Plasmatic carbonic anhydrase IX as a diagnostic marker for clear cell renal cell carcinoma.

Carbonic anhydrase (CA, EC 4.2.1.1) IX is regarded as a tumour hypoxia marker and CA inhibitors have been proposed as a new class of antitumor agents, with one such agent in Phase II clinical trials. The expression of some CAs, in particular the isoforms CA IX and CA XII, has been correlated with tumour aggressiveness and progression in several cancers. The aim of this study was to evaluate the possibility that CA IX could represent a marker related to clear cell Renal Cell Carcinoma (ccRCC). Bcl-2 and Bax, and the activity of caspase-3, evaluated in tissue biopsies from patients, were congruent with resistance to apoptosis in ccRCCs with respect to healthy controls, respectively. In the same samples, the CA IX and pro-angiogenic factor VEGF expressions revealed that both these hypoxia responsive proteins were strongly increased in ccRCC with respect to controls. CA IX plasma concentration and CA activity were assessed in healthy volunteers and patients with benign kidney tumours and ccRCCs. CA IX expression levels were found strongly increased only in plasma from ccRCC subjects, whereas, CA activity was found similarly increased both in plasma from ccRCC and benign tumour patients, compared to healthy volunteers. These results show that the plasmatic level of CA IX, but not the CA total activity, can be considered a diagnostic marker of ccRCCs. Furthermore, as many reports exist relating CA IX inhibition to a better outcome to anticancer therapy in ccRCC, plasma levels of CA IX could be also predictive for response to therapy.

General Practitioner-Performed Compression Ultrasonography for Diagnosis of Deep Vein Thrombosis of the Leg: A Multicenter, Prospective Cohort Study.

BACKGROUND: Patients with suspected deep vein thrombosis (DVT) of the lower limb represent a diagnostic dilemma for general practitioners. Compression ultrasonography (US) is universally recognized as the best test of choice. We assessed the diagnostic accuracy of compression US performed by general practitioners given short training in the management of symptomatic proximal DVT. METHODS: From May 2014 to May 2016, we evaluated in a multicenter, prospective cohort study all consecutive outpatients with suspected DVT; bilateral proximal lower limb compression US was performed by general practitioners and by physicians expert in vascular US, each group blinded to the other's findings. In all examinations with a negative or nondiagnostic result, compression US was repeated by the same operator after 5 to 7 days. Inter-observer agreement and accuracy were calculated. RESULTS: We enrolled a total of 1,107 patients. The expert physicians diagnosed DVT in 200 patients, corresponding to an overall prevalence of 18.1% (95% CI, 15.8%-20.3%). The agreement between the trained general practitioners and the experts was excellent (Cohen κ = 0.86; 95% CI, 0.84-0.88). Compression US performed by general practitioners had a sensitivity of 90.0% (95% CI, 88.2%-91.8%) and a specificity of 97.1% (95% CI, 96.2%-98.1%) with a diagnostic accuracy for DVT of 95.8% (95% CI, 94.7%-97.0%). CONCLUSIONS: Our results suggest that, even in hands of physicians not expert in vascular US, compression US can be a reliable tool in the diagnosis of DVT. We found that the sensitivity achieved by general practitioners appeared suboptimal, however, so future studies should evaluate the implementation of proper training strategies to maximize skill.

Protective effects of novel metal-nonoates on the cellular components of the vascular system.

At the cardiovascular level, nitric oxide (NO) controls smooth muscle functions, maintains vascular integrity, and exerts an antihypertensive effect. Metal-nonoates are a recently discovered class of NO donors, with NO release modulated through the complexation of the N-aminoethylpiperazine N-diazeniumdiolate ligand to metal ions, and thus representing a significant innovation with respect to the drugs traditionally used. In this study, we characterized the vascular protective effects of the most effective compound of this class, Ni(PipNONO)Cl, compared with the commercial N-diazeniumdiolate group derivate, diethylenetriamine/nitric oxide (DETA/NO). Ni(PipNONO)Cl induced a concentration-dependent relaxation of precontracted rat aortic rings. The ED50 was 0.67 µM, compared with 4.3 µM obtained with DETA/NO. When tested on cultured microvascular endothelial cells, Ni(PipNONO)Cl exerted a protective effect on the endothelium, promoting cell proliferation and survival in the picomolar range. The administration of Ni(PipNONO)Cl to vascular smooth muscle cells reduced the cell number, promoting their apoptosis at a high concentration (10 µM). Inhibition of smooth muscle cell migration, a hallmark of atherosclerosis, was accompanied by cytoskeletal rearrangement and loss of lamellipodia. When added to isolated platelets, Ni(PipNONO)Cl significantly reduced ADP-induced aggregation. Since atherosclerosis is accompanied by an inflammatory environment, cultured endothelial cells were exposed to interleukin (IL)-1ß. In the presence of IL-1ß, Ni(PipNONO)Cl inhibited cyclooxygenase-2 and inducible nitric oxide synthase upregulation, and reduced endothelial permeability and the platelet and monocyte adhesion markers CD31 and CD40 at the plasma membrane. Overall, these data indicate that Ni(PipNONO)Cl exerts vascular protective effects relevant for vascular dysfunction and prevention of atherosclerosis and thrombosis.

Efficacy and Safety of Nirmatrelvir/Ritonavir, Molnupiravir, and Remdesivir in a Real-World Cohort of Outpatients with COVID-19 at High Risk of Progression: The PISA Outpatient Clinic Experience.

INTRODUCTION: Different antivirals are available for the treatment of outpatients with COVID-19. Our aim was to describe a real-world experience of outpatient management of COVID-19 subjects at high risk of progression. METHODS: This prospective observational study conducted in the University Hospital of Pisa (January 2022-July 2022) included consecutive COVID-19 outpatients with at least one risk factor for disease progression. Patients received nirmatrelvir/ritonavir, molnupiravir, or 3-day remdesivir, according to the Italian Medicines Agency (AIFA) indications. All patients were followed up until 30 days from the first positive nasopharyngeal swab. The primary endpoint was a composite of death or hospitalization. Secondary endpoints were occurrence of adverse events and a negative test within 10 days from the first positive test. Multivariable analysis was performed to identify factors associated with death or hospitalization. RESULTS: Overall, 562 outpatients were included: 114 (20.3%) received molnupiravir, 252 (44.8%) nirmatrelvir/ritonavir, and 196 (34.9%) 3-day remdesivir. The composite endpoint occurred in 2.5% of patients and was more frequent in patients treated with remdesivir (5.1%) compared with molnupiravir (1.8%) or nirmatrelvir/ritonavir (0.8%, ANOVA among groups p = 0.012). On multivariable Cox regression analysis, presence of ≥ 3 comorbidities, hematological disease, gastrointestinal symptoms, and each-day increment from symptoms onset were factors associated with death or hospitalization, while antiviral treatment was not a predictor. Adverse events occurred more frequently in the nirmatrelvir/ritonavir group (49.2%). Nirmatrelvir/ritonavir compared with remdesivir was associated with a higher probability of having a negative test within 10 days from the first positive one. CONCLUSION: Death or hospitalization did not differ among high-risk COVID-19 outpatients treated with currently available antivirals. Safety and time to a negative test differed among the three drugs.

Pro-Inflammatory and Pro-Oxidative Changes During Nilotinib Treatment in CML Patients: Results of a Prospective Multicenter Front-Line TKIs Study (KIARO Study).

Tyrosine kinase inhibitors (TKI) may offer a normal life expectancy to Chronic Myeloid Leukemia (CML) patients. However, a higher than expected incidence of arterial occlusive events (AOEs) was observed during treatment with nilotinib. We previously showed an "inflammatory status" during nilotinib that may explain the increased incidence of AOEs. Thus, we conducted this prospective KIARO study involving 186 CML patients (89 imatinib, 59 nilotinib, 38 dasatinib). Interleukin 6 (IL6), interleukin 10 (IL10), Tumor Necrosis Factor-α (TNFα), oxLDL, and high-sensitivity C-reactive protein (hs-CRP) plasma levels were measured at diagnosis and during treatment, with the aim to investigate changes in the inflammatory status favoring AOEs of each patient. Clinical and biochemical pro-atherothrombotic profiles and the 10-year SCORE chart were also evaluated. We showed a pro-inflammatory/pro-oxidative milieu increasing along treatment with nilotinib compared with imatinib or dasatinib, as demonstrated by higher hs-CRP and oxLDL levels and increased IL6/IL10 and TNFα/IL10 ratios only in nilotinib cohort. After median follow-up of 23.3 months starting from TKI, 10/186 patients (5.4%) suffered an AOE. Approximately 5/10 (50%) AOEs occurred during nilotinib treatment despite a lower 10-year SCORE and a lower median age in this subgroup. A longer follow-up is needed to further confirm the active role of nilotinib in AOEs pathogenesis.

Selective inhibition of carbonic anhydrase IX decreases cell proliferation and induces ceramide-mediated apoptosis in human cancer cells.

Recently, carbonic anhydrase (CA) inhibitors have been proposed as a potential new class of antitumor agents. The aim of this study was to evaluate the antitumor activity of three CA inhibitors, namely acetazolamide (AZ) and two newly synthesized aromatic sulfonamides with high affinity for CA IX, 2-(4-sulfamoylphenyl-amino)-4,6-dichloro-1,3,5-triazine (TR1) and 4-[3-(N,N-dimethylaminopropyl)thioreidophenylsulfonylaminoethyl]benzenesulfonamide (GA15), against human tumor cells. The effects of AZ, TR1, and GA15 on cell proliferation and apoptosis were evaluated in CA IX-positive HeLa and 786-O cells and CA IX-negative 786-O/von Hippel-Lindau (VHL) cells. We also investigated whether the potential antitumor activity of these molecules might be mediated by an increase in ceramide production. AZ, TR1, and GA15 could significantly reduce cell proliferation and induce apoptosis in HeLa and 786-O cells. Moreover, all three inhibitors could decrease intracellular pH (pH(i)) and increase ceramide production in the same cells. Treatment with the ceramide synthase inhibitor fumonisin B1 prevented the apoptotic effects of the three CA inhibitors. In all experiments, the effects of aromatic sulfonamides were more pronounced than those of AZ. The three inhibitors did not show any antitumor activity in CA IX-negative 786-O/VHL cells and failed to lower pH(i) or increase intracellular ceramide levels in the same cells. In conclusion, CA inhibition can decrease cell proliferation and induce apoptosis in human tumor cells. The ability of CA inhibitors to decrease pH(i) might trigger cell apoptosis through mediation of ceramide synthesis. Activation of this apoptotic cascade probably is mediated by inhibition of the CA IX isoform.

Pro/Anti-inflammatory cytokine imbalance in postischemic left ventricular remodeling.

OBJECTIVES: Cytokines play an important role in left ventricular remodeling consequent to myocardial ischemia. The aim of this study was to correlate cytokine production and lymphocyte apoptosis to post-ischemic left ventricular remodeling in patients affected by acute myocardial infarction (AMI) undergoing primary cutaneous angioplasty (PCI). METHODS: In 40 patients, affected by AMI and undergoing PCI, we evaluated peripheral blood mononuclear cells (PBMCs), tumor necrosis factor-alpha (TNF-alpha) and interleukin 10 (IL10) production and apoptosis on day 1, day 3, day 7, 1 month and 6 months after PCI. Patients were divided into two subgroups of remodeling or not remodeling by echocardiographic criteria. RESULTS: In the subgroup of remodeling patients, at each timepoint TNF-alpha production was increased significantly in comparison with the subgroup of not remodeling patients. IL10 production was statistically lower in remodeling subjects than in not remodeling ones 1 and 6 months after reperfusion. There were no differences between the two groups as regards lymphomonocyte apoptosis. CONCLUSIONS: We found an increased production of pro-inflammatory cytokine TNF-alpha and a corresponding decrease of anti-inflammatory/regulatory cytokine IL10 in remodeling patients and we concluded that this cytokine imbalance resulted in pro-inflammatory effects which might contribute to the progression of left ventricular remodeling.

Investigations of the esterase, phosphatase, and sulfatase activities of the cytosolic mammalian carbonic anhydrase isoforms I, II, and XIII with 4-nitrophenyl esters as substrates.

The esterase, phosphatase, and sulfatase activities of carbonic anhydrase (CA, EC 4.2.1.1) isozymes, CA I, II, and XIII with 4-nitrophenyl esters as substrates was investigated. These enzymes show esterase activity with 4-nitrophenyl acetate as substrate, with second order rate constants in the range of 753-7706M(-1)s(-1), being less effective as phosphatases (k(cat)/K(M) in the range of 14.89-1374.40M(-1)s(-1)) and totally ineffective sulfatases. The esterase/phosphatase activities were inhibited by sulfonamide CA inhibitors, proving that the zinc-hydroxide mechanism responsible for the CO(2) hydrase activities of CAs is also responsible for their esterase/phosphatase activity. CA XIII was the most effective esterase and phosphatase. CA XIII might catalyze other physiological reactions than CO(2) hydration, based on its relevant phosphatase activity.

Residual Peripheral Blood CD26+ Leukemic Stem Cells in Chronic Myeloid Leukemia Patients During TKI Therapy and During Treatment-Free Remission.

Chronic myeloid leukemia (CML) patients in sustained "deep molecular response" may stop TKI treatment without disease recurrence; however, half of them lose molecular response shortly after TKI withdrawing. Well-defined eligibility criteria to predict a safe discontinuation up-front are still missing. Relapse is probably due to residual quiescent TKI-resistant leukemic stem cells (LSCs) supposedly transcriptionally low/silent and not easily detectable by BCR-ABL1 qRT-PCR. Bone marrow Ph+ CML CD34+/CD38- LSCs were found to specifically co-express CD26 (dipeptidylpeptidase-IV). We explored feasibility of detecting and quantifying CD26+ LSCs by flow cytometry in peripheral blood (PB). Over 400 CML patients (at diagnosis and during/after therapy) entered this cross-sectional study in which CD26 expression was evaluated by a standardized multiparametric flow cytometry analysis on PB CD45+/CD34+/CD38- stem cell population. All 120 CP-CML patients at diagnosis showed measurable PB CD26+ LSCs (median 19.20/µL, range 0.27-698.6). PB CD26+ LSCs were also detectable in 169/236 (71.6%) CP-CML patients in first-line TKI treatment (median 0.014 cells/µL; range 0.0012-0.66) and in 74/112 (66%), additional patients studied on treatment-free remission (TFR) (median 0.015/µL; range 0.006-0.76). Notably, no correlation between BCR-ABL/ABLIS ratio and number of residual LSCs was found both in patients on or off TKIs. This is the first evidence that "circulating" CML LSCs persist in the majority of CML patients in molecular response while on TKI treatment and even after TKI discontinuation. Prospective studies evaluating the dynamics of PB CD26+ LSCs during TKI treatment and the role of a "stem cell response" threshold to achieve and maintain TFR are ongoing.

Therapeutic ureteral occlusion with Ifabond cyanoacrylate glue: an interesting solution.

OBJECTIVE: The aim of this study was to present a novel approach for complete and permanent ureteral occlusion using a percutaneous injection of Ifabond cyanoacrylate glue. METHODS: We describe in detail all the steps of our surgery, performed on a 79-year-old patient with urinary leakage from ureteral stump following radical cystectomy. N-hexyl-cyanoacrylate glue (Ifabond) was used to occlude the distal ureter and solve the leakage. RESULTS: Our approach was successful, sparing our already frail patient further surgical procedures. Six months pyelography confirmed the complete ureteral blockage with absence of extravasation. CONCLUSIONS: In complicated scenarios with urinary leakages and frail patients, synthetic glues such as Ifabond might represent an interesting therapeutic option to solve the fistulas, leading to durable success with a minimally invasive approach.

Brain natriuretic peptide and other risk markers for outcome assessment in patients with non-ST-elevation coronary syndromes and preserved systolic function.

Several emerging cardiac markers constitute strong predictors among patients with coronary artery disease. In particular, brain natriuretic peptide (BNP), troponin T (TnT), and C-reactive protein (CRP) are related to increased risk of recurrent ischemic events and death. However, little is known about the utility of these biomarkers in combination. This study examined risk assessment in patients with coronary artery disease and preserved systolic function. We studied 208 consecutive patients (138 men, 70 women) with stable angina, unstable angina, and non-Q-wave myocardial infarction whose plasma BNP, TnT, and CRP levels were measured at hospital admission. All recruited patients underwent echocardiographic examination, and selective coronary angiography was performed. After adjusting for clinical presentation, age, gender, and common risk factors, BNP was demonstrated as a strong predictor of heart failure (6 months, odds ratio [OR] 2.03, 95% confidence interval [CI] 1.24 to 2.9, p <0.01; 12 months, OR 2.65, 95% CI 1.69 to 3.5, p <0.001) and mortality at 3, 6, and 12 months (p <0.001). BNP was also significantly related to extent of coronary artery disease and left anterior descending artery involvement (p <0.01). Patients with a BNP level >80 pg/ml in all 3 groups had a significantly poorer prognosis with increased incidence of heart failure and death. CRP was related to recurrent ischemic events (infarct or recurrent angina, OR 1.4, 95% CI 1.14 to 2.08, p <0.01) and was associated with major cardiac revascularization at 12 months (OR 1.51, 95% CI 1.29 to 1.73, p <0.001). TnT demonstrated a mild correlation with recurrent infarct or angina at 12 months (OR 1.1, 95% CI 0.96 to 1.22, p <0.05) but appeared related to multivessel coronary artery disease (OR 1.47, 95% CI 1.05 to 1.99, p <0.01). In conclusion, BNP appears to be associated with a long-term increased risk of mortality and heart failure in patients with apparently mild risk. BNP is also associated with a larger extent and greater severity of myocardial ischemia. Early BNP measurement could provide incremental information to TnT and CRP, and it may be the strongest independent predictor of cardiac outcome in subjects without left ventricular dysfunction or enlargement.

Genetic predisposition and induced pro-inflammatory/pro-oxidative status may play a role in increased atherothrombotic events in nilotinib treated chronic myeloid leukemia patients.

Several reports described an increased risk of cardiovascular (CV) events, mainly atherothrombotic, in Chronic Myeloid Leukemia (CML) patients receiving nilotinib. However, the underlying mechanism remains elusive. The objective of the current cross-sectional retrospective study is to address a potential correlation between Tyrosine Kinase Inhibitors (TKIs) treatment and CV events. One hundred and 10 chronic phase CML patients in complete cytogenetic response during nilotinib or imatinib, were screened for CV events and evaluated for: traditional CV risk factors, pro/anti-inflammatory biochemical parameters and detrimental ORL1 gene polymorphisms (encoding for altered oxidized LDL receptor-1). Multivariate analysis of the whole cohort showed that the cluster of co-existing nilotinib treatment, dyslipidaemia and G allele of LOX-1 polymorphism was the only significant finding associated with CV events. Furthermore, multivariate analysis according to TKI treatment confirmed IVS4-14 G/G LOX-1 polymorphism as the strongest predictive factor for a higher incidence of CV events in nilotinib patients. Biochemical assessment showed an unbalanced pro-inflammatory cytokines network in nilotinib vs imatinib patients. Surprisingly, pre-existing traditional CV risk factors were not always predictive of CV events. We believe that in nilotinib patients an induced "inflammatory/oxidative status", together with a genetic pro-atherothrombotic predisposition, may favour the increased incidence of CV events. Prospective studies focused on this issue are ongoing.

3'UTR/T polymorphism of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is associated with modified anti-platelet activity of atorvastatin in hypercholesterolemic subjects.

Oxidized-low density lipoproteins (ox-LDL) and the specific receptor LOX-1 are involved in atherogenesis and atherothrombosis. LOX-1 downregulation is associated with the anti-platelet action of atorvastatin. 3'UTR/T LOX-1 polymorphism has been associated with increased risk of coronary artery disease. This study was planned to determine whether LOX-1 genetic variations could affect anti-platelet action of atorvastatin. We studied by platelet P-selectin (P-sel), CD36 and LOX-1 expression (cytofluorimetric detection) whether differences in cellular activation could be suitable in 109 3'UTR/T carriers out of 201 hypercholesterolemic subjects treated with atorvastatin 20mg/day. Hyperactivated platelets (P-sel in resting cells and % variation upon thrombin activation, p<0.001) were detected at baseline in patients without significant differences between T or C carriers. P-sel and platelet-associated ox-LDL, were significantly decreased (all p<0.001) in C carriers after one week of treatment before LDL reduction. In 3'UTR/T carriers P-sel was reduced (p<0.01) after 6 weeks of treatment according to LDL and ox-LDL reduction. In 3'UTR/T carriers atorvastatin reduced platelet activity by LDL and ox-LDL lowering and not by rapid CD36 and LOX-1 downregulation as in C carriers. Such data suggest that in T carriers LDL lowering is needed to achieve anti-platelet action.

Mechanisms for antiplatelet action of statins.

Hydroxymethyl-glutaryl coenzyme A reductase inhibitors (statins) offer important benefits for the large populations of individuals at high risk for coronary heart and cerebrovascular disease. the overall clinical benefits observed with statin therapy appear to be greater than what might be expected from changes in lipid profile alone, suggesting that the beneficial effects of such drugs may extend beyond their effects on serum cholesterol. Platelet hyperactivity is a key step in atherothrombosis and experimental data suggest that statins could exert an antiplatelet effect which could be involved in their protective action. In the present review we report of the major studies in humans showing the effect of statins on platelets, especially by the more sensitive methods to explore platelet function such as cytofluorymetric detection of specific proteins. Moreover we describe the putative mechanisms involved in platelet deactivation with particular regard to the effects related to cholesterol reduction or beyond lipid-lowering. Indeed, data from several studies suggest some differences among compounds in terms of timing of action by modulation of several activating pathways which could take part either in the early, cholesterol-lowering independent, effects in the acute phase of vascular disease or during chronic treatment.