Alfredo Vannacci

Nuclear poly(ADP-ribose) polymerase-1 rapidly triggers mitochondrial dysfunction.

To obtain further information on time course and mechanisms of cell death after poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation, we used HeLa cells exposed for 1 h to the DNA alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine. This treatment activated PARP-1 and caused a rapid drop of cellular NAD(H) and ATP contents, culminating 8–12 h later in cell death. PARP-1 antagonists fully prevented nucleotide depletion and death. Interestingly, in the early 60 min after challenge with N-methyl-N′-nitro-N-nitrosoguanidine, mitochondrial membrane potential and superoxide production significantly increased, whereas cellular ADP contents decreased. Again, these events were prevented by PARP-1 inhibitors, suggesting that PARP-1 hyperactivity leads to mitochondrial state 4 respiration. Mitochondrial membrane potential collapsed at later time points (3 h), when mitochondria released apoptosis-inducing factor and cytochrome c. Using immunocytochemistry and targeted luciferase transfection, we found that, despite an exclusive localization of PARP-1 and poly(ADP-ribose) in the nucleus, ATP levels first decreased in mitochondria and then in the cytoplasm of cells undergoing PARP-1 activation. PARP-1 inhibitors rescued ATP (but not NAD(H) levels) in cells undergoing hyper-poly(ADP-ribosyl)ation. Glycolysis played a central role in the energy recovery, whereas mitochondria consumed ATP in the early recovery phase and produced ATP in the late phase after PARP-1 inhibition, further indicating that nuclear poly(ADP-ribosyl)ation rapidly modulates mitochondrial functioning. Together, our data provide evidence for rapid nucleus-mitochondria cross-talk during hyper-poly(ADP-ribosyl)ation-dependent cell death.

Inducible Nitric Oxide Synthase Expression in Human Colorectal Cancer : Correlation with Tumor Angiogenesis

To investigate the potential involvement of the nitric oxide (NO) pathway in colorectal carcinogenesis, we correlated the expression and the activity of inducible nitric oxide synthase (iNOS) with the degree of tumor angiogenesis in human colorectal cancer. Tumor samples and adjacent normal mucosa were obtained from 46 surgical specimens. Immunohistochemical expression of iNOS, vascular endothelial growth factor (VEGF), and CD31 was analyzed on paraffin-embedded tissue sections. iNOS activity and cyclic GMP levels were assessed by specific biochemical assays. iNOS protein expression was determined by Western blot analysis. iNOS and VEGF mRNA levels were evaluated using Northern blot analysis. Both iNOS and VEGF expressions correlated significantly with intratumor microvessel density (r(s) = 0.31, P = 0.02 and r(s) = 0.67, P < 0.0001, respectively). A significant correlation was also found between iNOS and VEGF expression (P = 0.001). iNOS activity and cyclic GMP production were significantly higher in the cancer specimens than in the normal mucosa (P < 0.0001 and P < 0.0001, respectively), as well as in metastatic tumors than in nonmetastatic ones (P = 0.002 and P = 0.04, respectively). Western and Northern blot analyses confirmed the up-regulation of the iNOS protein and gene in the tumor specimens as compared with normal mucosa. NO seems to play a role in colorectal cancer growth by promoting tumor angiogenesis.

Stillbirths: recall to action in high-income countries

Variation in stillbirth rates across high-income countries and large equity gaps within high-income countries persist. If all high-income countries achieved stillbirth rates equal to the best performing countries, 19,439 late gestation (28 weeks or more) stillbirths could have been avoided in 2015. The proportion of unexplained stillbirths is high and can be addressed through improvements in data collection, investigation, and classification, and with a better understanding of causal pathways. Substandard care contributes to 20-30% of all stillbirths and the contribution is even higher for late gestation intrapartum stillbirths. National perinatal mortality audit programmes need to be implemented in all high-income countries. The need to reduce stigma and fatalism related to stillbirth and to improve bereavement care are also clear, persisting priorities for action. In high-income countries, a woman living under adverse socioeconomic circumstances has twice the risk of having a stillborn child when compared to her more advantaged counterparts. Programmes at community and country level need to improve health in disadvantaged families to address these inequities.

Inhibition of 5-lipoxygenase by MK886 augments the antitumor activity of celecoxib in human colon cancer cells

Cyclooxygenase (COX)-2 and 5-lipoxygenase (5-LOX) are key enzymes involved in arachidonic acid metabolism. Their products, prostaglandins and leukotrienes, are involved in colorectal tumor development. We aimed at evaluating whether combined blocking of the COX-2 and 5-LOX pathways might have additive antitumor effects in colorectal cancer. The expression/activity of COX-2 and 5-LOX were assessed in 24 human colorectal cancer specimens. The effects of the COX-2 inhibitor celecoxib and the 5-LOX inhibitor MK886 on prostaglandin E2 and cysteinyl leukotriene production, tumor cell proliferation, cell apoptosis, and Bcl-2/Bax expression were evaluated in the Caco-2 and HT29 colon cancer cells. We also investigated the effect of the enzymatic inhibition on mitochondrial membrane depolarization, one of the most important mechanisms involved in ceramide-induced apoptosis. Up-regulation of the COX-2 and 5-LOX pathways was found in the tumor tissue in comparison with normal colon mucosa. Inhibition of either COX-2 or 5-LOX alone resulted in activation of the other pathway in colon cancer cells. Combined treatment with 10 μmol/L celecoxib and MK886 could prevent this activation and had additive effects on inhibiting tumor cell proliferation, inducing cell apoptosis, decreasing Bcl-2 expression, increasing Bax expression, and determining mitochondrial depolarization in comparison with treatment with either inhibitor alone. The administration of the ceramide synthase inhibitor fumonisin B1 could prevent some of these antineoplastic effects. In conclusion, our study showed that inhibition of 5-LOX by MK886 could augment the antitumor activity of celecoxib in human colorectal cancer. [Mol Cancer Ther 2006;5(11):2716–26]

Nuclear poly (ADP-ribose) polymerase-1 radpidly triggers mitochondrial dysfunction

To obtain further information on time course and mechanisms of cell death after poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation, we used HeLa cells exposed for 1 h to the DNA alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine. This treatment activated PARP-1 and caused a rapid drop of cellular NAD(H) and ATP contents, culminating 8–12 h later in cell death. PARP-1 antagonists fully prevented nucleotide depletion and death. Interestingly, in the early 60 min after challenge with N-methyl-N′-nitro-N-nitrosoguanidine, mitochondrial membrane potential and superoxide production significantly increased, whereas cellular ADP contents decreased. Again, these events were prevented by PARP-1 inhibitors, suggesting that PARP-1 hyperactivity leads to mitochondrial state 4 respiration. Mitochondrial membrane potential collapsed at later time points (3 h), when mitochondria released apoptosis-inducing factor and cytochrome c. Using immunocytochemistry and targeted luciferase transfection, we found that, despite an exclusive localization of PARP-1 and poly(ADP-ribose) in the nucleus, ATP levels first decreased in mitochondria and then in the cytoplasm of cells undergoing PARP-1 activation. PARP-1 inhibitors rescued ATP (but not NAD(H) levels) in cells undergoing hyper-poly(ADP-ribosyl)ation. Glycolysis played a central role in the energy recovery, whereas mitochondria consumed ATP in the early recovery phase and produced ATP in the late phase after PARP-1 inhibition, further indicating that nuclear poly(ADP-ribosyl)ation rapidly modulates mitochondrial functioning. Together, our data provide evidence for rapid nucleus-mitochondria cross-talk during hyper-poly(ADP-ribosyl)ation-dependent cell death.

Eating disorders and body image disturbances among ballet dancers, gymnasium users and body builders.

Background: Eating disorders are frequent among elite performers of certain sports or physical activities; however, little is known about non-professional performers. Method: 113 female non-elite ballet dancers, 54 female gymnasium users, 44 male non-competitive body builders, 105 female controls and 30 male controls were evaluated using the Body Uneasiness Test, the State-Trait Anxiety Inventory, the Beck Depression Inventory, and the Eating Disorder Examination 12th edition (EDE-12). Results: Non-elite ballet dancers reported the highest prevalence of eating disorders (anorexia nervosa 1.8%; bulimia nervosa 2.7%; eating disorders not otherwise specified 22.1%), followed by gymnasium users (anorexia nervosa 2.6%; eating disorders not otherwise specified 18%). Significant differences (p < 0.01) between athletes and their controls were found in the following parameters (median values): Beck Depression Inventory (female dancers 5.7, gymnasium users 6.1, female controls 2.8, body builders 1.6, and male controls 1.3), Body Uneasiness Test (female dancers 1.08, gymnasium users 0.62, female controls 0.54, body builders 0.35, and male controls 0.27), EDE total scores (female dancers 1.6, gymnasium users 1.7, female controls 1.0, body builders 1.0, and male controls 0.4), EDE – restraint subscale scores (female dancers 0.8, gymnasium users 1.6, female controls 0.0, body builders 0.8, and male controls 0.0), EDE – eating concern subscale scores (female dancers 0.4, gymnasium users 0.2, female controls 0.0, body builders 0.0, and male controls 0.0), EDE – weight concern subscale scores (female dancers 2.1, gymnasium users 2.1, female controls 1.6, body builders 1.4, and male controls 0.5), and EDE – shape concern subscale scores (female dancers 2.7, gymnasium users 2.8, female controls 2.0, body builders 2.1, and male controls 0.9). EDE scores were highly related to Body Uneasiness Test scores, especially in non-elite ballet dancers and in non-competitive body builders (p < 0.01). Conclusion: Non-professional performers of sports emphasising thinness or muscularity, such as ballet and body-building, show a high degree of body uneasiness and inappropriate eating attitudes and behaviours.

Carbon monoxide: the bad and the good side of the coin, from neuronal death to anti-inflammatory activity.

Abstract.The double origin of carbon monoxide (CO) as an atmospheric pollutant or as an endogenous gaseous modulator of many pathophysiological processes prompted us to review some aspects of the bad side and of the good side of coin among the pleiotropic effects of CO.On the bad side of the coin, we focus on the interval form in acute CO poisoning, discussing experimental evidence suggesting that the delayed neuropathology after CO poisoning is a free radical-driven event. In this context, we challenge the mandatory place of hyperbaric oxygen therapy (HBO) in CO poisoning as a possible summation of oxy-radicals generated by HBO and the free radical cascade set in motion during the reoxygenation phase of acute CO-poisoning. We also discuss an opposing view, which provides evidence suggesting that HBO therapy actually decreases the load of free radicals in acute CO-poisoning and may be beneficial in preventing delayed neuropsychiatric sequelae.On the good side of the coin, we briefly outline the endogenous generation of CO and the leading role of heme–oxygenases (HO) in relation to the place of CO in biology and medicine.The main focus of this section is on the growing literature on CO and inflammation. Here we report on in-vitro and in-vivo studies on the modulation afforded by exogenously administered/endogenously produced CO in a variety of experimental and clinical settings of inflammation.Our recent studies on experimental models of allergic inflammation are also discussed, and the CO-releasing molecules envisaged as potential anti-inflammatory drugs suitable for clinical use.

Use, Attitudes and Knowledge of Complementary and Alternative Drugs (CADs) Among Pregnant Women: a Preliminary Survey in Tuscany.

To explore pregnant womens use, attitudes, knowledge and beliefs of complementary and alternative drugs (CADs) defined as products manufactured from herbs or with a natural origin. A preliminary survey was conducted among 172 pregnant women in their third trimester of pregnancy, consecutively recruited in two obstetrical settings; 15 women were randomly selected to compute a test-to-retest analysis. Response rate was 87.2%. Test-to-retest analysis showed a questionnaires reproducibility exceeding a K-value of 0.7 for all items. Mean age was 32.4 ± 0.4 years; most women were nulliparae (62.7%). The majority of subjects (68%) declared to have used one or more CADs during their lifetime; 48% of pregnant women reported taking at least one CAD previously and during the current pregnancy. Womens habitual use of CADs meant they were at higher risk of taking CADs also during pregnancy (adjusted odds ratio = 10.8; 95% confidence interval: 4.7–25.0). Moreover, 59.1% of the subjects were unable to correctly identify the type of CADs they were using. The majority of women resorted to gynecologists as the primary information source for CADs during pregnancy, while they mainly referred to herbalists when not pregnant. Habitual use of CADs seems to be a strong predictor for their ingestion also during pregnancy; in addition most subjects were unable to correctly identify the products they were taking. In the light of the scanty data concerning the safety of CADs during pregnancy, these preliminary results confirm the need to investigate thoroughly the situation of pregnant women and CADs consumption.

Amatoxin poisoning: A 15-year retrospective analysis and follow-up evaluation of 105 patients

Introduction. Fatalities due to mushroom poisonings are increasing worldwide, with more than 90% of deaths resulting from ingestion of amatoxin-containing species. Methods. A retrospective evaluation of the history and clinical outcome of each patient treated from 1988 to 2002 in the Toxicological Unit of Careggi General Hospital (University of Florence, Italy) for amatoxin poisoning. Data included the biological parameters monitored, the treatment protocols used (intensive fluid and supportive therapy, restitution of the altered coagulation factors, multiple-dose activated charcoal, mannitol, dexamethasone, glutathione, and penicillin G), and outpatient follow-up evaluations. Results. The clinical data of 111 patients were evaluated; their biological parameters were monitored every 12–24 hours until discharge. Two patients died; both were admitted to the hospital more than 60 hours after mushroom ingestion. Of all the laboratory parameters evaluated, the evolution of hepatic transaminases and prothrombin activity over four days were the most predictive indicators of recovery or death. Our follow-up evaluation of 105 patients demonstrated that our survivors recovered completely. Conclusions. Our experience indicates that the protocol used in our Toxicologicy Unit is effective for amatoxin poisoning, and that all patients treated within 36 hours after mushroom ingestion were cured without sequelae.

Heme Oxygenase-1 and the Ischemia-Reperfusion Injury in the Rat Heart

Carbon monoxide (CO) is a signaling gas produced intracellularly by heme oxygenase (HO) enzymes using heme as a substrate. During heme breakdown, HO-1 and HO-2 release CO, biliverdin, and Fe2+. In this study, we investigated the effects of manipulation of the HO-1 system in an in vivo model of focal ischemia–reperfusion (FIR) in the rat heart. Male Wistar albino rats, under general anesthesia and artificial ventilation, underwent thoracotomy, the pericardium was opened, and a silk suture was placed around the left descending coronary artery; ischemia was induced by tightening the suture and was monitored for 30 min. Subsequently, the ligature was released to allow reperfusion lasting for 60 min. The first group of rats was sham operated and injected intraperitoneally (ip) with saline. The second group underwent FIR. The third group was treated ip 18 hr before FIR with hemin (4 mg/kg). The fourth group was pretreated ip 24 hr before FIR and 6 hr before hemin with zinc protoporphyrin IX (ZnPP-IX, 50 μg/kg). Specimens of the left ventricle were taken for determination of HO expression and activity, infarct size, malonyldialdehyde (MDA) production, and tissue calcium content. FIR led to a significant increase in the generation of MDA and notably raised tissue calcium levels. Induction of HO-1 by hemin significantly decreased infarct size, incidence of reperfusion arrhythmias, MDA generation, and calcium overload induced by FIR. These effects were prevented by the HO-1 inhibitor ZnPP-IX. The present experiments show that the concerted actions of CO, iron, and biliverdin/bilirubin modulate the FIR-induced myocardial injury.