International Tailored Chemotherapy Adjuvant (ITACA) trial, a phase III multicenter randomized trial comparing adjuvant pharmacogenomic-driven chemotherapy versus standard adjuvant chemotherapy in completely resected stage II-IIIA non-small-cell lung cancer.

BACKGROUND: Several strategies have been investigated to improve the 4% survival advantage of adjuvant chemotherapy in early-stage non-small-cell lung cancer (NSCLC). In this investigator-initiated study we aimed to evaluate the predictive utility of the messenger RNA (mRNA) expression levels of excision repair cross complementation group 1 (ERCC1) and thymidylate synthase (TS) as assessed in resected tumor. PATIENTS AND METHODS: Seven hundred and seventy-three completely resected stage II-III NSCLC patients were enrolled and randomly assigned in each of the four genomic subgroups to investigator's choice of platinum-based chemotherapy (C, n = 389) or tailored chemotherapy (T, n = 384). All anticancer drugs were administered according to standard doses and schedules. Stratification factors included stage and smoking status. The primary endpoint of the study was overall survival (OS). RESULTS: Six hundred and ninety patients were included in the primary analysis. At a median follow-up of 45.9 months, 85 (24.6%) and 70 (20.3%) patients died in arms C and T, respectively. Five-year survival for patients in arms C and T was of 65.4% (95% CI (confidence interval): 58.5% to 71.4%) and 72.9% (95% CI: 66.5% to 78.3%), respectively. The estimated hazard ratio (HR) was 0.77 (95% CI: 0.56-1.06, P value: 0.109) for arm T versus arm C. HR for recurrence-free survival was 0.89 (95% CI: 0.69-1.14, P value: 0.341) for arm T versus arm C. Grade 3-5 toxicities were more frequently reported in arm C than in arm T. CONCLUSION: In completely resected stage II-III NSCLC tailoring adjuvant chemotherapy conferred a non-statistically significant trend for OS favoring the T arm. In terms of safety, the T arm was associated with better efficacy/toxicity ratio related to the different therapeutic choices in the experimental arm.

ALK gene copy number gains in non-small-cell lung cancer: prognostic impact and clinico-pathological correlations.

BACKGROUND: The correlation between ALK gene copy number gain (ALK-CNG) and prognosis in the context of advanced non-small-cell lung cancer (NSCLC) remains a controversial issue. This study aimed to evaluate the association among ALK-CNG according to Fluorescent In Situ Hybridization (FISH), clinical characteristics and survival in resectable and advanced NSCLC. METHODS: Clinical and pathological data of patients with resectable and advanced NSCLC were retrospectively collected. Tumor tissues were analyzed for ALK-CNG by FISH, and patients were divided in 3 groups/patterns on the basis of ALK signals: disomic [Pattern A], 3-7 signals [Pattern B], >7 signals [Pattern C]. The association between clinical and pathological features and ALK-CNG patterns was evaluated. Disease/progression-free and overall survival (DFS/PFS and OS) were estimated using the Kaplan-Meyer method. RESULTS: A number of 128 (76.6 %) out of the 167 eligible patients were evaluable for ALK-CNG, displaying pattern A, B and C in 71 (42.5 %), 42 (25.1 %) and 15 (9 %) patients, respectively. Gains in ALK-CNG appear to be more frequent in smokers/former smokers than in non-smokers (74.2 % versus 20.4 %, respectively, p = 0.03). Pattern A and C seem more frequently associated with higher T-stage (T3-4), while pattern B appears more represented in lower T-stage (T 1-2) (p = 0.06). No significant differences in survival rate were observed among the above groups. CONCLUSIONS: A high ALK-CNG pattern might be associated with smoking status and theoretically it might mirror genomic instability. The implications for prognosis should be prospectively investigated and validated in larger patients' series. TRIAL REGISTRATION: We confirm that all the study was performed in accordance with relevant guidelines and regulations and that all the protocol (part of a larger project MFAG 2013 N.14282) was approved by the local Ethics Committee of the Azienda Ospedaliera Universitaria Integrata of Verona on November 11st, 2014.

Selective inhibition of TNFR1 reduces osteoclast numbers and is differentiated from anti-TNF in a LPS-driven model of inflammatory bone loss.

The treatment of autoimmune disorders has been revolutionised by the introduction of biologics such as anti-tumour necrosis factor (anti-TNF). Although in rheumatoid arthritis patients a bone sparing effect of anti-TNF has been shown, the mechanism is not fully understood. Anti-TNF molecules block tumour necrosis factor (TNF) and prevent signalling via both TNF receptor 1 (TNFR1; p55) and TNF receptor 2 (TNFR2; p75). However, signalling via TNFR2 is reported to have protective effects in a number of cell and organ systems. Hence we set out to investigate if pharmacological inhibition of TNFR1 had differential effects compared to pan-TNF inhibition in both an in vitro cell-based model of human osteoclast activity and an in vivo mouse model of lipopolysaccharide (LPS)-induced osteolysis. For the in vitro experiments the anti-human TNFR1 domain antibody (dAb) DMS5541 was used, whereas for the in vivo mouse experiments the anti-mouse TNFR1 dAb DMS5540 was used. We show that selective blocking of TNFR1 signalling reduced osteoclast formation in the presence of TNF. Subcutaneous LPS injection over the calvaria leads to the development of osteolytic lesions within days due to inflammation driven osteoclast formation. In this model, murine TNFR2 genetically fused with mouse IgG1 Fc domain (mTNFR2.Fc), an anti-TNF, did not protect from bone loss in contrast to anti-TNFR1, which significantly reduced lesion development, inflammatory infiltrate, and osteoclast number and size. These results support further exploring the use of TNFR1-selective inhibition in inflammatory bone loss disorders such as osteomyelitis and peri-prosthetic aseptic loosening.

A revised energy partitioning approach to assess the yields of non-photochemical quenching components.

Non-photochemical quenching (NPQ) is a complex and still unclear mechanism essential for higher plants. The intensive research on this subject has highlighted three main components of NPQ: energy-dependent process (qE); state transitions to balance the excitation of PSII and PSI (qT); and photoinhibitory processes (qI). Recently, these components have been resolved as quantum yields according to the energy partitioning approach that takes into account the rate constants of every process involved in the quenching mechanisms of excited chlorophylls. In this study a fully extended quantum yield approach and the introduction of novel equations to assess the yields of each NPQ component are presented. Furthermore, a complete analysis of the yield of NPQ in Beta vulgaris exposed to different irradiances has been carried out. In agreement with experimental results here it is shown that the previous approach may amplify the yield of qE component and flatten the quantitative results of fluorescence analysis. Moreover, the significance of taking into account the physiological variability of NPQ for a correct assessment of energy partitioning is demonstrated.

Association between Bone Mineral Density and Nutritional Status, Body Composition and Bone Metabolism in Older Adults.

PURPOSE: To identify an association between bone mineral density (BMD) and nutritional status, body composition and bone metabolism in older patients. METHODS: Cross-sectional study, involving older adults, with osteopenia/osteoporosis and with normal BMD. The mineral density of the lumbar spine from L1 to L4 and the proximal region of the femur was assessed using dual energy X-ray absorptiometry. Biochemical analyzes were performed of 25(OH)-D, calcium and parathormone. Weight, knee height, and abdominal (AC), mid-upper arm (MUAC) and calf (CC) circumferences were measured. The percentage of body fat (%BF) and Fat-Free Mass (FFM) were quantified by electrical bioimpedance analysis. The Body Mass Index (BMI) was calculated. The statistical analysis used bivariate and multivariate, parametric and/or non-parametric tests, and was considered significant when p <0.05. RESULTS: Of the total 51 older adults assessed, 30 of them (58.8%) were diagnosed with osteopenia/osteoporosis. Body weight (p = 0.001), BMI (p = 0.001), % BF (p = 0.030) and serum concentrations of 25(OH)-D (p = 0.003) were higher in the group without changes in BMD. BMI and serum levels of 25(OH)-D demonstrated a positive correlation with the BMD of all bone compartments and the AC displayed a positive correlation with the lumbar vertebrae. In the logistic regression models, adjusted for sex and age, the BMI and the serum concentration of 25(OH)-D were presented as a protective factor against osteopenia/osteoporosis. CONCLUSIONS: Higher body weight, BMI, AC and %BF, and sufficient serum levels of vitamin D, were shown to be promoters of BMD.

Semiquantitative analysis of interim 18F-FDG PET is superior in predicting outcome in Hodgkin lymphoma patients compared to visual analysis.

PURPOSE: To investigate the prognostic value of interim PET (PETi) in adult HL patients, comparing visual with semiquantitative analysis. MATERIAL AND METHODS: Retrospective analysis of Hodgkin's lymphoma (HL) patients diagnosed between 2012 and 2016 in the Onco-hematology Department of Instituto Português de Oncologia - Porto (median follow-up: 46.5 months [2.6-66.4]). Fifty-eight patients with available PET at diagnosis (PET0) and PETi data were included. PETi scans were analyzed according to Deauville 5-point scale (5-PS), and cut-off values for changes in maximum standardized uptake value [SUVmax], peak SUV [SUVpeak], metabolic tumour volume [MTV] and total lesion glycolysis index [TLG] between PETi and PET0 were computed using ROC analysis. Visual and semiquantitative data were compared with each other in the prediction of patient outcomes. RESULTS: Semiquantitative analysis obtained a higher sensitivity for persistent/relapsed disease compared to the 5-PS (70% vs. 10%, respectively), but lower specificity. It also demonstrated better predictive performance for response to first-line therapy (negative predictive value >92%). The positive predictive value was similar for all five measurements. At 60 months of follow-up, there was a significant difference between the progression free survival (PFS) curves of patients with positive and negative PETi according to ΔSUVmax (56.9% vs. 88.0%, p<0.05), ΔSUVpeak (55.9% vs. 88.1%, p<0.05), ΔMTV (35.3% vs. 88.7%, p<0.05), and ΔTLG (42.4% vs. 88.1%, p<0.05). Statistical significance was not reached when considering 5-PS results. DISCUSSION: PETi interpretation according to a semiquantitative approach appears to discriminate HL patients better than the visual 5-PS analysis. This could allow better detection of persistent or early relapsed disease, while a negative PETi result could support de-escalating therapy intensity.

Mapping of Texture and Phase Fractions in Heterogeneous Stress States during Multiaxial Loading of Biomedical Superelastic NiTi.

Thermoelastic deformation mechanisms in polycrystalline biomedical-grade superelastic NiTi are spatially mapped using in situ neutron diffraction during multiaxial loading and heating. The trigonal R-phase is formed from the cubic phase during cooling to room temperature and subsequently deforms in compression, tension, and torsion. The resulting R-phase variant microstructure from the variant reorientation and detwinning processes are equivalent for the corresponding strain in tension and compression, and the variant microstructure is reversible by isothermal loading. The R-phase variant microstructure is consistent between uniaxial and torsional loading when the principal stress directions of the stress state are considered (for the crystallographic directions observed here). The variant microstructure evolution is tracked and the similarity in general behavior between uniaxial and torsional loading, in spite of the implicit heterogeneous stress state associated with torsional loading, pointed to the ability of the reversible thermoelastic transformation in NiTi to accommodate stress and strain mismatch with deformation. This ability of the R-phase, despite its limited variants, to accommodate stress and strain and satisfy strain incompatibility in addition to the existing internal stresses has significance for reducing irrecoverable deformation mechanisms during loading and cycling through the phase transformation.

Trends in systemic lupus erythematosus mortality rates in the state of Sao Paulo, Brazil from 1985 to 2004.

OBJECTIVES: To estimate mortality rates and mortality trends from SLE in the state of São Paulo, Brazil. MATERIAL AND METHODS: The official data bank was used to study all deaths occurred from 1985 to 2004 in which SLE was mentioned as the underlying cause of death. Besides the overall mortality rate, the annual gender- and age-specific mortality rates were estimated for each calendar year by age bracket (0-19 years, 20-39 years, 40-59 years and over 60 years) and for the sub-periods 1985-1995 (first) and 1996-2004 (second), by decades. Chi-square test was used to compare the mortality rates between the two periods, as well the mortality rates according to educational level considering years of study. Pearson correlation coefficient test was used to analyse mortality trends. The crude rates were adjusted for age by the direct method, using the standard Brazilian population in 2000. RESULTS: A total of 2,601 deaths (90% female) attributed to SLE were analysed. The mean age at death was significantly higher in the second than in the first sub-period (36.6+/-15.6 years vs. 33.9+/-14.0 years; p<0.001). The overall adjusted mortality rate was 3.8 deaths/million habitants/year for the entire period and 3.4 deaths/million inhabitants/year for the first and 4.0 deaths/million inhabitants/year for the second sub-period (p<0.001). In each calendar year, the mortality rate was significantly lower for the better educated group. Throughout the period, there was a significant increase in mortality rates only among women over 40. CONCLUSIONS: SLE patients living in the state of São Paulo still die at younger ages than those living in developed countries. Our data do not support the theory that there was an improvement in the SLE mortality rate in the last 20 years in the state of Sao Paulo. Socio-economic factors, such as the difficulty to get medical care and adequate treatment, may be the main factors to explain the worst prognosis for our patients.

Dental status of Portuguese HIV+ patients and related variables: a multivariate analysis.

OBJECTIVE: The aim of this cross-sectional study was to evaluate the dental status of 101 Portuguese HIV+ subjects aged 22-71 years (mean = 39) and its association with clinical, socioeconomic, and behavioral variables. MATERIALS AND METHODS: A calibrated dentist performed clinical examination and collected data on dental caries, periodontal status, dental plaque levels, prosthetic conditions, and need. The volunteers completed questionnaires on socioeconomic and behavioral variables as well as the Oral Health Impact Profile (OHIP-14) questionnaire. Univariate and multiple logistic regression (MLR) analyses were performed. RESULTS: The mean number of decayed, missing or filled teeth index (DMFT index) was 16.44, standard deviation (s.d.) = 8.42. MLR demonstrated that salaried employee and those with OHIP-14 median (=17). As regards prosthetic status, 28.8% of the examined individuals used dental prosthesis. MLR demonstrated that HIV+ with DMFT >17 or those who knew they were HIV-positive for longer than 5 years were more prone to need dental prostheses. The mean OHIP-14 index was 5.83 (s.d. = 7.79). CONCLUSIONS: The dental health status of HIV-infected Portuguese patients was unsatisfactory and related to clinical, socioeconomic, and behavioral variables.

Metronomic oral vinorelbine for the treatment of advanced non-small cell lung cancer: a multicenter international retrospective analysis.

PURPOSE: Metronomic oral vinorelbine (MOV) could be a treatment option for unfit patients with advanced non-small cell lung cancer (NSCLC) based on its safety profile and high patient compliance. METHODS: We retrospectively collected data on 270 patients [median age 76 (range 48-92) years, M/F 204/66, PS 0 (27)/1 (110)/≥ 2 (133), median of 3 serious comorbidities] with stage IIIB-IV NSCLC treated with MOV as first (T1) (67%), second (T2) (19%) or subsequent (T3) (14%) line. Schedules consisted of vinorelbine 50 mg (138), 40 mg (68) or 30 mg (64) three times a week continuously. RESULTS: Patients received an overall median of 6 (range 1-25) cycles with a total of 1253 cycles delivered. The overall response rate was 17.8% with 46 partial and 2 complete responses and 119 patients (44.1%) experienced stable disease > 12 weeks with an overall disease control rate of 61.9%. Median overall time to progression was 5 (range 1-21) months [T1 7 (1-21), T2 5.5 (1-19) and T3 4 (1-19) months] and median overall survival 9 (range 1-36) months [T1 10 (1-31), T2 8 (1-36) and T3 6.5 (2-29) months]. Treatment was extremely well tolerated with 2% (25/1253) G3/4 toxicity (mainly G3 fatigue and anemia) and no toxic deaths. We observed the longer OS 14 (range 7-36) months in a subset of squamous NSCLC patients receiving immunotherapy after metronomic oral vinorelbine. CONCLUSION: We confirmed MOV as an extremely safe treatment in a large real world population of advanced NSCLC with an interesting activity mainly consisting of long-term disease stabilization. We speculate the possibility of a synergistic effect with subsequent immunotherapy.

Paraheliotropism in Robinia pseudoacacia L.: an efficient strategy to optimise photosynthetic performance under natural environmental conditions.

We assessed the contribution of leaf movements to PSII photoprotection against high light and temperature in Robinia pseudoacacia. Gas exchange and chlorophyll a fluorescence measurements were performed during the day at 10:00, 12:00, 15:00 and 18:00 hours on leaves where paraheliotropic movements were restrained (restrained leaves, RL) and on control unrestrained leaves (UL). RL showed a strong decrease of net photosynthesis (A(n)), stomatal conductance (g(sH2O)), quantum yield of electron transport (PhiPSII), percentage of photosynthesis inhibited by O2 (IPO) and photochemical quenching (q(P)) in the course of the day, whereas, a significant increase in C(i)/C(a) and NPQ was observed. Contrary to RL, UL had higher photosynthetic performance that was maintained at elevated levels throughout the day. In the late afternoon, A(n), g(sH2O), PhiPSII and q(P) of RL showed a tendency to recovery, as compared to 15:00 hours, even if the values remained lower than those measured at 10:00 hours and in UL. In addition, contrary to UL, no recovery was found in F(v)/F(m) at the end of the study period in RL. Data presented suggest that in R. pseudoacacia, leaf movements, by reducing light interception, represent an efficient, fast and reversible strategy to overcome environmental stresses such as high light and temperature. Moreover, paraheliotropism was able to protect photosystems, avoiding photoinhibitory damage, leading to a carbon gain for the plant.

Viability and function in lymphocytes cultured from the horse, chicken, and mouse: effects of different leukocyte enrichment techniques.

Methods of lymphocyte enrichment tend to vary across species, with the most common techniques employed being density-gradient separation and erythrocyte lysis buffer enrichment. In this study, we assessed lymphocyte viability and proliferation of avian, equine, and murine lymphocytes enriched by a commercial density-gradient technique and under identical, standardized culture conditions. The results of this study clearly show that, under identical enrichment and culture conditions, lymphocyte viability and function can be quite different among the equine, bird, and mouse species. Secondly, the type of enrichment technique employed in the mouse can impact the quality of the immune data generated.

Inter- and intra-variability of seed germination traits of Carpobrotus edulis N.E.Br. and its hybrid C. affine acinaciformis.

Invasions by alien Carpobrotus spp. have been recognised as one of the most severe threats to Mediterranean climate coastal ecosystems, and Carpobrotus is considered one of the most widespread invasive alien genera in the Mediterranean Basin. The aims of this study were to characterise seed germination of both C. edulis and its hybrid C. aff. acinaciformis, in terms of photoperiod, temperature and salinity. Inter- and intra-specific variability in the responses to photoperiod (12/12 h light and total darkness), constant temperatures (5, 10, 15, 20, 25 °C) and an alternating temperature regime (25/10 °C), salt stress (0, 125, 250, 500 mm NaCl) and the recovery of seed germination were evaluated for two seed lots of C. edulis and two of its hybrid C. aff. acinaciformis. All the tested seed lots achieved higher germination percentages in the light, with respect to total darkness. In relation to temperature, the two C. edulis seed lots did not show a preference, while the two C. aff. acinaciformis seed lots differed in their germination response, one germinating more at the lowest temperatures (5 and 10 °C) and one at the highest (20 and 25 °C). For all seed lots, highest germination occurred without NaCl (0 mm) and germination decreased with increasing salinity. Different germination requirements in a saline substrate were not detected for C. edulis, while they were observed for C. aff. acinaciformis. Marked differences were detected in recovery responses between the two taxa. C. edulis has the ability to germinate over a wide time window throughout the year. This study identified significant differences in seed production, seed mass, germination requirements (temperature) and salinity tolerance for both C. edulis and C. aff. acinaciformis. Our results indicate the extreme versatility of the hybrid forms to germinate in a wide range of natural conditions and habitats.

Leptin induces tissue factor expression in human peripheral blood mononuclear cells: a possible link between obesity and cardiovascular risk?

BACKGROUND: Obesity is a major modifiable risk factor for cardiovascular disease. Leptin, the hormone synthesized and released primarily by adipose tissue and found increased in obese individuals, has been implicated in the regulation of inflammation and arterial and venous thrombosis. OBJECTIVE: To investigate the role of tissue factor (TF), the pivotal agonist of the clotting cascade, as a link between obesity and cardiovascular disease. METHODS AND RESULTS: In 15 obese patients, plasma levels of leptin and TF as well as TF expression in resting and endotoxin-stimulated mononuclear leukocytes (MN) were increased when compared with healthy donors. In a selected sample of obese patients, loss of body weight led to decreased circulating leptin levels, accompanied by a reduction in plasma TF as well as in TF expression, both in resting and endotoxin-stimulated MN. In subsequent in vitro experiments, leptin was incubated with MN from healthy subjects. Leptin induced TF activity and antigen in a dose-dependent fashion, as assessed by clotting assay and ELISA, respectively. Increased migration of c-Rel/p65 into the nucleus, as determined by EMSA, and development of TF mRNA in monocytes, as assessed by RT-PCR, were observed. Experiments with mitogen-activated protein kinase (MAPK) inhibitors, indicated the involvement of p38 and ERK1/2 pathways. CONCLUSIONS: The presence of TF-expressing MN in blood from obese subjects and the in vitro induction of TF by pharmacologic concentrations of leptin in MN from healthy subjects suggest that TF expression by leptin-stimulated monocytes may contribute to the cardiovascular risk associated with obesity.

Effects of NaCl stress on seed germination and seedling development of Brassica insularis Moris (Brassicaceae).

Brassica insularis is a protected plant that grows on both coastal and inland cliffs in the western Mediterranean Basin. The objective of this study was to test if any variability exists in the salt stress response during seed germination and seedling development in this species relative to its provenance habitat. Variability among three populations in the salt stress effects on seed germination and recovery under different temperatures was evaluated. The effect of nebulisation of a salt solution on seedling development was evaluated between populations growing at different distances from the sea. Seeds of B. insularis could germinate at NaCl concentrations up to 200 mm. Seed viability was negatively affected by salt, and recovery ability decreased with increasing temperature or salinity. Inter-population variability was detected in salt response during the seed germination phase, as well as in seedling salt spray tolerance. The inland population seedlings had drastically decreased survival and life span and failed to survive to the end of the experiment. In contrast, at least 90% of the coastal seedlings survived, even when sprayed at the highest frequency with salt solution. This study allowed investigation of two natural factors, soil salinity and marine aerosols, widely present in the B. insularis habitat, and provided the first insights into ecology of this protected species and its distribution in the Mediterranean. These results might be useful in understanding the actual distributions of other species with the same ecology that experience these same abiotic parameters.

Pain-motor integration in the primary motor cortex in Parkinson's disease.

BACKGROUND: In Parkinson's disease (PD), the influence of chronic pain on motor features has never been investigated. We have recently designed a technique that combines nociceptive system activation by laser stimuli and primary motor cortex (M1) activation through transcranial magnetic stimulation (TMS), in a laser-paired associative stimulation design (Laser-PAS). In controls, Laser-PAS induces long-term changes in motor evoked potentials reflecting M1 long-term potentiation-like plasticity, arising from pain-motor integration. OBJECTIVE: We here examined the possible influence of chronic pain on motor responses to Laser-PAS in patients with PD, with and without chronic pain. METHODS: We compared motor responses to Laser-PAS in healthy subjects and in patients with PD, with and without chronic pain. RESULTS: Unlike controls, we found reduced responses to Laser-PAS in patients with PD, with and without pain. Patients off and on dopaminergic therapy had similar responses to Laser-PAS. When comparing responses to Laser-PAS in patients with and without pain, the two patients' subgroups had similar abnormalities. When we compared patients with pain in the body region investigated with Laser-PAS, with those with pain in other body regions, we found prominent changes in patients with homotopic pain. Finally, when comparing Laser-PAS with the original PAS protocol, which combines electric peripheral nerve stimuli and TMS, in patients without pain and those with homotopic pain, we found similar responses to both techniques in patients without pain, whereas Laser-PAS induced greater abnormalities than PAS in patients with pain. CONCLUSIONS: In PD, chronic pain degrades response to Laser-PAS through abnormal pain-motor integration.

Angiotensin-converting enzyme inhibitors downregulate tissue factor synthesis in monocytes.

Angiotensin-converting enzyme (ACE) inhibitors reduce the risk of recurrent myocardial infarction in patients with left ventricular dysfunction. Tissue factor (TF), the initiator of blood coagulation, plays a pivotal role in arterial thrombosis that occurs after atherosclerotic plaque fissuring. Because monocytes synthesize TF and contain several components of the renin-angiotensin system, we investigated the possibility that ACE inhibitors could modulate monocyte TF expression. Mononuclear leukocytes from healthy volunteers were incubated with endotoxin in the presence or absence of different ACE inhibitors. Captopril reduced TF expression in endotoxin-stimulated mononuclear leukocytes, as measured by a 1-stage clotting assay and ELISA analysis, by approximately 60%. The effect was dose-dependent and was attributable to ACE inhibition, given that other ACE inhibitors, such as idrapril or fosinopril, and losartan, an antagonist of the angiotensin II AT(1) receptor, caused a comparable reduction in TF activity. Reverse transcriptase-polymerase chain reaction indicated that endotoxin-mediated increased levels of TF mRNA were inhibited by ACE inhibitors. Moreover, endotoxin-induced nuclear factor-kappaB translocation to the promoter region of the gene encoding for TF was markedly inhibited by captopril. The finding that ACE inhibitors and angiotensin II AT(1) antagonists can potentially modulate TF expression by mononuclear cells has important biological and therapeutic implications for the evolution of thrombi. Our results suggest that the anti-ischemic effect of these drugs might be explained, at least in part, by their ability to reduce TF expression in monocytes.

Roflumilast inhibits leukocyte-platelet interactions and prevents the prothrombotic functions of polymorphonuclear leukocytes and monocytes.

UNLABELLED: ESSENTIALS: Thrombosis is a major comorbidity in patients with chronic obstructive pulmonary disease (COPD). Roflumilast is a selective phosphodiesterase type-4 (PDE4) inhibitor approved for treatment of severe COPD. PDE4 blockade by roflumilast inhibits prothrombotic functions of neutrophils and monocytes. PDE4 inhibitors may reduce thrombotic risk in COPD as well as in other vascular diseases. BACKGROUND: Roflumilast, an oral selective phosphodiesterase type 4 inhibitor, is approved for the treatment of severe chronic obstructive pulmonary disease (COPD). A recent meta-analysis of trials on COPD revealed that treatment with roflumilast was associated with a significant reduction in the rate of major cardiovascular events. The mechanisms of this effect remain unknown. OBJECTIVES: We tested the hypothesis that roflumilast N-oxide (RNO), the active metabolite of roflumilast, curbs the molecular mechanisms required for leukocyte-platelet (PLT) interactions and prevents the prothrombotic functions of polymorphonuclear leukocytes (PMNs) and monocytes (MNs). METHODS: Using well-characterized in vitro models, we analysed the effects of RNO on: (i) PMN adhesiveness; (ii) the release of neutrophil extracellular traps (NETs); and (iii) tissue factor expression in MNs. Key biochemical events underlying the inhibitory effects of RNO were defined. RESULTS AND CONCLUSIONS: In PMNs, RNO prevented phosphoinositide 3-kinase (PI3K)-dependent phosphorylation of Akt on Ser473, and Src family kinase (SFK)-mediated Pyk2 phosphorylation on Tyr579-580, while inducing protein kinase A-mediated phosphorylation of C-terminal Src kinase, the major negative regulator of SFKs. Modulation of these signaling pathways by RNO resulted in a significant impairment of PMN adhesion to activated PLTs or human umbilical vein endothelial cells, mainly mediated by inhibition of the adhesive function of Mac-1. Moreover RNO curbed SFK/PI3K-mediated NET release by PMNs adherent on fibrinogen-coated surfaces. In MNs interacting with activated PLTs, RNO curbed PI3K-mediated expression of tissue factor. The efficacy of RNO was significantly potentiated by formoterol, a long acting ß-adrenergic receptor agonist. This study reveals novel antithrombotic activities by which roflumilast may exert protective effects against cardiovascular comorbodities in COPD.

Cytofluorimetric evaluation of DNA ploidy in lung cancer: a bronchoscopic study.

The study of the biological characteristics of lung cancer is gaining more and more interest both because of their potential role as prognostic indicators and for therapeutic reasons. The DNA content estimated by flow cytometry in surgical samples of non-small cell lung cancer (NSCLC) has already been demonstrated to be correlated with survival in these patients. From July 1990 to February 1992 we analyzed the DNA distribution of bronchoscopic biopsies from 88 patients with lung cancer (18 small cell lung cancer, SCLC, and 68 NSCLC, two unspecified histology). Twenty-eight tumors (34.6%) had a diploid DNA distribution, while 53 were aneuploid (65.4%). A correlation was found between DNA ploidy and survival. Evaluation of the DNA content in bronchoscopic samples in a large series of patients could determine the role of this analysis prior to surgery in NSCLC and its value as a marker with respect to prognosis and response to therapy in SCLC.