Showing papers by "University of Perugia published in 2006"

Analysis of apoptosis by propidium iodide staining and flow cytometry

Abstract: Since its introduction, the propidium iodide (PI) flow cytometric assay has been widely used for the evaluation of apoptosis in different experimental models. It is based on the principle that apoptotic cells, among other typical features, are characterized by DNA fragmentation and, consequently, loss of nuclear DNA content. Use of a fluorochrome, such as PI, that is capable of binding and labeling DNA makes it possible to obtain a rapid (the protocol can be completed in about 2 h) and precise evaluation of cellular DNA content by flow cytometric analysis, and subsequent identification of hypodiploid cells. The original protocol enhanced the capacity for a rapid, quantitative measure of cell apoptosis. For this reason, since its publication, the PI assay has been widely used, as demonstrated by the large number of citations of the original paper and/or the continuous use of the method in many laboratories.

The Combined Effects of Tryptophan Starvation and Tryptophan Catabolites Down-Regulate T Cell Receptor ζ-Chain and Induce a Regulatory Phenotype in Naive T Cells

Abstract: Tryptophan catabolism is a tolerogenic effector system in regulatory T cell function, yet the general mechanisms whereby tryptophan catabolism affects T cell responses remain unclear. We provide evidence that the short-term, combined effects of tryptophan deprivation and tryptophan catabolites result in GCN2 kinase-dependent down-regulation of the TCR zeta-chain in murine CD8+ T cells. TCR zeta down-regulation can be demonstrated in vivo and is associated with an impaired cytotoxic effector function in vitro. The longer-term effects of tryptophan catabolism include the emergence of a regulatory phenotype in naive CD4+CD25- T cells via TGF-beta induction of the forkhead transcription factor Foxp3. Such converted cells appear to be CD25+, CD69-, CD45RBlow, CD62L+, CTLA-4+, BTLAlow and GITR+, and are capable of effective control of diabetogenic T cells when transferred in vivo. Thus, both tryptophan starvation and tryptophan catabolites contribute to establishing a regulatory environment affecting CD8+ as well as CD4+ T cell function, and not only is tryptophan catabolism an effector mechanism of tolerance, but it also results in GCN2-dependent generation of autoimmune-preventive regulatory T cells.

The endocrine function of adipose tissue: an update.

Abstract: Summary Adipose tissue secretes bioactive peptides, termed ‘adipokines’, which act locally and distally through autocrine, paracrine and endocrine effects. In obesity, increased production of most adipokines impacts on multiple functions such as appetite and energy balance, immunity, insulin sensitivity, angiogenesis, blood pressure, lipid metabolism and haemostasis, all of which are linked with cardiovascular disease. Enhanced activity of the tumour necrosis factor and interleukin 6 are involved in the development of obesity-related insulin resistance. Angiotensinogen has been implicated in hypertension and plasminogen activating inhibitor-1 (PAI-1) in impaired fibrinolysis. Other adipokines like adiponectin and leptin, at least in physiological concentrations, are insulin sparing as they stimulate beta oxidation of fatty acids in skeletal muscle. The role of resistin is less understood. It is implicated in insulin resistance in rats, but probably not in humans. Reducing adipose tissue mass, through weight loss in association with exercise, can lower TNF- α and IL-6 levels and increase adiponectin concentrations, whereas drugs such as thiazolinediones increase endogenous adiponectin production. In-depth understanding of the pathophysiology and molecular actions of adipokines may, in the coming years, lead to effective therapeutic strategies designed to protect against atherosclerosis in obese patients

The epidemiology of fungal infections in patients with hematologic malignancies: the SEIFEM-2004 study.

Abstract: BACKGROUND AND OBJECTIVES: The aim of this study was to evaluate the incidence and outcome of invasive fungal infections (IFI) in patients with hematologic malignancies. DESIGN AND METHODS: This was a retrospective cohort study of patients admitted between 1999 and 2003 to 18 hematology wards in Italy. Each participating center provided information on all patients with newly diagnosed hematologic malignancies admitted during the survery period and on all episodes of IFI experienced by these patients. RESULTS: The cohort was formed of 11,802 patients with hematologic malignacies: acute leukemia (myeloid 3012, lymphoid 1173), chronic leukemia (myeloid 596, lymphoid 1104), lymphoma (Hodgkin's 844, non-Hodgkin's 3457), or multiple myeloma (1616). There were 538 proven or probable IFI (4.6%); 373 (69%) occurred in patients with acute myeloid leukemia. Over half (346/538) were caused by molds (2.9%), in most cases Aspergillus spp. (310/346). The 192 yeast infections (1.6%) included 175 cases of candidemia. Overall and IFI-attributable mortality rates were 2% (209/11802) and 39% (209/538), respectively. The highest IFI-attributable mortality rates were associated with zygomycosis (64%) followed by fusariosis (53%), aspergillosis (42%), and candidemia (33%). INTERPRETATION AND CONCLUSIONS: Patients with hematologic malignancies are currently at higher risk of IFI caused by molds than by yeasts, and the incidence of IFI is highest among patients with acute myeloid leukemia. Aspergillus spp are still the most common pathogens, followed by Candida spp. Other agents are rare. The attributable mortality rate for aspergillosis has dropped from 60-70% to approximately 40%. Candidemia-related mortality remains within the 30-40% range reported in literature although the incidence has decreased.

Relationship of plasma polyunsaturated fatty acids to circulating inflammatory markers.

Abstract: Aims: Persons with high intake of polyunsaturated fatty acids (PUFAs) have lower cardiovascular morbidity and mortality. The protective effect of PUFAs is mediated by multiple mechanisms, including their antiinflammatory properties. The association of physiological PUFA levels with pro- and antiinflammatory markers has not been established. Methods and Results: In 1123 persons (aged 20–98 yr), we examined the relationship between relative concentration of fatty acids in fasting plasma and level of inflammatory markers. Adjusting for age, sex, and major confounders, lower arachidonic and docosahexaenoic acids were associated with significantly higher IL-6 and IL-1ra and significantly lower TGF. Lower -linolenic acid was associated with higherC-reactiveproteinandIL-1ra,andlowereicosapentaenoicacid was associated with higher IL-6 and lower TGF. Lower docosahexaenoic acid was strongly associated with lower IL-10. Total n-3 fatty acids were associated with lower IL-6 (P 0.005), IL-1ra (P 0.004), and TNF (P 0.040) and higher soluble IL-6r (P 0.001), IL-10 (P 0.024), and TGF (P 0.0012). Lower n-6 fatty acid levels weresignificantlyassociatedwithhigherIL-1ra(P0.026)andlower TGF(P0.014).Then-6ton-3ratiowasastrong,negativecorrelate of IL-10. Findings were similar in participants free of cardiovascular diseases and after excluding lipids from covariates. Conclusions: In this community-based sample, PUFAs, and especially total n-3 fatty acids, were independently associated with lower levels of proinflammatory markers (IL-6, IL-1ra, TNF, C-reactive protein) and higher levels of antiinflammatory markers (soluble IL6r, IL-10, TGF) independent of confounders. Our findings support the notion that n-3 fatty acids may be beneficial in patients affected by diseases characterized by active inflammation. (J Clin Endocrinol Metab 91: 439–446, 2006)

A clinical outcome-based prospective study on venous thromboembolism after cancer surgery: the @RISTOS project.

Abstract: Summary Background Data: The epidemiology of venous thromboembolism (VTE) after cancer surgery is based on clinical trials on VTE prophylaxis that used venography to screen deep vein thrombosis (DVT). However, the clinical relevance of asymptomatic venography-detected DVT is unclear, and the population of these clinical trials is not necessarily representative of the overall cancer surgery population. Objective: The aim of this study was to evaluate the incidence of clinically overt VTE in a wide spectrum of consecutive patients undergoing surgery for cancer and to identify risk factors for VTE. Methods: @RISTOS was a prospective observational study in patients undergoing general, urologic, or gynecologic surgery. Patients were assessed for clinically overt VTE occurring up to 30 5 days after surgery or more if the hospital stay was longer than 35 days. All outcome events were evaluated by an independent Adjudication Committee. Results: A total of 2373 patients were included in the study: 1238 (52%) undergoing general, 685 (29%) urologic, and 450 (19%) gynecologic surgery. In-hospital prophylaxis was given in 81.6% and postdischarge prophylaxis in 30.7% of the patients. Fifty patients (2.1%) were adjudicated as affected by clinically overt VTE (DVT, 0.42%; nonfatal pulmonary embolism, 0.88%; death 0.80%). The incidence of VTE was 2.83% in general surgery, 2.0% in gynecologic surgery, and 0.87% in urologic surgery. Forty percent of the events occurred later than 21 days from surgery. The overall death rate was 1.72%; in 46.3% of the cases, death was caused by VTE. In a multivariable analysis, 5 risk factors were identified: age above 60 years (2.63, 95% confidence interval, 1.21‐5.71), previous VTE (5.98, 2.13‐16.80), advanced cancer (2.68, 1.37‐5.24), anesthesia lasting more than 2 hours (4.50, 1.06‐19.04), and bed rest longer than 3 days (4.37, 2.45‐7.78). Conclusions: VTE remains a common complication of cancer surgery, with a remarkable proportion of events occurring late after surgery. In patients undergoing cancer surgery, VTE is the most common cause of death at 30 days after surgery.

d-Dimer Testing to Determine the Duration of Anticoagulation Therapy

Abstract: Background The optimal duration of oral anticoagulation in patients with idiopathic venous thromboembolism is uncertain. Testing of d-dimer levels may play a role in the assessment of the need for prolonged anticoagulation. Methods We performed d-dimer testing 1 month after the discontinuation of anticoagulation in patients with a first unprovoked proximal deep-vein thrombosis or pulmonary embolism who had received a vitamin K antagonist for at least 3 months. Patients with a normal d-dimer level did not resume anticoagulation, whereas those with an abnormal d-dimer level were randomly assigned either to resume or to discontinue treatment. The study outcome was the composite of recurrent venous thromboembolism and major bleeding during an average follow-up of 1.4 years. Results The d-dimer assay was abnormal in 223 of 608 patients (36.7%). A total of 18 events occurred among the 120 patients who stopped anticoagulation (15.0%), as compared with 3 events among the 103 patients who resumed anticoagulation (2.9%), for an adjusted hazard ratio of 4.26 (95% confidence interval [CI], 1.23 to 14.6; P = 0.02). Thromboembolism recurred in 24 of 385 patients with a normal d-dimer level (6.2%). Among patients who stopped anticoagulation, the adjusted hazard ratio for recurrent thromboembolism among those with an abnormal d-dimer level, as compared with those with a normal d-dimer level, was 2.27 (95% CI, 1.15 to 4.46; P = 0.02). Conclusions Patients with an abnormal d-dimer level 1 month after the discontinuation of anticoagulation have a significant incidence of recurrent venous thromboembolism, which is reduced by the resumption of anticoagulation. The optimal course of anticoagulation in patients with a normal d-dimer level has not been clearly established. (ClinicalTrials.gov number, NCT00264277.)

Search for neutral MSSM Higgs bosons at LEP

Abstract: The four LEP collaborations, ALEPH, DELPHI, L3 and OPAL, have searched for the neutral Higgs bosons which are predicted by the Minimal Supersymmetric standard model (MSSM). The data of the four collaborations are statistically combined and examined for their consistency with the background hypothesis and with a possible Higgs boson signal. The combined LEP data show no significant excess of events which would indicate the production of Higgs bosons. The search results are used to set upper bounds on the cross-sections of various Higgs-like event topologies. The results are interpreted within the MSSM in a number of “benchmark” models, including CP-conserving and CP-violating scenarios. These interpretations lead in all cases to large exclusions in the MSSM parameter space. Absolute limits are set on the parameter cosβ and, in some scenarios, on the masses of neutral Higgs bosons.

Uric acid and inflammatory markers

Abstract: Aims The role of uric acid (UA) in the process of atherosclerosis and atherotrombosis is controversial. Epidemiological studies have recently shown that UA may be a risk factor for cardiovascular diseases and a negative prognostic marker for mortality in subjects with pre-existing heart failure. Methods and results We evaluate a relationship between UA levels and several inflammatory markers in 957 subjects, free of severe renal failure, from a representative Italian cohort of persons aged 65–95. Plasma levels of UA and white blood cell (WBC) and neutrophil count, C-reactive protein, interleukin-1 receptor antagonist (IL-1ra), interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6r), interleukin-18 (IL-18), and tumor necrosis factor-α (TNF-α) were measured. Complete information on potential confounders was collected using standard methods. WBC ( P =0.0001), neutrophils ( P <0.0001), C-reactive protein ( P <0.0001), IL-1ra ( P <0.0001), IL-6 ( P =0.0004), sIL-6r ( P =0.002), IL-18 ( P <0.0001), TNF-α ( P =0.0008), and the percentage of subjects with abnormally high levels of C-reactive protein ( P =0.004) and IL-6 ( P =<0.0001) were significantly higher across UA quintiles. After adjustment for age, sex, behaviour- and disease-related confounders, results were virtually unchanged. In subjects with UA within the normal range, UA was significantly and independently associated with neutrophils count, C-reactive protein, IL-6, IL-1ra, IL-18, and TNF-α, whereas non-significant trends were observed for WBC ( P =0.1) and sIL-6r ( P =0.2). Conclusion A positive and significant association between UA and several inflammatory markers was found in a large population-based sample of older persons and in a sub-sample of participants with normal UA. Accordingly, the prevalence of abnormally high levels of C-reactive protein and IL-6 increased significantly across UA quintiles.

A convergent model for cognitive dysfunctions in Parkinson's disease: the critical dopamine–acetylcholine synaptic balance

Abstract: Summary Parkinson's disease is classically characterised as a motor neurodegenerative disorder. Motor symptoms in the disorder are secondary to an altered dopamine–acetylcholine balance due to reduced striatal dopaminergic tone and subsequent cholinergic overactivity. In the past, anticholinergic drugs were given to improve motor aspects of the disease. There is now an increasing interest in the cognitive and non-motor symptoms of Parkinson's disease and in cholinesterase-inhibitor therapy for dementia associated with Parkinson's disease. In this Personal View, we reconsider the dopamine–acetylcholine balance theory and look at recent clinical findings and the possible cooperative role of dopamine and acetylcholine in the induction and maintenance of the long-lasting changes of striatal and cortical synaptic plasticity. We also discuss a convergent versus parallel model to explain cognitive dysfunctions in Parkinson's disease according to dopamine–acetylcholine dependent alterations in synaptic plasticity.

Microencapsulated Pancreatic Islet Allografts Into Nonimmunosuppressed Patients With Type 1 Diabetes First two cases

Abstract: Time-related decline of human islet allograft (TX) function in generally immunosuppressed type 1 diabetic patients (1–5) has led us, after years of preclinical study (6), to initiate a phase 1 pilot clinical trial of microencapsulated TX into 10 nonimmunosuppressed patients with type 1 diabetes, under permission and surveillance by the Italian Ministry of Health (file no. 19382, PRE 805, 5 September 2003). ### Human islet procurement Human islets were isolated from single-donor pancreases according to the Edmonton protocol (1). Only preparations complying with standard quality control criteria (1) were considered for TX. The islets were cultured for 24 h in HAM F12 (Celbio, Milano, Italy), supplemented with antibiotics and 1.25% human albumin (Kedrion Spa, Milano, Italy) at 37°C in 95% air/CO2. ### Microencapsulation The islets were washed and thoroughly mixed with 1.6% endotoxin- and pyrogen-free sodium alginate (Stern Italia, Milan, Italy) that had been highly purified according to U.S. Pharmacopeia. Upon extrusion …

Quantitative assessment of minimal residual disease in acute myeloid leukemia carrying nucleophosmin (NPM1) gene mutations

Abstract: Mutations in exon 12 of the nucleophosmin (NPM1) gene occur in about 60% of adult AML with normal karyotype. By exploiting a specific feature of NPM1 mutants, that is insertion at residue 956 or deletion/insertion at residue 960, we developed highly sensitive, real-time quantitative (RQ) polymerase chain reaction (PCR) assays, either in DNA or RNA, that are specific for various NPM1 mutations. In all 13 AML patients carrying NPM1 mutations at diagnosis, cDNA RQ-PCR showed >30 000 copies of NPM1-mutated transcript. A small or no decrease in copies was observed in three patients showing partial or no response to induction therapy. The number of NPM1-mutated copies was markedly reduced in 10 patients achieving complete hematological remission (five cases: <100 copies; five cases: 580-5046 copies). In four patients studied at different time intervals, the number of NPM1 copies closely correlated with clinical status and predicted impending hematological relapse in two. Thus, reliable, sensitive RQ-PCR assays for NPM1 mutations can now monitor and quantify MRD in AML patients with normal karyotype and NPM1 gene mutations.

Evidence That Hydrogen Sulfide Exerts Antinociceptive Effects in the Gastrointestinal Tract by Activating KATP Channels

Abstract: Hydrogen sulfide (H(2)S) functions as a neuromodulator, but whether it modulates visceral perception and pain is unknown. Cystathionine beta-synthase (CBS) and cystathionine-gamma-lyase (CSE) mediate enzymatic generation of H(2)S in mammalian cells. Here we have investigated the role of H(2)S in modulating nociception to colorectal distension, a model that mimics some features of the irritable bowel syndrome. Four graded (0.4-1.6 ml of water) colorectal distensions (CRDs) were produced in conscious rats (healthy and postcolitic), and rectal nociception was assessed by measuring the behavioral response during CRD. Healthy rats were administered with sodium hydrogen sulfide (NaHS) (as a source of H(2)S), L-cysteine, or vehicle. In a second model, we investigated nociception to CRD in rats recovering from a chemically induced acute colitis. We found that CBS and CSE are expressed in the colon and spinal cord. Treating rats with NaHS resulted in a dose-dependent attenuation of CRD-induced nociception with the maximal effect at 60 micromol/kg (p < 0.05). Administration of L-cysteine, a CSE/CBS substrate, reduced rectal sensitivity to CRD (p < 0.05). NaHS-induced antinociception was reversed by glibenclamide, a ATP-sensitive K(+) (K(ATP)) channel inhibitor, and N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME), a nitric-oxide (NO) synthase inhibitor. The antinociceptive effect of NaHS was maintained during the resolution of colon inflammation induced by intrarectal administration of a chemical irritant. In summary, these data show that H(2)S inhibits nociception induced by CRD in both healthy and postcolitic rats. This effect is mediated by K(ATP) channels and NO. H(2)S-releasing drugs might be beneficial in treating painful intestinal disorders.

Chemistry and biological activity of natural and synthetic prenyloxycoumarins.

Abstract: Prenyloxycoumarins and prenyloxyfuranocoumarins (isopentenyloxy-, geranyloxy-, linear and cyclic sesquiterpenyloxy compounds and their biosynthetic derivatives) represent a family of secondary metabolites that have been considered for years just as intermediates of other coumarin-based compounds. Only in the last two decades these secondary metabolites have been recognized as interesting and valuable biologically active natural products. Up to now more than 160 compounds have been isolated from plants mainly belonging to the families of Rutaceae and Umbelliferae, comprising common edible vegetables and fruits like lemons, oranges and grapefruits. In view of the biological activity of some natural prenyloxycoumarins, very recently syntheses of structurally related analogs aimed to establish detailed structure-activity relationships have also been carried out. Many of the isolated prenyloxy- and prenyloxyfuranocoumarins and their semisynthetic derivatives were shown to exert in vitro and in vivo remarkable antitumoral, anti-inflammatory and anti-viral effects. The object of this review is to examine in detail the different types of prenyloxycoumarins and prenyloxyfuranocoumarins from the chemical, phytochemical and biological point of view.

Age effects on diffusion tensor magnetic resonance imaging tractography measures of frontal cortex connections in schizophrenia.

Abstract: Diffusion tensor magnetic resonance imaging (DT-MRI) has previously been used to investigate white matter tracts in schizophrenia, with inconsistent results The aim of the study was to use a novel method for tract-specific measurements of fronto-temporal fasciculi in early-onset schizophrenia We hypothesized that by making tract-specific measurements, clear diffusion abnormalities would be revealed in specific fasciculi in schizophrenia Measurements of diffusion anisotropy and mean diffusivity were localized within fronto-temporal fasciculi by forming 3-D reconstructions of the cingulum, uncinate, superior longitudinal, and inferior fronto-occipital fasciculi using diffusion tensor tractography We were limited in our ability to test our hypothesis by the important and surprising finding that age affected DT-MRI-based measures in schizophrenia patients in a different way from comparison subjects, most notably in the left superior longitudinal fasciculus The youngest schizophrenia patients that we studied had lower diffusion anisotropy than age-matched comparison subjects, but this difference diminished with increasing age The main conclusion of this study was that direct comparisons of absolute DT-MRI-based measures between individuals with schizophrenia and comparison subjects may be problematic and misleading because of underlying age-related differences in brain maturation between groups

Proinflammatory cytokines, adhesion molecules, and lymphocyte integrin expression in the internal jugular blood of migraine patients without aura assessed ictally.

Abstract: Objective.—The aim of the present research was to verify the levels of the soluble adhesion molecules sL- and sE-selectins, intercellular adhesion molecule (sICAM)-1, and vascular cell adhesion molecule-1 in serial samples of internal jugular venous blood taken from migraine patients without aura (MWoA) during attacks. The expression of leukocyte function antigen (LFA)-1 and very late activation antigen (VLA)-4 was also assessed on lymphocytes obtained from jugular venous blood. Levels of certain proinflammatory cytokines (tumor necrosis factor-α[TNF-α], interleukin-1β[IL-1β], IL-4, and IL-6) were also determined and correlated with those of adhesion molecules. Patients and Methods.—Seven MWoA patients were admitted in the hospital during attacks and blood samples were taken immediately after catheter insertion, at 1, 2, and 4 hours after attack onset, and within 2 hours after its termination. The levels of adhesion molecules and cytokines were measured with ELISA method. The expression of LFA-1 and VLA-4 was assessed by flow cytometry. Results.—A parallel transient increase of sICAM-1, TNF-α, and IL-6 was observed in the first 2 hours after attack onset compared with the time of catheter insertion (P < .0001, <.001, and <.003, respectively). The proportion of CD4+ and CD8+ T-cells expressing high levels of LFA-1 showed instead a progressive down-regulation with significantly lower percentages at 2 and 4 hours after attack onset (P < .01 and <.022, respectively). No variation in the percentage of VLA-4 expressing cells was observed at any time of the study. Conclusions.—The transient increase in sICAM-1 and TNF-α found in the internal jugular blood of MWoA patients assessed ictally can be induced by sensory neuropeptides released from activated trigeminal endings. The progressive decrease in sICAM-1 levels during attacks and the down-regulation of LFA-1 expression by lymphocytes could antagonize their transvascular migration, supporting the hypothesis of sterile inflammation in the dura mater during migraine attacks.

Comparison of the effects of continuous subcutaneous insulin infusion (CSII) and NPH-based multiple daily insulin injections (MDI) on glycaemic control and quality of life: results of the 5-nations trial

Abstract: Aims The goal of the study was to determine whether continuous subcutaneous insulin infusion (CSII) differs from a multiple daily injection (MDI) regimen based on neutral protamine hagedorn (NPH) as basal insulin with respect to glycaemic control and quality of life in people with Type 1 diabetes. Methods The 5-Nations trial was a randomized, controlled, crossover trial conducted in 11 European centres. Two hundred and seventy-two patients were treated with CSII or MDI during a 2-month run-in period followed by a 6-month treatment period, respectively. The quality of glycaemic control was assessed by HbA1c, blood glucose values, and the frequency of hypoglycaemic events. For the evaluation of the quality of life, three different self-report questionnaires have been assessed. Results CSII treatment resulted in lower HbA1c (7.45 vs. 7.67%, P < 0.001), mean blood glucose level (8.6 vs. 9.4 mmol/l, P < 0.001) and less fluctuation in blood glucose levels than MDI (± 3.9 vs. ± 4.3 mmol/l, P < 0.001). There was a marked reduction in the frequency of hypoglycaemic events using CSII compared with MDI, with an incidence ratio of 1.12 [95% confidence interval (CI): 1.08–1.17] and 2.61 (95% CI: 1.59–4.29) for mild and severe hypoglycaemia, respectively. The overall score of the diabetes quality of life questionnaire was higher for CSII (P < 0.001), and an improvement in pump users’ perception of mental health was detected when using the SF-12 questionnaire (P < 0.05). Conclusion CSII usage offers significant benefits over NPH-based MDI for individuals with Type 1 diabetes, with improvement in all significant metabolic parameters as well as in patients’ quality of life. Additional studies are needed to compare CSII with glargine- and detemir-based MDI.

Sequential BCG and electromotive mitomycin versus BCG alone for high-risk superficial bladder cancer: a randomised controlled trial

Abstract: Summary Background The rationale for combining anticancer drugs has not been applied consistently to use of intravesical agents for treatment of superficial bladder cancer, for which immunotherapeutic BCG and chemotherapeutic mitomycin seem to be a potentially effective combination. We aimed to do a prospective, randomised comparison of BCG alone with that of sequential BCG and electromotive mitomycin in patients with stage pT1 bladder cancer. Methods After transurethral resection and multiple biopsies, 212 patients with stage pT1 bladder cancer were randomly assigned to: 81 mg BCG infused over 120 min once a week for 6 weeks (n=105); or to 81 mg BCG infused over 120 min once a week for 2 weeks, followed by 40 mg electromotive mitomycin (intravesical electric current 20 mA for 30 min) once a week as one cycle for three cycles (n=107). Complete responders underwent maintenance treatment: those assigned BCG alone had one infusion of 81 mg BCG once a month for 10 months, and those assigned BCG and mitomycin had 40 mg electromotive mitomycin once a month for 2 months, followed by 81 mg BCG once a month as one cycle for three cycles. The primary endpoint was disease-free interval; secondary endpoints were time to progression; overall survival; and disease-specific survival. Analyses were done by intention to treat. This trial has been submitted for registration at the US National Cancer Institute website http://clinicaltrials.gov. Findings Median follow-up was 88 months (IQR 63–110). Patients assigned sequential BCG and electromotive mitomycin had higher disease-free interval than did those assigned BCG alone (69 months [95% CI 55–86] vs 21 months [15–54]; difference between groups 48 months [42–54], log-rank p=0·0012). Patients assigned sequential BCG and electromotive mitomycin also had lower recurrence (41·9% [32·7–51·5] vs 57·9% [48·7–67·5]; difference between groups 16·0% [2·7–29·3], log-rank p=0·0012); progression (9·3% [3·8–14·8] vs 21·9% [17·9–25·9]; difference between groups 12·6% [3·0–22·2], log-rank p=0·004); overall mortality (21·5% [13·5–29·5] vs 32·4% [23·4–41·4], difference between groups 10·9% [0·6–21·2], log-rank p=0·045); and disease-specific mortality (5·6% [1·2–10·0] vs 16·2% [6·1–23·3], difference between groups 10·6% [2·5–18·7], log-rank p=0·01). Side-effects were mainly localised to the bladder. Interpretation BCG-induced inflammation might increase the permeability of the bladder mucosa such that mitomycin can reach the target tissue more easily and exert its anticancer effect.

Correlation between B-RAFV600E mutation and clinico-pathologic parameters in papillary thyroid carcinoma: data from a multicentric Italian study and review of the literature.

Abstract: Recently, a somatic point mutation of the B-RAF gene (V600E) has been identified as the most common genetic event in papillary thyroid carcinoma (PTC), with a prevalence variable among different series. Since discordant data on the clinico-pathologic features of B-RAF mutated PTC are present in the literature, the aim of the present co-operative study was to establish the prevalence of this genetic alteration and to perform a genotype-phenotype correlation in a large cohort of patients with PTC. To this purpose, a series of 260 sporadic PTCs with different histological variants were included in the study. The mutational analysis of the B-RAF gene was performed either by RT-PCR followed by single-stranded conformational polymorphism or by PCR and direct sequencing. Statistical analyses were obtained by means of chi2/Fisher's exact test and t-test. Overall, a heterozygous T > A transversion at nucleotide 1799 (V600E) was found in 99 out of 260 PTCs (38%). According to the histological type of the tumor, the B-RAF (V600E) mutation was present in 48.3% of cases of classic PTCs (85 out of 176), in 17.6% (nine out of 51) of follicular variants of PTCs, in 21.7% (five out of 23) in other PTC variants and in none of the ten poorly differentiated tumors. B-RAF (V600E) was significantly associated with the classic variant of PTC (P = 0.0001) and with an older age at diagnosis (P = 0.01). No statistically significant correlation was found among the presence of B-RAF (V600E) and gender, tumor node metastasis (TNM), multicentricity of the tumor, stage at diagnosis and outcome. In conclusion, the present study reports the prevalence of B-RAF (V600E) (38%) in the largest series of sporadic PTCs, including 260 cases from three different Italian referring centers. This prevalence is similar to that calculated by pooling together all data previously reported, 39.6% (759 out of 1914 cases), thus indicating that the prevalence of this genetic event lies around 38-40%. Furthermore, B-RAF (V600E) was confirmed to be associated with the papillary growth pattern, but not with poorer differentiated PTC variants. A significant association of B-RAF mutation was also found with an older age at diagnosis, the mutation being very rare in childhood and adolescent PTCs. Finally, no correlation was found with a poorer prognosis and a worse outcome after a median follow-up of 72 months.

Immunity and tolerance to Aspergillus involve functionally distinct regulatory T cells and tryptophan catabolism

Abstract: The inherent resistance to diseases caused by Aspergillus fumigatus suggests the occurrence of regulatory mechanisms that provide the host with adequate defense without necessarily eliminating the fungus or causing unacceptable levels of host damage. In this study, we show that a division of labor occurs between functionally distinct regulatory T cells (Treg) that are coordinately activated by a CD28/B-7-dependent costimulatory pathway after exposure of mice to Aspergillus conidia. Early in infection, inflammation is controlled by the expansion, activation and local recruitment of CD4+CD25+ Treg capable of suppressing neutrophils through the combined actions of IL-10 and CTLA-4 on indoleamine 2,3-dioxygenase. The levels of IFN-gamma produced in this early phase set the subsequent adaptive stage by conditioning the indoleamine 2,3-dioxygenase-dependent tolerogenic program of dendritic cells and the subsequent activation and expansion of tolerogenic Treg, which produce IL-10 and TGF-beta, inhibit Th2 cells, and prevent allergy to the fungus. The coordinate activation of Treg may, however, be subverted by the fungus, as germinating conidia are capable of interfering with anti-inflammatory and tolerogenic Treg programs. Thus, regulation is an essential component of the host response in infection and allergy to the fungus, and its manipulation may allow the pathogen to overcome host resistance and promote disease.

Electronic transitions involved in the absorption spectrum and dual luminescence of tetranuclear cubane [Cu4I4(pyridine)4] cluster: a density functional theory/time-dependent density functional theory investigation.

Abstract: We present a combined density functional theory (DFT)/time-dependent density functional theory (TDDFT) study of the geometry, electronic structure, and absorption and emission properties of the tetranuclear "cubane" Cu4I4py4 (py = pyridine) system. The geometry of the singlet ground state and of the two lowest triplet states of the title complex were optimized, followed by TDDFT excited-state calculations. This procedure allowed us to characterize the nature of the excited states involved in the absorption spectrum and those responsible for the dual emission bands observed for this complex. In agreement with earlier experimental proposals, we find that while in absorption the halide-to-pyridine charge-transfer excited state (XLCT*) has a lower energy than the cluster-centered excited state (CC*), a strong geometrical relaxation on the triplet cluster-centered state surface leads to a reverse order of the excited states in emission.