When your MR linac is down: Can an automated pipeline bail you out of trouble?

PURPOSE: The unique treatment delivery technique provided by magnetic resonance guided radiotherapy (MRgRT) can represent a significant drawback when system fail occurs. This retrospective study proposes and evaluates a pipeline to completely automate the workflow necessary to shift a MRgRT treatment to a traditional radiotherapy linac. MATERIAL AND METHODS: Patients undergoing treatment during the last MRgRT system failure were retrospectively included in this study. The core of the proposed pipeline was based on a tool able to mimic the original MR linac dose distribution. The so obtained dose distribution (AUTO) has been compared with the distribution obtained in the conventional radiotherapy linac (MAN). Plan comparison has been performed in terms of time required to obtain the final dose distribution, DVH parameters, dosimetric indices and visual analogue scales scoring by radiation oncologists. RESULTS: AUTO plans generation has been obtained within 10 min for all the considered cases. All AUTO plans were found to be within clinical tolerance, showing a mean target coverage variation of 1.7% with a maximum value of 4.3% and a minimum of 0.6% when compared with MAN plans. The highest OARs mean variation has been found for rectum V60 (6.7%). Dosimetric indices showed no relevant differences, with smaller gradient measure in favour of AUTO plans. Visual analogue scales scoring has confirmed comparable plan quality for AUTO plans. CONCLUSION: The proposed workflow allows a fast and accurate generation of automatic treatment plans. AUTO plans can be considered equivalent to MAN ones, with limited clinical impact in the worst-case scenario.

VMAT-like plans for magnetic resonance guided radiotherapy: Addressing unmet needs.

PURPOSE: VMAT delivery technique is currently not applicable to Magnetic Resonance-guided radiotherapy (MRgRT) hybrid systems. Aim of this study is to evaluate an innovative VMAT-like (VML) delivery technique. MATERIAL AND METHODS: First, planning and dosimetric evaluation of the MRgRT VML treatment have been performed on 10 different disease sites and the results have been compared with the corresponding IMRT plans. Then, in the second phase, 10 of the most dosimetrically challenging locally advanced pancreas treatment plans have been retrospectively re-planned using the VML approach to explore the potentiality of this new delivery technique. Finally, VML robustness was evaluated and compared with the IMRT plans, considering a lateral positioning error of ± 5 mm. RESULTS: In phase one, all VML plans were within constraint for all OARs. When PTV coverage is considered, in the 50% of the cases VML PTV coverage is equal or higher than in IMRT plan. In the remaining 50%, the highest target under coverage difference in comparison with IMRT plan is -1.71%. The mean and maximum treatment time differences (VML-IMRT) is 0.2 min and 3.1 min respectively. In phase two, the treatment time variation (VML-IMRT), shows a mean, maximum and minimum variations of 1.3, 4.6 and -0.6 min respectively. All VML plans have a better target coverage if compared with IMRT plans, keeping in any case the OARs constraints within tolerance. VML doesn't increase plan robustness. CONCLUSION: VMAT-like treatment approach appeared to be an efficient planning solution and it was decided to clinically implement it in daily practice, especially in the frame of hypo fractionated treatments.

Dosimetric accuracy of dual isocenter irradiation in low magnetic field resonance guided radiotherapy system for extended abdominal tumours.

PURPOSE: Due to limited field size of Magnetic Resonance Linear Accelerators (MR-Linac), some treatments could require a dual-isocenter planning approach to achieve a complete target coverage and thus exploit the benefits of the online adaptation. This study evaluates the dosimetric accuracy of the dual-isocenter intensity modulated radiation therapy (IMRT) delivery technique for MR-Linac. MATERIAL AND METHODS: Dual-isocenter multi leaf collimator (MLC) and couch accuracy tests have been performed to evaluate the delivery accuracy of the system. A mono-isocenter plan delivered in clinical practice has then been retrospectively re-planned with dual-isocenter technique. The dual-isocenter plan has been re-calculated and delivered on a 3-dimensional (3D) ArcCHECK phantom and 2-dimensional (2D) films to assess its dosimetric accuracy in terms of gamma analysis. Clinical and planning target volume (CTV and PTV respectively) coverage robustness was then investigated after the introduction of ± 2 mm and ± 5 mm positioning errors by shifting the couch. RESULTS: MLC and couch accuracy tests confirmed the system accuracy in delivering a dual-isocenter irradiation. 2D/3D gamma analysis results occurred always to be above 95% if considered a gamma criteria 1%/2 mm and 1%/1 mm respectively for the 2D and 3D analysis. The mean variations for CTV D98% and PTV V95% were 0.2% and 1.1% respectively when positioning error was introduced separately in each direction, while the maximum observed variations were 0.9% (CTV) and 3.7% (PTV). CONCLUSION: The dosimetric accuracy of dual-isocenter irradiation has been verified for MR-Linac, achieving accurate and robust treatment strategy and improving dose conformality also in presence of targets whose extension exceeds the nominal maximum field size.

Antioxidant activity of phenolic acids and their metabolites: synthesis and antioxidant properties of the sulfate derivatives of ferulic and caffeic acids and of the acyl glucuronide of ferulic acid.

The main metabolites of caffeic and ferulic acids (ferulic acid-4'-O-sulfate, caffeic acid-4'-O-sulfate, and caffeic acid-3'-O-sulfate), the most representative phenolic acids in fruits and vegetables, and the acyl glucuronide of ferulic acid were synthesized, purified, and tested for their antioxidant activity in comparison with those of their parent compounds and other related phenolics. Both the ferric reducing antioxidant power (FRAP) assay and the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging method were used. Ferulic acid-4'-O-sulfate and ferulic acid-4'-O-glucuronide exhibited very low antioxidant activity, while the monosulfate derivatives of caffeic acid were 4-fold less efficient as the antioxidant than caffeic acid. The acyl glucuronide of ferulic acid showed strong antioxidant action. The antioxidant activity of caffeic acid-3'-O-glucuronide and caffeic acid-4'-O-glucuronide was also studied. Our results demonstrate that some of the products of phenolic acid metabolism still retain strong antioxidant properties. Moreover, we first demonstrate the ex vivo synthesis of the acyl glucuronide of ferulic acid by mouse liver microsomes, in addition to the phenyl glucuronide.

Absorption of phenolic acids in humans after coffee consumption.

Despite extensive literature describing the biological effects of polyphenols, little is known about their absorption from diet, one major unresolved point consisting of the absorption of the bound forms of polyphenols. In this view, in the present work we studied the absorption in humans of phenolic acids from coffee, a common beverage particularly rich in bound phenolic acids, such as caffeic acid, ferulic acid, and p-coumaric acid. Coffee brew was analyzed for free and total (free + bound) phenolic acids. Chlorogenic acid (5'-caffeoylquinic acid), a bound form of caffeic acid, was present in coffee at high levels, while free phenolic acids were undetectable. After alkaline hydrolysis, which released bound phenolic acids, ferulic acid, p-coumaric acid, and high levels of caffeic acid were detected. Plasma samples were collected before and 1 and 2 h after coffee administration and analyzed for free and total phenolic acid content. Two different procedures were applied to release bound phenolic acids in plasma: beta-glucuronidase treatment and alkaline hydrolysis. Coffee administration resulted in increased total plasma caffeic acid concentration, with an absorption peak at 1 h. Caffeic acid was the only phenolic acid found in plasma samples after coffee administration, while chlorogenic acid was undetectable. Most of caffeic acid was present in plasma in bound form, mainly in the glucuronate/sulfate forms. Due to the absence of free caffeic acid in coffee, plasma caffeic acid is likely to be derived from hydrolysis of chlorogenic acid in the gastrointestinal tract.

Inhibition of neutrophil apoptosis by acrolein: a mechanism of tobacco-related lung disease?

Cigarette smoking is known to contribute to inflammatory diseases of the respiratory tract by promoting recruitment of inflammatory-immune cells such as neutrophils and perhaps by altering neutrophil functional properties. We investigated whether acrolein, a toxic unsaturated aldehyde found in cigarette smoke, could directly affect neutrophil function. Exposure of freshly isolated human neutrophils to acrolein markedly inhibited spontaneous neutrophil apoptosis as indicated by loss of membrane asymmetry and DNA fragmentation and induced increased neutrophil production of the chemokine interleukin-8 (IL-8). Acrolein (1--50 microM) was found to induce marked activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases (MAPKs), and inhibition of p38 MAPK activation by SB-203580 prevented acrolein-induced IL-8 release. However, inhibition of either ERK or p38 MAPK did not affect acrolein-dependent inhibition of apoptosis. Acrolein exposure prevented the activation of caspase-3, a crucial step in the execution of neutrophil apoptosis, presumably by direct inhibition of the enzyme. Our results indicate that acrolein may contribute to smoke-induced inflammatory processes in the lung by increasing neutrophil recruitment and reducing neutrophil clearance by apoptosis.

Modulation of ceramide-induced NF-kappaB binding activity and apoptotic response by caffeic acid in U937 cells: comparison with other antioxidants.

Ceramide acts as second messenger in the signal transduction triggered by a variety of stress stimuli and extracellular agents. Stress response through ceramide is involved in the development of many human diseases, such as atherosclerosis, inflammation, neurodegenerative disorders, and acquired immunodeficiency syndrome. Dietary polyphenols have been reported to exert a beneficial effect on the onset and development of most of these human chronic-degenerative pathologies. However, the mechanisms underlying this beneficial effect are mostly not understood at the present. To investigate the ability of polyphenols in modulating fundamental cellular functions, we studied the effect of caffeic acid, a widespread phenolic acid largely present in human diet, in the modulation of ceramide-induced signal transduction pathway leading to apoptosis in U937 cells, in comparison with other established antioxidants of nutritional interest (N-acetylcysteine, d-alpha-tocopherol acetate and ascorbic acid). Our results indicate that caffeic acid efficiently inhibits both ceramide-induced NF-kappaB binding activity and apoptosis at micromolar concentration. Other antioxidants tested are totally ineffective in inhibiting apoptosis, although affecting NF-kappaB activation. Caffeic acid was found to inhibit protein tyrosine kinase activity, suggesting that this mechanism can be on the basis of the inhibition of apoptosis. Our results suggest that dietary caffeic acid might modulate ceramide-induced signal transduction pathway and NF-kappaB activation through either antioxidant and nonantioxidant mechanisms.

In vitro inhibition of the activity of phosphorylase kinase, protein kinase C and protein kinase A by caffeic acid and a procyanidin-rich pine bark (Pinus marittima) extract.

Caffeic acid (CA) is a common constituent of human diet while pine bark extract (PBE) is utilized either as nutritional supplement or as phytochemical remedy for different diseases. CA and PBE, are reported as efficient antioxidants and more recently have been described to modulate cellular response to oxidative challenge and to possess many other biological activities, i.e. anti-inflammatory, antimutagenic, antitumoral effects. In order to investigate in depth the mechanism of action of these polyphenols, the effects of CA and PBE on the activity of some protein kinases involved in the regulation of fundamental cellular processes were studied in vitro: phosphorylase kinase (PhK), protein kinase A (PKA), protein kinase C (PKC). PBE at the concentration of 20 microg/ml (corresponding to 69 microM catechin equivalents) inhibited PKA, PhK and PKC by about 90, 59, 57%, respectively, while 100 microM CA inhibited by 37, 52 and 54%, respectively. Considerable inhibitions have been still observed at even lower concentrations of CA and PBE. For PhK and PKA, the inhibition follows a non-competitive mechanism. CA also inhibits PKC activity in a partially purified cellular extract. The results suggest a possible involvement of CA and PBE in modulation of cellular functions.

Benzoic and cinnamic acid derivatives as antioxidants: structure-activity relation.

The antioxidant activity of four derivatives of benzoic acid was systematically compared with the activity of the four homologous derivatives of cinnamic acid. The couples of compounds differed for the kind of aromatic substitution (p-hydroxy, p-hydroxymethoxy, p-hydroxydimethoxy, dihydroxy). The antioxidant activity was measured using (i) a competition kinetic test, to measure the relative capacity to quench peroxyl radical and (ii) the in vitro oxidative modification of human low-density lipoprotein (LDL), initiated by 2,2'-azobis(amidinopropane) dihydrochloride or catalyzed by Cu(II). In both models, cinnamic acids were more efficient than their benzoic counterparts. As for the influence of the aromatic substitution, in the kinetic test the antioxidant activity increased in the sequence p-hydroxy < p-hydroxymethoxy < dihydroxy < p-hydroxydimethoxy. In contrast, in the LDL system, the dihydroxy acids had an antioxidant capacity equal to or higher than that of the p-hydroxydimethoxy acids.

Effect of caffeic acid on tert-butyl hydroperoxide-induced oxidative stress in U937.

Nonvitamin phenolic compounds are ubiquitous in food plants and therefore potentially present in human plasma in a diet-dependent concentration. The aim of this study was to evaluate the ability of caffeic acid, a phenolic acid with antioxidant activity, to affect cellular response in U937 human monocytic cells to t-butyl hydroperoxide-induced oxidative stress. In our experimental conditions caffeic acid was incorporated into cells without any cytotoxic effect. Caffeic acid-treated cells showed an increased resistance to oxidative challenge, as revealed by an higher percent of survival and the maintenance of an higher proliferative capacity in respect to control cells. This effect seems to be due to the ability of caffeic acid to reduce glutathione depletion and to inhibit lipid peroxidation during tBOOH treatment. It can be concluded that caffeic acid exerts an antioxidant action inside the cell, responsible for the observed modulation of the cellular response to oxidative challenge. Due to its presence in the diet, therefore, caffeic acid may play a role in the modulation of oxidative processes in vivo.

Beer affects oxidative stress due to ethanol in rats.

The relationship between chronic moderate beer consumption and oxidative stress was studied in rats. Animals were fed three different isocaloric diets for six weeks: a beer-containing diet (30% w/w), an ethanol-supplemented diet (1.1 g/100 g, the same as in the beer diet) and an alcohol-free basal diet. At the end of the feeding period, rats were analyzed for plasma and liver oxidative status. Some livers were isolated and exposed to ischemia-reperfusion to assess the additional oxidative stress determined by reperfusion. No significant differences in plasma antioxidant status were found among the three dietary groups. Lipoproteins from the beer group, however, showed a greater propensity to resist lipid peroxidation. Ischemia caused a decrease in liver energy and antioxidant status in all groups. Nevertheless, ATP was lower in the livers of rats exposed to the ethanol diet. During reperfusion, lipoperoxidation increased significantly in all groups. However, livers obtained from ethanol-treated rats showed the higher formation of lipoperoxides. In conclusion, a moderate consumption of beer in a well-balanced diet did not appear to cause oxidative stress in rats; moreover, probably through its minor components, beer could attenuate the oxidative action of ethanol by itself.

Antioxidant Activity of Different Phenolic Fractions Separated from an Italian Red Wine.

Using liquid/liquid extraction, three fractions were obtained from an Italian red wine containing single polyphenolic subfractions: (1) phenolic acids and quercetin-3-glucuronide, (2) catechins and quercetin-3-glucoside, and (3) anthocyanins. Beside the scavenging capacity of the different fractions against hydroxyl and peroxyl radicals, the in vitro inhibition of low density lipoprotein oxidation and platelet aggregation (two main events in the pathogenesis of atherosclerosis) were tested. The antioxidant activity of the fractions has been compared with that of the original red wine before and after dealcoholization. The anthocyanin fraction was the most effective both in scavenging reactive oxygen species and in inhibiting lipoprotein oxidation and platelet aggregation. This higher activity can be explained by both its high concentration in red wine and its antioxidant efficiency, which, at least for peroxyl radical scavenging, was three times as high as that of the other two fractions. Our results suggest that anthocyanins could be the key component in red wine in light of the protection against cardiovascular diseases, although this hypothesis needs in vivo evidence.

Effect of caffeic acid dietary supplementation on the antioxidant defense system in rat: an in vivo study.

Dietary supplementation of caffeic acid (0.2 and 0.8% w/w) in rats resulted in a statistically significant increase of alpha-tocopherol both in plasma and lipoprotein. While caffeic acid was not detectable in plasma under fasting conditions, in postprandial plasma it was present at micromole concentrations, doubling plasma total antioxidant capacity. Lipoproteins from caffeic acid-fed rats were more resistant than control to Cu2+-catalyzed oxidation, despite the lack of incorporation of caffeic acid in the particles. No significant effects on plasma and liver copper concentration, nor the increase in liver of Mn-superoxide dismutase reported in copper deficiency, were detected. These results demonstrate the physiological relevance of caffeic acid and its antioxidant action in vivo, through both a direct contribution to the antioxidant defense system and a sparing effect on alpha-tocopherol.

Reptile heme protein structure: X-ray crystallographic study of the aquo-met and cyano-met derivatives of the loggerhead sea turtle (Caretta caretta) myoglobin at 2.0 A resolution.

The X-ray crystal structures of the aquo-met and cyano-met derivatives of the loggerhead sea turtle (Caretta caretta) myoglobin have been determined at 2.0 A resolution (R = 0.182, and 0.178, respectively). The results here reported, representing the first reptile globin solved by X-ray crystallography, have been analyzed in parallel with data for related monomeric hemoproteins, and indicate a strong overall structural similarity between the loggerhead sea turtle and mammalian myoglobins, reflected by the 63% amino acid identity of their primary structures. The root-mean-square deviation between the entire polypeptide backbones of loggerhead sea turtle and sperm whale myoglobins, after structure superposition, is 0.83 A. Upon cyanide binding to the protein distal site, the iron-bound water molecule present in the aquo-met form is displaced by the incoming ligand. Cyanide is oriented towards the inner part of the heme distal site forming a Fe-C-N angle of 133 degrees.

Detection of cystathionine and lanthionine ketimines in human urine.

A recently developed HPLC procedure for the determination of cystathionine ketimine (CK) and lanthionine ketimine (LK) has been applied to the detection of these compounds in human urine. The assay has taken advantage of the selective production of an absorbance at 380 nm, not seen with other amino acids, when the two ketimines are reacted with phenylisothiocyanate. Coelution with authentic phenylthiohydantoin derivatives of CK and LK and the identical absorption spectra establish the identity of the compounds found in the urine with the synthetic products. Quantitation of the two ketimines by HPLC indicates that the excretion of CK and LK is respectively 606 micrograms and 84 micrograms per g of creatinine as mean values of 10 healthy subjects of both sexes, 20-40 years old, in the early morning voided urine.

Bovine brain ketimine reductase.

We report the purification from bovine brain of an NAD(P)H-dependent reductase which actively reduces a new class of cyclic unsaturated compounds, named ketimines. Ketimines arise from the transamination of some sulphur-containing amino acids, such as L-cystathionine, S-aminoethyl-L-cysteine and L-lanthionine. The enzyme also reduces delta 1-piperidine 2-carboxylate, the carbon analog of aminoethylcysteine ketimine. Some kinetic and molecular properties of this enzyme have been determined. Subcellular localization and regional brain distribution have also been studied. The ketimine reductase activity was found to be associated with the soluble fraction, and was located prevalently in the cerebellum and cerebral cortices. Cyclothionine and 1,4-thiomorpholine-3,5-dicarboxylic acid, the enzymatic reduction products of cystathionine ketimine and lanthionine ketimine, respectively, have been detected in bovine brain, thus suggesting a role of this enzyme in their biosynthesis.

Purification and characterization of a ketimine-reducing enzyme.

An NAD(P)H-dependent reductase able to reduce a new class of cyclic unsaturated compounds named ketimines has been detected and purified 2500-fold from pig kidney. Some molecular and kinetic properties of this enzyme have been determined. The enzymatic reduction proceeds with a classical ping-pong mechanism and some results suggest that the true substrate has the ketiminic structure and is in equilibrium with the enaminic and keto-open forms. As previously described, ketimines arise from the deamination of a number of sulfur-containing amino acids, i.e. L-cystathionine, L-lanthionine and S-aminoethyl-L-cysteine, catalyzed by a widespread mammalian transaminase. The enzymatic reduction products of ketimines have been identified as cyclothionine, 1,4-thiomorpholine 3,5-dicarboxylic acid and 1,4-thiomorpholine 3-carboxylic acid. Some of these compounds have been detected in mammals, thus suggesting a possible role of this enzyme in their biosynthesis.

Italian multicenter study of reversible cerebral ischemic attacks. Part 5. Risk factors and cerebral atherosclerosis.

As part of a prospective study, the influence of several premorbid and environmental factors on the presence, extent and severity of cerebral vessel atherosclerosis was studied in 462 patients with clinical diagnosis of RIA who underwent cerebral angiography. The extent and severity of atherosclerosis of the cerebral vessels was quantified using extracranial and intracranial cerebrovascular scores (ECS, ICS) based on the number and severity of the lesions in 11 extracranial and 21 intracranial arterial segments. Results of univariate and multivariate analyses indicate that the presence of atherosclerotic changes of cerebral vessels, as shown by angiography, was strongly related with age in both sexes. The lesions were more frequent in males, in particular under age 55. Elevated cholesterol was associated with a higher incidence of atherosclerotic lesions. Smoking was associated with a higher incidence of extracranial lesions. Age, smoking and history of hypertension were the best predictors of the extent and severity of cerebral vessel atherosclerosis.

Cysteamine oxygenase: possible involvement of superoxide ion in the catalytic mechanism.

The reaction catalyzed by cysteamine oxygenase on cysteamine in the presence of phenazine methosulphate as cofactor like compound is inhibited by nitroblue tetrazolium, a scavenger of superoxide ions. The reaction is not inhibited by superoxide dismutase and allyl alcohol and it is not activated by superoxide ions produced in solution. Nitroblue tetrazolium is reduced by cysteamine or mercaptoethanol and phenazine methosulphate. This reaction is completely inhibited by superoxide dismutase. In the presence of cysteamine oxygenase the reduction with mercaptoethanol is greatly enhanced and it is only partially inhibited by superoxide dismutase. According to these data a reaction mechanism is proposed in which superoxide ions and thiyl radicals are produced at the active site during catalysis.

The transamination of L-cystathionine, L-cystine and related compounds by a bovine kidney transaminase.

An enzyme which actively transaminates L-cystathionine, L-cystine, L-lanthionine and S-aminoethyl-L-cysteine has been purified from bovine kidney. The transaminase appears to be pure up to 90% and probably consists of two subunits of similar molecular mass of about 47 kDa. The enzymatic products arising from the transamination of L-cystathionine and related compounds spontaneously cyclize into ketiminic structures, which are the immediate precursors of unusual imino acids recovered in biological materials. The specificity towards other amino acid and oxo acid acceptors is similar to the specificity exhibited by rat kidney glutamine transaminase. This suggests that the sulfur amino acid transaminations that have been described could be performed by the bovine kidney glutamine transaminase.