a chronic (auto)-inflammatory disorder characterized by different clusters of symptoms, including mucocutaneous and ocular involvements. Interleukin-1 inhibitors anakinra (ANA), canakinumab (CAN), and gevokizumab (GEV) represent a promising therapeutic alternative in BS. To date, evidence on the use of ANA, CAN, and GEV is mainly based on small isolated studies or case series, and the real place of anti-IL1 agents in the treatment of BS is still unclear. We performed a systematic review of current evidence on the efficacy and safety of anti-IL1 agents in BS. The PubMed search yielded a total of 398 references, from which we retrieved 24 studies for inclusion (4 clinical trials, 6 observational studies, 14 case reports, case series or letters to the editor). Four studies evaluated the overall efficacy of IL-1 inhibitors, 15 studies focused on the specific efficacy of ANA, whereas efficacy of CAN and GEV was evaluated in 8 and 3 studies, respectively. Both ANA and CAN were associated with good control of mucocutaneous and ocular manifestations. ANA resulted effective also for osteoarticular manifestations. GEV was studied only for ocular manifestations, but gave contrasting results. Discordant evidence supports the use of ANA and CAN in pediatric setting and for first-line treatment of general BS manifestations. Most frequent side effects were local or diffuse cutaneous reactions and injection site reactions, particularly for ANA treatment. Blocking the IL-1 pathway could be an effective therapeutic strategy in particular BS involvements.

Effects of Cadmium on ZO-1 Tight Junction Integrity of the Blood Brain Barrier.

Cadmium (Cd) is a highly toxic environmental pollutant released from the smelting and refining of metals and cigarette smoking. Oral exposure to cadmium may result in adverse effects on a number of tissues, including the central nervous system (CNS). In fact, its toxicity has been related to neurological disorders, as well as neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Under normal conditions, Cd barely reaches the brain in adults because of the presence of the blood-brain barrier (BBB); however, it has been demonstrated that Cd-dependent BBB alteration contributes to pathogenesis of neurodegeneration. However, the mechanism underlying Cd-dependent BBB alteration remain obscure. Here, we investigated the signaling pathway of Cd-induced tight junction (TJ), F-actin, and vimentin protein disassembly in a rat brain endothelial cell line (RBE4). RBE4 cells treated with 10 µM cadmium chloride (CdCl2) showed a dose- and time-dependent significant increase in reactive oxygen species (ROS) production. This phenomenon was coincident with the alteration of the TJ zonula occludens-1 (ZO-1), F-actin, and vimentin proteins. The Cd-dependent ROS increase elicited the upregulation of GRP78 expression levels, a chaperone involved in endoplasmic reticulum (ER) stress that induces caspase-3 activation. Further signal profiling by the pannexin-1 (PANX1) specific inhibitor 10Panx revealed a PANX1-independent increase in ATP spillage in Cd-treated endothelial cells. Our results point out that a ROS-dependent ER stress-mediated signaling pathway involving caspase-3 activation and ATP release is behind the BBB morphological alterations induced by Cd.

A Khorasan Wheat-Based Replacement Diet Improves Risk Profile of Patients With Nonalcoholic Fatty Liver Disease (NAFLD): A Randomized Clinical Trial.

OBJECTIVE: KAMUT khorasan is an ancient grain with widely acclaimed health benefits. The aim of this study was to investigate the effects of a replacement diet with ancient khorasan wheat products in patients with NAFLD, in comparison to a similar replacement diet with control products made from organic semi-whole-grain modern wheat. METHODS: Forty NAFLD patients (12 M/28 F; age 55.2 ± 10.4 years) with mild to moderate liver steatosis were included. The experimental design was a randomized, double-blind, parallel-arm study with 20 participants assigned to consume either KAMUT khorasan or control wheat products (pasta, bread, crackers, biscuits) over a 3-month period. Anthropometric measurements, blood analyses, and ultrasonography examination were performed at both the beginning and end of each dietary intervention. RESULTS: After the implementation of a general linear model for repeated measurements adjusted for baseline demographic details, risk factors, and medication, alanine aminotransferase (ALT) was significantly reduced by 12%, aspartate aminotransferase (AST) by 14%, alkaline phosphatase (ALP) by 8%, and cholesterol by 6% only in the khorasan group (p < 0.05 for all). Similarly, significant reductions in circulating proinflammatory tumor necrosis factor-alpha by 50%, interleukin l-receptor antagonist-alpha by 37%, interleukin-8 by 24%, and interferon gamma by 24% were evident only in participants who consumed the khorasan products (p < 0.05 for all). Finally, significant improvements in the liver steatosis grading, Doppler perfusion index values, and reactive oxygen species (ROS) production were evident after consumption of both the khorasan and control products. CONCLUSIONS: This study suggests that a short-term replacement diet with ancient KAMUT khorasan products is most effective in reducing metabolic risk factors and ameliorating the liver profile in patients with NAFLD.

Sirt1 Protects against Oxidative Stress-Induced Apoptosis in Fibroblasts from Psoriatic Patients: A New Insight into the Pathogenetic Mechanisms of Psoriasis.

Psoriasis, a multisystem chronic disease characterized by abnormal keratinocyte proliferation, has an unclear pathogenesis where systemic inflammation and oxidative stress play mutual roles. Dermal fibroblasts, which are known to provide a crucial microenvironment for epidermal keratinocyte function, represented the selected experimental model in our study which aimed to clarify the potential role of SIRT1 in the pathogenetic mechanisms of the disease. We firstly detected the presence of oxidative stress (lipid peroxidation and total antioxidant capacity), significantly reduced SIRT1 expression level and activity, mitochondrial damage and apoptosis (caspase-3, -8 and -9 activities) in psoriatic fibroblasts. Upon SIRT1 activation, redox balance was re-established, mitochondrial function was restored and apoptosis was no longer evident. Furthermore, we examined p38, ERK and JNK activation, which was strongly altered in psoriatic fibroblasts, in response to SIRT1 activation and we measured caspase-3 activity in the presence of specific MAPK inhibitors demonstrating the key role of the SIRT1 pathway against apoptotic cell death via MAPK modulation. Our results clearly demonstrate the involvement of SIRT1 in the protective mechanisms related to fibroblast injury in psoriasis. SIRT1 activation exerts an active role in restoring both mitochondrial function and redox balance via modulation of MAPK signaling. Hence, SIRT1 can be proposed as a specific tool for the treatment of psoriasis.

A Biochemical Approach to Detect Oxidative Stress in Infertile Women Undergoing Assisted Reproductive Technology Procedures.

Oxidative stress plays a major role in critical biological processes in human reproduction. However, a reliable and biologically accurate indicator of this condition does not yet exist. On these bases, the aim of this study was to assess and compare the blood and follicular fluid (FF) redox status of 45 infertile subjects (and 45 age-matched controls) undergoing in vitro fertilization (IVF), and explore possible relationships between the assessed redox parameters and IVF outcomes. Reactive Oxygen Species (ROS) production, assessed by flow cytometry analysis in blood leukocytes and granulosa cells, significantly increased (p < 0.05) in infertile patients. Also, oxidative stress markers-ThioBarbituric Acid-Reactive Substances (TBARS) as an index of lipid peroxidation, and Oxygen Radical Absorbance Capacity (ORAC) to account for total antioxidant capacity, both assayed by fluorometric procedures-in blood and FF were significantly (p < 0.001) modified in infertile patients compared to the control group. Moreover, a significant correlation between blood redox markers and FF redox markers was evident. An ORAC/TBARS ratio, defined as the redox index (RI), was obtained in the plasma and FF of the patients and controls. In the patients, the plasma RI was about 3.4-fold (p < 0.0001) lower than the control, and the FF RI was about six-fold (p < 0.0001) lower than the control. Interestingly, both the plasma RI and FF RI results were significantly correlated (p < 0.05) to the considered outcome parameters (metaphase II, fertilization rate, and ongoing pregnancies). Given the reported findings, a strict monitoring of redox parameters in assisted reproductive techniques and infertility management is recommended.

Neutrophil Activation Promotes Fibrinogen Oxidation and Thrombus Formation in Behçet Disease.

BACKGROUND: Behçet disease (BD) is a systemic vasculitis with a broad range of organ involvement, characterized by a multisystemic, immune-inflammatory disorder involving vessels of all sizes and often complicated by thrombosis. Systemic redox imbalance and circulating neutrophil hyperactivation have been observed in BD patients and are thought to be responsible for impaired coagulation. We here focused on the pathogenetic mechanisms potentially linking immune cell activation and thrombosis, and specifically examined whether neutrophil activation can affect fibrinogen modifications and consequently elicit thrombosis. METHODS AND RESULTS: Blood samples were collected from 98 consecutive BD patients attending our dedicated Center and from 70 age- and sex-matched healthy controls; in all patients fibrinogen function and structure, fibrin susceptibility to plasmin-lysis, plasma redox status, leukocyte oxidative stress markers, and possible reactive oxygen species sources were examined. Thrombin-catalyzed fibrin formation and fibrin susceptibility to plasmin-induced lysis were significantly impaired in BD patients (P<0.001). These findings were associated with increased plasma oxidative stress markers (P<0.001) and with a marked carbonylation of fibrinogen (P<0.001), whose secondary structure appeared deeply modified. Neutrophils displayed an enhanced NADPH oxidase activity and increased reactive oxygen species production (P<0.001), which significantly correlated with fibrinogen carbonylation level (r(2)=0.33, P<0.0001), residual ß-band intensity (r(2)=0.07, P<0.01), and fibrinogen clotting ability (r(2)=0.073, P<0.01) CONCLUSIONS: In BD patients, altered fibrinogen structure and impaired fibrinogen function are associated with neutrophil activation and enhanced reactive oxygen species production whose primary source is represented by neutrophil NADPH oxidase.

A khorasan wheat-based replacement diet improves risk profile of patients with type 2 diabetes mellitus (T2DM): a randomized crossover trial.

PURPOSES: The aim of the present study was to examine whether a replacement diet with products made with organic ancient khorasan wheat could provide additive protective effects in reducing glucose, insulin, lipid and inflammatory risk factors, and in restoring blood redox balance in type 2 diabetes mellitus (T2DM) patients compared to diet with product made with modern organic wheat. METHODS: We conducted a randomized, double-blinded crossover trial with two intervention phases on 21 T2DM patients (14 females, 7 males). The participants were assigned to consume products (bread, pasta, crackers and biscuits) made using semi-whole flour from organic wheat that was either from ancient khorasan wheat or modern control wheat for 8 weeks in a random order. An 8-week washout period was implemented between the interventions. Laboratory analyses were performed both at the beginning and at the end of each intervention phase. RESULTS: The metabolic risk profile improved only after the khorasan intervention period, as measured by a reduction in total and LDL cholesterol (mean reduction: -3.7 and -3.4 %, respectively), insulin (-16.3 %) and blood glucose (-9.1 %). Similarly, there was a significant reduction in circulating levels of reactive oxygen species (ROS), vascular endothelial growth factor (VEGF) and interleukin-1ra, and a significant increase of total antioxidant capacity (+6.3 %). No significant differences from baseline were noted after the modern control wheat intervention phase. The change (from pre- to post-intervention) between the two intervention arms was significantly different (p < 0.05) for total and LDL-c, insulin and HOMA index. CONCLUSIONS: A replacement diet with ancient khorasan wheat consumption provided additive protection in reducing total and LDL cholesterol, insulin, blood glucose, ROS production, and some inflammatory risk factors, which are all key factors warranting of control in secondary prevention of T2DM compared to a diet with products made with modern wheat.

Certolizumab Pegol treatment in Behcet's disease with different organ involvement: A multicenter retrospective observational study.

OBJECTIVES: The purpose of the present study was to describe our experience with the recombinant Fab' antibody fragment against TNF-α Certolizumab Pegol (CZP) in patients with Behçet's disease (BD) refractory to standardized therapies and previous biologic agents. METHODS: Retrieved data including demographic characteristics, clinical manifestations, and previous treatments were collected in three different specialized Rheumatologic Units in Italy. In order to evaluate disease activity, the BD current activity form (BDCAF) has been used before starting CZP therapy and at each visit during treatment. RESULTS: Thirteen BD patients (mean age 42.6 ± 8.8 years) with a disease duration of 8.80 ± 6.9 years, underwent CZP treatment for 6.92 ± 3.52 months. Six patients (46.15%) experienced a worsening of symptoms after 4.16 ± 1.21 months, whereas a satisfactory response was achieved in seven patients (53.84%) who were still on CZP therapy at the last follow-up visit (after 9.28 ± 3.03 months of treatment). The mean decrease of BDCAF between the first and last visit was 0.308 ± 1.84 without reaching significant difference (mean 8.3 ± 1.3 and 8 ± 2.08, respectively; p= .51). During the whole study period, CZP was well tolerated in all patients except one who developed a generalized cutaneous reaction after the third administration. CONCLUSIONS: These results suggest that despite an improvement of clinical manifestations has been observed in more than half of the patients, it is not possible to draw firm conclusions about the effectiveness of CZP in BD and further studies with larger cohorts of patients are warranted. Whether the increase of CZP dosage may ensure a better clinical response remains an unsolved issue that needs to be considered.

Protection from cigarette smoke-induced vascular injury by recombinant human relaxin-2 (serelaxin).

Smoking is regarded as a major risk factor for the development of cardiovascular diseases (CVD). This study investigates whether serelaxin (RLX, recombinant human relaxin-2) endowed with promising therapeutic properties in CVD, can be credited of a protective effect against cigarette smoke (CS)-induced vascular damage and dysfunction. Guinea pigs exposed daily to CS for 8 weeks were treated with vehicle or RLX, delivered by osmotic pumps at daily doses of 1 or 10 µg. Controls were non-smoking animals. Other studies were performed on primary guinea pig aortic endothelial (GPAE) cells, challenged with CS extracts (CSE) in the absence and presence of 100 ng/ml (17 nmol/l) RLX. In aortic specimens from CS-exposed guinea pigs, both the contractile and the relaxant responses to phenylephrine and acetylcholine, respectively, were significantly reduced in amplitude and delayed, in keeping with the observed adverse remodelling of the aortic wall, endothelial injury and endothelial nitric oxide synthase (eNOS) down-regulation. RLX at both doses maintained the aortic contractile and relaxant responses to a control-like pattern and counteracted aortic wall remodelling and endothelial derangement. The experiments with GPAE cells showed that CSE significantly decreased cell viability and eNOS expression and promoted apoptosis by sparkling oxygen free radical-related cytotoxicity, while RLX counterbalanced the adverse effects of CSE. These findings demonstrate that RLX is capable of counteracting CS-mediated vascular damage and dysfunction by reducing oxidative stress, thus adding a tile to the growing mosaic of the beneficial effects of RLX in CVD.