Data on animal models are very encouraging but the safety and efficacy of this approach in humans has not yet been investigated. Therefore, our objectives were to study the safety and efficacy of a rapid infusion of manganese chloride (MnCl2) for cardiovascular magnetic resonance (CMR) in humans.Manganese based agents are intracellular and accumulate inside myocytes allowing for different imaging strategies compared to gadolinium contrasts. While previous agents release manganese very slowly in the circulation, MnCl1 calculations. MnCl2 was infused over three minutes at a total dose of 5 μMol/kg. Immediately after the infusion, and at 15 and 30 minutes later, new images were obtained and cardiac function re-evaluated.Fifteen healthy volunteers underwent a CMR scan on a 1.5 T scanner. Before the infusion, cardiac function was calculated and images of a short axis mid-ventricular slice were obtained using a 2D and 3D gradient-echo inversion recovery (GRE-IR) sequence, a phase-sensitive IR sequence and a single breath-hold segmented IR prepared steady-state precession acquisition for T1 values compared to baseline, sustained up to 30 minutes after the MnCl2 infusion . The 2D and 3D GRE-IR sequence showed the greatest increase in signal-to-noise ratio compared to the other sequences . There was a slight increase in systolic pressure and heart rate after three and four minutes of the infusion with normalization of these parameters thereafter. Patients showed good tolerance to MnCl2 with no major adverse events, despite all reporting transient facial flush.There was a significant decrease in T2 appears safe for human use. It effectively decreases myocardium T1, maintaining this effect for a relatively long period of time and allowing for the development of new imaging strategies in CMR, especially in ischemia research.In the short term, MnCl Cardiovascular magnetic resonance (CMR) has become one of the most important diagnostic tools in cardiology, especially in infarct detection and myocardial perfusion in coronary artery disease ,2. Gadol+ and Cl- ions. The manganese ions, which are strongly paramagnetic, behave in vivo as a calcium analogue, and actively enter myocytes through voltage-gated calcium channels [2 allows for the detection of ischemic tissues as well as infarcted areas with great accuracy [2 has not been associated with any serious adverse short term events [2 releases Mn2+ much more rapidly than MnDPDP allowing for more rapid uptake of the free manganese ions by myocytes, it may in one hand promote deleterious effects on heart function due to direct competition with Ca2+ with possible decrease in inotropism [In solution, MnCl2 freely dissociates into Mn2channels ,6. Once accuracy -9. Howevaccuracy . Despitem events . The usem events . Howeverotropism  at the s2 for CMR in humans. We found that low-dose MnCl2 appears to offer a safe profile with significant enhancement of myocardial tissue in different pulse sequence protocols.Based on these observations, the purpose of this study was to investigate the safety and efficacy of a rapid infusion of MnClFifteen healthy volunteers of both sexes with ages from 18 to 60 were prospectively selected for the study. Inclusion criteria consisted of absence of previous cardiovascular disease or risk factors  and normal left ventricular function . We excluded from the study individuals taking any chronic medication, current smokers, and those with a history of liver diseases, arrhythmias detected in a resting electrocardiogram (ECG) or with known absolute contra-indications for undergoing a CMR study.1 calculations .After the initial ECG to assess PR, QRS and QT intervals, subjects underwent a CMR exam without any contrast in a 1.5 T scanner  with a four-element phased-array coil. After localizer sequences, cine images for ventricular function evaluation were obtained using previously described steady-state free-precession (SSFP) sequences in short axis, two-chamber and four-chamber views. After that, a short axis mid-ventricular slice was chosen and four different imaging protocols acquired: (1) 2D gradient-echo inversion recovery (GRE-IR) sequence ; (2) 3D GRE-IR sequence ; (3) phase-sensitive IR sequence (same parameters as the 2D GRE-IR) and (4) a single breath-hold segmented IR prepared steady-state precession (sIR-SSFP) acquisition for T2 was manually infused over three minutes (1.67 μMol/kg/min). MnCl2 was manufactured in 10 ml vials of 20 mg/mL  and the total amount of MnCl2 per subject was calculated, drawn from the vial and diluted to 10 mL with normal saline. During and ten minutes after the infusion, heart rate, arterial pressure and any arrhythmias were recorded . Subjects were questioned regarding symptoms throughout the study.After acquiring these images, subjects were removed from inside the magnet without moving from the table. Venous access was obtained and 5 μMol/kg of MnClImmediately after the infusion, subjects entered the scanner once again and the same single slice images were obtained using the four previously described sequences with cine images being acquired shortly after that. At fifteen and thirty minutes after the end of the infusion, new single slice images were again obtained as described, with new cine images at thirty minutes only. At the end of the study, a final ECG was performed before the subject left the facility. After six months, all subjects were contacted by telephone and questioned about the occurrence of clinical events that included: death by any cause, myocardial infarction, angina symptoms , heart failure symptoms  and neurological disturbances .2 in humans, great care was taken to assure safety and ethical considerations in applying the drug. The local ethics committee required that the lowest dose of the drug was to be used (5 μMol/kg) since this concentration of manganese had already been used in humans with the commercially approved contrast MnDPDP. This dose along was chosen along with an infusion rate significantly lower than the reported doses shown to cause any adverse effects on heart function in animals. The ethics committee therefore understood that this new agent would provide a total dose of manganese similar to the one approved for MnDPDP while releasing Mn2+ more rapidly but well below a perilous rate as previously described. Finally, patients were closely monitored before, during and after the infusions and stayed in the lab for two hours after the procedure. We also took the care of contacting each individual six months after the infusion to assess any potential complications not predicted in the day of the CMR study.The local institutional review board approved the study methods and all subjects provided written informed consent. Since this was the first reported use of MnCl2, a manual region of interest (ROI) was drawn including all the myocardium for each of the pulse sequences and the mean signal intensity was chosen for each ROI. Signal to noise ratio (SNR) was calculated as the ratio between the ROI mean and the standard deviation of background noise. Contrast to noise ratio (CNR) was calculated as the difference between precontrast SNRs and each postcontrast values [All images were anonymized prior to analysis for subject name and timing of the study. All images were analyzed by the same reader , in random order. The choice of a single reader for all images was done due to the blind reading form described for image analysis as well as for the rather objective values obtained from this analysis, requiring very little subjective assessment. Cine images were evaluated for diastolic volume, systolic volume and ejection fraction using commercial software . Volumes were determined semi-automatically by drawing endocardial borders in each slice at systole and diastole from base to apex excluding papillary muscles according to established references . For sigt values . To calct values .2 in animals which used from eight to thirty subjects [Sample size calculation was not performed previous to the study; rather, the number of subjects was selected according to other similar studies with MnClsubjects ,17,18. WAll the 15 subjects selected  completed the study. No subject suffered any major adverse effect during the CMR exam or within six months of completing the study.2 infusion there was a significant increase in mean SNR in all sequences analyzed except in the sIR-SSFP sequence  [2 seems to be much more important regarding acute safety than the total dose used since free manganese directly competes with calcium for voltage channels in the heart. At the same time, if one wants to look at uptake by myocytes during ischemia , it is important to guarantee a very fast delivery of Mn2+ to the heart. Having that in mind, our data suggests that the infusion rate observed in our study guarantees a short term safety profile without significant clinical changes in heart rate, arterial pressure and ventricular function in patients without heart disease, while still permitting delivery of MnCl2 at a rate where possible ischemia detection might prove achievable. Whether higher infusion rates or total doses of the drug might exhibit the same safety profile with better efficacy remains to be shown.However, to be useful for human studies, it is mandatory that safety issues are taken into account since previous animal studies have shown that in very large doses manganese can induce acute heart failure . In our μMol/kg)  with infμMol/kg) ,18,24,25μMol/kg) . In our 2 [+. Despite that, the delivery of Mn2+ to myocytes using MnDPDP is very slow while MnCl2 promotes rapid availability of the ion, a potential advantage especially for stress ischemia studies. Nevertheless, if MnCl2 is to be used in humans over the established MnDPDP remains to be proven in a larger study in patients with ischemic heart disease. The other manganese compound used in humans was EVP 1001-1, a compound with a short vascular half-life of 1.5 minutes with flow-dependent differences in uptake similar to the uses proposed for MnCl2 [2 with an adequate safety profile as well.The only other manganese based contrast agent to have been used in humans previously for the study of the heart is MnDPDP, which is a chelated agent, and has different pharmacokinetics and biological distribution from MnCl2 -30. Withor MnCl2 ,27. This+ to the heart. Therefore, we did not evaluate other possible effects of MnCl2 on the liver or kidneys, did not measure the concentration of the ion in the blood or urine or its interaction with iron or copper metabolism. While we did make contact with the subjects six months after the exam regarding clinical consequences of manganese toxicity on other organs, the number of subjects studied and the period of observation do not allow us to reach any conclusions regarding long-term safety. A publication studying methcathinone users with high levels of whole blood manganese has shown that the mean time between extrapyramidal symptoms  and druxication . There axication ,34. Neve+ [Another limitation of this study is that we only looked at normal individuals without left ventricular dysfunction or ischemic heart disease. It is not known if the safety and efficacy data presented here can be reproduced in patients who already have borderline cardiac function or previous ischemia. While the main cardiotoxicity of manganese is related more to the extracellular than the intracellular concentration of Mn2+  the poss2.Finally, this preliminary data might be considered limited to further advance the use of substance in humans, especially in some countries where other safety measures would be required to allow for subsequent trials with MnCl2, preventing us from addressing whether higher doses would be tolerated, more efficient or even allowing for the study of concentration and signal enhancement relationships. Nevertheless, it had been shown with MnDPDP that increasing the dose to 10 or 15 μMol/kg did not yield further myocardial enhancement thus only allowing for an increase in side effects [The study was limited to a fixed infusion rate and total amount of MnCl effects . Finally2 (5 μMol/kg) has a good short-term cardiac safety profile in humans promoting a significant decrease in myocardium T1 for a relatively long time. From this data, we believe that new strategies to study coronary heart disease with CMR can be designed with special focus on ischemia with higher resolution sequences and out of the magnet protocols, taking advantage of the longer-lasting changes in relaxation times produced by the agent.In conclusion, our data suggests that low-dose MnClThe authors declare that they have no competing interests.JLF has participated in all phases of the study including its design, acquisition and interpretation of data and drafting of the manuscript; PS participated in the interpretation of data and drafting of the manuscript; JAS, GSF and JMK participated in the acquisition of data; ORC has participated in the study design, interpretation of data and drafting of the manuscript. All authors have read approved the final manuscript.


Schistosomiasis is one of the most important neglected tropical diseases and an effective control is unlikely in the absence of improved sanitation and vaccination. A new approach of oral vaccination with alginate coated chitosan nanoparticles appears interesting because their great stability and the ease of target accessibility, besides of chitosan and alginate immunostimulatory properties. Here we propose a candidate vaccine based on the combination of chitosan-based nanoparticles containing the antigen SmRho and coated with sodium alginate.In vitro release studies of protein showed great stability of coated nanoparticles in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Further in vivo studies was performed with different formulations of chitosan nanoparticles and it showed that oral immunization was not able to induce high levels of antibodies, otherwise intramuscular immunization induced high levels of both subtypes IgG1 and IgG2a SmRho specific antibodies. Mice immunized with nanoparticles associated to CpG showed significant modulation of granuloma reaction. Mice from all groups immunized orally with nanoparticles presented significant levels of protection against infection challenge with S. mansoni worms, suggesting an important role of chitosan in inducing a protective immune response. Finally, mice immunized with nanoparticles associated with the antigen SmRho plus CpG had 38% of the granuloma area reduced and also presented 48% of protection against of S. mansoni infection.Our results showed an efficient performance of protein loading of nanoparticles before and after coating with alginate. Characterization of the resulting nanoparticles reported a size around 430 nm and a negative zeta potential. Taken together, this results support this new strategy as an efficient delivery system and a potential vaccine against schistosomiasis. S. mansoni infection. This vaccination strategy offers many technical advantages, including the possibility of administration by oral route, which makes the vaccine safer than injectable vaccines and facilitates its use mainly in underdeveloped areas. Chitosan nanoparticles were prepared and characterized; the results showed that the formulation has features suitable to be delivery orally. Immunization studies suggest that the combination of chitosan nanoparticles associated to the antigen SmRho and CpG is an efficient vaccine candidate against schistosomiasis, which was able to modulate the granuloma area, that represents the major pathological response in schistosomiasis and also to induce protection against infection of S. mansoni.Schistosomiasis is one of the most important neglected tropical diseases and an effective control is unlikely in the absence of improved sanitation and vaccine. The selection of a suitable delivery system and an adjuvant to aid in the stimulation of the appropriate immune response is a critical step in the path to the development and employment of successful anti-schistosome vaccines. Here we propose a candidate vaccine based on chitosan nanoparticles associated with the antigen SmRho and coated with alginate, as an alternative strategy to induce protection against  Schistosomiasis remains one of the most prevalent diseases in the world and so a significant public health problem, especially in developing countries Current schistosomiasis control strategies are mainly based on chemotherapy but, despite decades of mass treatment, the number of infected people remains constant S. mansoniS. mansoni Rho1 on the parenchymal cells surrounding the vitellaria adds support to this suggestion The current schistosoma vaccine candidates prove not to be the most effective, so it is important to identify new antigens and to explore alternative vaccination strategies, including new adjuvants to improve vaccine efficacy Considering that schistosome infection occurs predominantly in areas of rural poverty in sub-Saharan Africa, Southeast Asia and tropical regions of the Americas Chitosan is the deacetylated form of chitin and has many properties suitable for vaccine delivery. It is a mucoadhesive polymer, biodegradable and biocompatible. In particular, its ability to stimulate cells from the immune system has been shown in several studies Alginate coated chitosan nanoparticles was recently described in vitro characteristics of chitosan nanoparticles associated to protein, for which the antigen Rho1-GTPase of S. mansoni was chosen, to be used as a candidate oral vaccine against schistosomiasis. Once in vitro characterization showed favorable data, its in vivo role was evaluated through mouse immunization. Added to that, chitosan was evaluated not only for its performance as a delivery system but also for its contribution due to its adjuvant properties.Herein, we proposed to evaluate Additionally, since a mixed Th1/Th2 response seems to be optimal for a schistosomiasis vaccine, then bacterial CpG motifs (which induce the production of IL-12 by DCs and macrophages that express the appropriate TLR95) can be used as adjuvants to boost immunity and also with the aim of inducing a TH1-like immune response that can prevent the normal Th1 to Th2 transition. With this in mind we investigated the co-administration of synthetic unmethylated oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN), a TLR-9 ligand, with chitosan nanoparticles.With this in mind, on this work we will report a new strategy of vaccination against schistosomiasis based on chitosan nanoparticles associated to SmRho antigen plus the adjuvant CpG, and coated with sodium alginate.5 . The degree of acetylation determined by FT-IR according to Brugnerotto et al. 5′-tcgtcgttttcggcgc:gcgccg-3′) was purchased from InvivoGen . All the others reagents used were of analytical grade.Chitosan (CH)  was supplied by Medicarb, Sweden. CH was purified by filtration of an acidic chitosan solution and subsequent alkali precipitation (1 M NaOH). The purified polymer was characterized by gel permeation chromatography (GPC) and Fourier Transform-Infrared Spectroscopy (FT-IR). The average weight molecular weight of the material was found to be 1.2×10The SmRho cDNA sequence was amplified from an adult extract worm cDNA using specific oligonucleotides  designedEscherichia coli BL21 pRARE for protein expression. The recombinant protein was then purified using HiTrap Chelating HP according to the manufacturer instructions . The protein purity was assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).To produce a recombinant (r) SmRho, the full-length DNA sequence was cloned into the expression vector pDEST42 . The resulting plasmid was transformed into After obtaining the purified protein SmRho, we wanted to confirm the ability of protein to be recognized by and to active the immune system after parasite infection. For this propose, sera of infected patient that had received drug treatment or not, compared to non-infected patient serum, were pooled and tested, by ELISA and Western Blotting. In this last assay, soluble extracts of schistosomula, eggs (SEA – soluble egg antigens) and adult worms (SWAP – soluble worm antigen preparation) were also evaluated, together with the protein SmRho, about their recognition against the sera tested.The preparation of this delivery system contains three main steps, the manufacturing of the chitosan particles, their recombinant protein loading by adsorption and finally the coating with sodium alginate.3COONa buffer,pH 5.5 and a solution containing 0.625% (w/v) of Na2SO4, both previously heated at 55°C for 10 min. Nanoparticles were allowed to form overnight under stirring. In the following day, the suspension was centrifuged at 3200 g for 20 min at 18°C. The particles were resuspended in about 1/8 of the volume with ultrapure water .Nanoparticles were prepared by mixing, while vortexing, equal volumes of a 0.1% (w/v) of chitosan in 5 mM CHThe loading was done as described by Borges et al The drug loading efficiency (LE) were calculated from the following equation:2 solution and kept under agitation for another 10 min.The coating was performed as described by Borges and colleagues The evaluation of the protein desorption during the coating procedure was performed during the incubation of the particles with sodium alginate. Aliquots of the particle suspension were collected, centrifuged at 15700 g for 30 min and the protein in the supernatant was assayed with a BCA protein assay as described previously.SmRho+CpG ODN loaded alginate coated chitosan NPs were prepared by adding the CpG and protein at same time during the loading process and before coating the nanoparticles. The loading of CpG with chitosan particles was assessed by electrophoresis in 1% (w/v) agarose (Cambrex) gel, with 0.05 µg/mL of ethidium bromide (Q-BioGene) and also by measuring the OD of the nanoparticle supernatants at 260 nm and calculate CpG by the difference. To eliminate background interference, the supernatant of unloaded particles were treated of the same way.To characterize the size and zeta potential, suspensions of particles loaded with SmRho protein were prepared as described above, with and without coating, and diluted in ultrapure water  to a final volume of 1 mL. The zeta potential and mean hydrodynamic size of the nanoparticles were assessed using a Zetasizer Nano ZS . The Smoluchowski model was applied for determination of the zeta potential, and cumulant analysis was used for mean particle size determination. All measurements were performed in triplicate at 25°C.in vitro release study.The protein release from coated particles was performed in simulated intestinal fluid (SIF) and simulated gastric fluid (SGF). The nanoparticle suspensions were added (1∶4) to individual tubes containing the release medium previously equilibrated at 37°C and placed in a shaker bath adjusted to 100 rpm. At appropriate time intervals, samples from each tube were collected and centrifuged for 15 min at 15700 g. The protein in supernatant was assayed with a BCA-protein assay. For each time evaluated, the samples were prepared in triplicate and a coated blank (without protein) nanoparticles suspension was submitted to the same conditions and was used as a blank for correction on the BCA-protein assay. In addition, non-bound protein in the alginate coated particle suspension was determined by BCA-protein assay in order to calculate the initial quantity of protein encapsulated in the beginning of the Six-week-old female C57BL6 mice were purchased from the Federal University of Minas Gerais (UFMG) animal facility. Animals had free access to food and water, with 12 h light/dark cycle.S. mansoni cercariae were maintained routinely on Biomphalaria glabrata snails at the Center of Research Rene Rachou-Fiocuz (CPqRR). For infections, cercariae were prepared by exposing infected snails to light for 1 h to induce shedding. Cercariae numbers and viability were determined using a light microscope prior to infection.Six groups (n = 5) were submitted to different treatments, as is described in The different formulations were administered orally with a gavage-feeding needle (groups I–V) and intramuscularly with an injection in the tibialis anterior muscle (group VI). The primary immunization was followed by two immunizations with an interval of two weeks. Seven days after the last immunization mice were challenged and then, after 50 days mice were sacrificed.Blood samples were collected from tail veins from mice of each experimental group at two-week intervals and the sera were prepared by centrifugation and stored at −20°C until further analysis. Serum was collected from immunized and control mice to measure kinetics of SmRho specific antibodies.2O2 in each well; after 20 min reactions were stopped with the addition of 50 µl of 5% (v/v) of sulfuric acid per well. The absorbance was read at 450 nm in an ELISA plate reader (ELX 800 BIO-TEK Instruments Inc.).A measurement of specific anti-SmRho antibodies was performed using an indirect enzyme-linked immunoabsorbent assay (ELISA) as described elsewhere Seven days after the last immunization, mice were challenged through percutaneous exposure of abdominal skin to water containing 25 cercariae (LE strain) for one hour. 50 days after challenge, mice were sacrificed and adult worms were perfused from their portal veins Antigenic preparations were obtained from schistosome eggs and adult worms, prepared as soluble supernatant fluids from buffered saline homogenates of the respective life-cycle stages 6 cells per well in RPMI 1640 medium  supplemented with 10% FBS, 100 U/mL of penicillin G sodium, 100 µg/mL of streptomycin sulfate, and 250 ng/mL of amphotericin B. Splenocytes were maintained in culture with medium alone or stimulated with SmRho (25 µg/mL), SWAP (25 µg/mL) or SEA (25 µg/mL). The 96-well plates  were maintained in an incubator at 37°C with 5% CO2. Culture supernatants were collected after 72 h for IL-10, TGF-β and IFN-γ analysis. IL-10, IFN-γ and TGF-β concentrations were measured using Duoset ELISA kit  according to the manufacturer instructions.Cytokine experiments were performed using splenocyte cultures from individual mice of control and experimental groups. Splenocytes were isolated from macerated spleens of individual mice at day 50 post-infection. Cells were washed twice with sterile PBS and were plated at a concentration of 1×10Liver fragments from mice (5 mice per group) of control and experimental groups immunized and infected, were collected 50 days post-infection in order to evaluate the effect of immunization on granuloma formation. Livers fragments were fixed in 10% paraformaldehyde. Fragments processed for paraffin embedding and histopathological sections were cut using a microtome at 5 µm. Sections were stained on a slide with hematoxilin-eosin (HE). The areas of individual granulomas were obtained through the MacBiophotonics ImageJ software analyzer. Fifteen granulomas from each mouse with a single well-defined egg were randomly chosen using a microscope with the 4× objective lens; granulomas were then scanned using JVC TK-1270/RGB microcamera. Using a digital pad, the total area of each granuloma measured, and the results were expressed in square micrometers.Statistical analysis was performed using the ANOVA test using the Graph Pad Prism 5 software package. The Bonferroni test was used to compare subgroups with the level of significance set at p<0.05.The studies regarding sera samples taken from patients were approved by Research Ethics Committee (COEP) of UFMG, the protocol number 523/07, and a written informed consent was obtained from each patient before blood collection. Experiments with animals were performed in compliance with the guidelines of the Institutional Animal Care and Committee on Ethics of Animal Experimentation  from Universidade Federal de Minas Gerais (UFMG); protocol number 204/2009 was approved on 24/03/2010.S. mansoni cDNA encoding SmRho was obtained from an adult worm cDNA library using PCR with specific oligonucleotides. The resulting full-length cDNA displayed an ORF of 579 bp, encoding a protein of 193 amino acids with a predicted molecular mass of approximately 21.8 kDa and an isoelectric point of 5.70. BlastP comparisons of the deduced protein sequence in GenBank exhibit a complete identity and similarity to S. mansoni Rho-GTPase protein (EMBL-Bank CDS: AAD31508.1)(data not shown).The full-length sequence of the E. coli BL21 pRARE strain. Protein extract of transformed bacteria showed a band at ∼26 kDa when induced with IPTG, since the plasmid add a C-terminal hexahistidine tag in the protein expressed. The bacteria were then lysed and the lysate separated into soluble and insoluble fractions. The inclusion bodies were shown to contain the majority of the recombinant protein .The size of chitosan-SmRho particles, before and after coating, was determined by light scattering technique. One of the major currently described drawbacks of this methodology of particle preparation is the high polydispersity of the obtained nanoparticles These particles were also characterized in terms of zeta potential . In the Release studies were performed to evaluate the stability of the coated nanoparticles and the profiles of SmRho protein desorption from these nanoparticles in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF), at 37°C. It can be observed in in vivo studies were performed to investigate the potential utility of rSmRho loaded chitosan-based nanoparticles in eliciting the production of antibodies or modulating the immune response following intramuscular or oral administration of the suspension of the particles. To evaluate the levels of SmRho-specific IgG antibodies serum samples from vaccinated animals from each group were tested by ELISA. The measures of IgG antibodies showed that nanoparticles were able to induce the production of specific SmRho antibodies mainly in the experimental group vaccinated with coated SmRho-chitosan nanoparticles by intramuscular route, which showed high levels of IgG that appeared at the day 45 (5 weeks after the first immunization) and presented the highest level at the day 75, compared to the control group  or SWAP (soluble worm antigen preparation). The highest levels of the immunomodulatory cytokine IL-10 were produced by SEA-stimulated splenocytes from mice immunized with coated SmRho-CpG-chitosan nanoparticles, compared with the control-stimulated splenocytes. In the others experimental groups significant levels of IL-10 were also observed, although their levels were not so high compared with those above described . SignifiSchistosomiasis is one of the most important neglected tropical diseases and an effective control is unlikely in the absence of improved sanitation and vaccine. The antigens tested, so far as vaccine candidates, prove not to be so effective as desirable, consequently, it is important to continue identifying new target antigens The selection of a suitable delivery system and an adjuvant to aid in the stimulation of the appropriate immune response is a critical step in the path to the development and employment of successful anti-schistosome vaccines, and a number of approaches are being tested, with some success S. mansoni infection. This vaccination strategy offers many technical advantages, including the possibility of administration by oral route, which makes the vaccine safer than injectable vaccines and facilitates its use mainly in underdeveloped areas Here we proposed a candidate vaccine formulation based on SmRho antigen loaded chitosan nanoparticles, coated with alginate, as an alternative strategy to induce protection against E. coli pRARE lineage, which co-expresses rare tRNAs required for the synthesis of some eukaryotic proteins. The expression and purification of the protein of interest was obtained with high yield and showed a protein with approximately 26 kDa of molecular mass. The immunogenicity of SmRho protein was confirmed through the reaction with sera of patient infected with S. mansoni. Besides that, the presence of SmRho in soluble egg and adult worms extracts was verified, which is supported by Vermeire and co-workers reports The recombinant expression of SmRho was optimized using an The methodology for the preparation of coated chitosan nanoparticles was successfully adapted from Borges and co-workers In vitro release studies were performed in SGF and SIF medium at 37°C in order to evaluate the release profiles of protein SmRho and also to assess the stability of the designed delivery system when submitted to medium with different pH, ionic strength at physiological temperature. In SGF, there was a gradual release of the protein, and after 2 h, about 30% of the protein had been released. After this period, it is believed that the particles have passed through the stomach therefore, it can be deducted that nanoparticles are quite resistant to the influence of acid environment. Despite using a similar system, Borges et co-workers (2006) S. mansoni. Our results demonstrated that an anti-pathological or a protective response induced against infection with cercarie are not necessary correlated with high levels of specific antibodies. This can be presumed because only the group intramuscularly immunized with coated chitosan nanoparticles presented high levels of SmRho specific IgG1 and IgG2a antibodies, and it did not show any significant reduction in granuloma area or in worm burden. On the other hand, SmRho-CpG-chitosan nanoparticles administered by oral route reduced liver granuloma area by 38.4% and were not able to induce systemic specific antibodies. This result confirm our previous work, recently published It is a consensus that the development of new and safe adjuvants is necessary not only for parenteral vaccination but also for the more challenging mucosal routes of administration in order to maximize the effectiveness of new antigens as well as those already available With respect to sodium alginate, some studies show that alginate microparticles are internalized by M cells of the mucosa S. mansoni infection. This is likely due to the induction of INF- γ production that can prevent the normal Th1 to Th2 transition that occurs in infected hosts after the onset of egg production by the parasites, and therefore acts preventing the development of severe chronic morbidity S. mansoni.Pathology resulting from granuloma formation around the eggs in murine schistosomiasis is characterized by Th2-type of immune response and the granuloma size can be reduced by neutralization of IL-4 S. mansoni infection, immunized mice were challenged with cercariae and worm burdens were assessed. All groups immunized with coated chitosan nanoparticles by oral route showed a significant reduction in worm burden and even the group immunized with chitosan nanoparticles without protein showed a 47% of protection. It suggests that chitosan has an important role in inducing a protective immune response against schistosomiasis that is likely due to its immunostimmulatory properties. Nevertheless, the mechanism behind the ability of chitosan in inducing protection is still unknown. Its role is more likely related to trigger an innate immune response, since chitin, which is a progenitor to chitosan, has been demonstrated to function as a PAMP and it is linked with the accumulation of innate cells including alternatively activated macrophages, eosinophils and basophils in vitro and in vivo, by a TLR-2 dependent mechanism that utilizes a MyD88-dependent pathway to induce IL-17 elaboration and enhance the expression of the IL-17AR. These studies also demonstrated that this novel innate immune pathway plays an essential role in the regulation of macrophage cytokine production and the induction of acute inflammation S. mansoni infection, however it needs to be further investigated.To determine whether coated chitosan nanoparticles conferred protection against Mice immunized with coated chitosan nanoparticles associated or not with CpG also showed high percentage of protection 48% and 55%, respectively. The antigen and CpG seems not to have a high contribution in conferring protection against worm infection, nevertheless, they demonstrated important roles in granuloma down-modulation, as discussed before.S. mansoni. It is important to highlight that these results were obtained with oral administration of the formulation and can be compared to those obtained from conventional routes of administration. Comparing with our previous work, in which DNA-chitosan nanoparticles were explored, this system achieved better results to be used as a vaccine because induced both protection as well as reduced the granulomatous reaction, while the first presented just an anti-pathological effect with granuloma modulation. Chitosan-based nanoparticles were also found to play an important role as adjuvant and this characteristic should be more explored with other antigens. Furthermore, the role of chitosan in inducing a protective immune response against schistosomiasis deserves special attention and requires more studies to confirm and understand this feature. Taken together, these results support this new strategy to find a safe and efficacious vaccine against schistosomiasis.As a final conclusion of this work, we believe that the combination of chitosan nanoparticles associated to the antigen SmRho plus CpG is an efficient vaccine formulation candidate against schistosomiasis in light of the data obtained from murine studies, which was able to modulate the granuloma area, that represents the major pathological response in schistosomiasis and also to induce protection against infection of


Sputum induction was performed the day before and 24 h after exposure to the offending agent. Total and differential cell counts were assessed. LTB4 level (p = 0.0078) in patients with OA due to HMW agents. An increase in the percentage of sputum neutrophils after SIC (p = 0.0040) was observed in subjects without OA exposed to LMW agents. IL-8 levels after SIC were higher in patients without OA compared with patients with OA (p = 0.0146).Thirty-four patients tested positive to SIC and were diagnosed with OA (in 10 due to HMW agents and in 24 to LMW agents). SIC was negative in 17 subjects. As compared to baseline an increase was found in the percentage of sputum eosinophils and neutrophils, and in IL-10 concentration after SIC , and a decrease was seen in LTBWhen conducting airway inflammation studies in OA, patients should be divided according to the causal agent (HMW or LMW). In OA patients exposed to HMW agents, an increase in the number of neutrophils can be found in parallel to the increase of eosinophils, although this does not contradict an IgE-mediated mechanism. Exposure to LMW agents can result in increased neutrophilic inflammation in patients with airway diseases unrelated to OA. There is variability in the responses observed in patients with OA exposed to LMW agents. As its name indicates, work-related asthma (WRA) is a type of asthma in which the symptoms occur in relation to work Specific inhalation challenge (SIC) may be an effective test for establishing an accurate diagnosis of the different types of WRA 4 (LTB4) and a panel of Th1/Th2 cytokines. Information in this line can help to clarify the pathophysiological mechanisms involved in the genesis of OA, in particular OA caused by LMW agents.The aim of this study was to establish the sputum inflammatory cell profile in subjects with suspected OA following SIC to HMW and LMW agents and to determine possible changes in the levels of leukotriene B2-agonists were stopped at least 24 h and 6 h before SIC, respectively.The study included all individuals older than 18 years of age referred to our center for SIC to investigate possible OA in the period of 2005 to 2010, and from whom adequate sputum samples could be obtained before and after SIC. All subjects had a medical history consistent with OA and a workplace agent was considered the probable cause of their respiratory symptoms. Concurrent treatment with anti-inflammatory drugs was maintained at the same level during the study. None of the patients were receiving leukotriene modifiers or non-steroidal anti-inflammatory drugs. Long- and short-acting βThe local Ethics Committee approved the study  19/2005), and all subjects signed informed consent documents for participation.1 every 10 minutes during the first hour after exposure and then every hour to complete 15 hours after inhalation. Response was considered positive when FEV1 fell more than 20% of the baseline value in the absence of any change to placebo.In each subject, SIC was performed with the suspected offending agent Subjects testing positive to SIC were diagnosed with OA. Subjects negative on SIC underwent various tests based on clinical suspicion to establish a definite diagnosis. Those who presented symptoms and abnormal findings on pulmonary function study in relation to work received a diagnosis of WEA Sputum induction was performed using the method described by Pizzichini et al Sputum samples were examined and processed within 2 h, as described by Pizzichini et al. Differential cell count of macrophages, neutrophils, lymphocytes, and eosinophils was carried out by optic microscopy and expressed as a percentage of the total of non-squamous cells. The specimen was considered adequate if total and differential cell counts could be obtained; this required as little as 50 mg of selected material. Results are expressed as the absolute number of cells in millions per mL of sputum sample.4 concentration in sputum supernatant samples was determined by a commercially available LTB4 enzyme immunoassay kit . The LTB4 assay has a detection limit of 13 pg/mL.LTBA panel of 10 cytokines, including interferon-gamma (IFN-γ), interleukin (IL)-1beta (β), IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, and tumor necrosis factor-alpha (TNF-α) and -beta (TNF-β), were analyzed using a commercially available kit .p value of ≤0.05. The variable “difference” (VD) for differential cell count, cytokines, and LTB4 was calculated by subtracting the value before SIC from the value after SIC. SPSS release 17.0 for Windows  and GraphPad InStat4  were used for the statistical analyses.The characteristics of the subjects are expressed as the median and range. A one-sample Kolmogorov-Smirnov test, calculated to assess normality, showed non-normal distribution of the parameters studied. Between-group differences were analyzed by the Mann-Whitney test and within-group differences by the Wilcoxon signed rank test. Differences were considered significant at a Of the original 175 individuals who were referred for suspected OA, adequate sputum samples before and after SIC were obtained in 51, yielding a success rate of 49% in positive SICs (n = 70) and 16% in negative SICs (n = 105) . The bas1 tested <70% the theoretical value following SIC. The percentages of methacholine-positive tests before exposure and 24 h after exposure were higher in the OA Group than NoOA . PC20 values before exposure and 24 h after exposure differed significantly between the OA group and NoOA group, with lower values in OA . A PC20 decrease of at least two-fold at 24 h occurred in 43.5% of patients in the OA group and no patients in the NoOA Group (p = 0.007).Comparison of the demographic data and clinical characteristics of the study subjects with and without OA are summarized in Total and differential cell counts (percentage of sputum eosinophils and neutrophils) in the two groups studied (OA/NoOA) are summarized in Patients with OA due to HMW agents showed a significant increase in percentage of sputum eosinophils and neutrophils after SIC . This increase was not observed in OA patients exposed to LMW agents . No signThe single subject in this group exposed to a HMW agent was not included in the analysis . In subj4 and cytokine levels in the study population. Following SIC, IL-2 and IL-10 concentrations were higher in patients with OA compared to those without , whereas IL-8 levels were higher in NoOA patients compared to OA (p = 0.0146) ( 0.0146) .4 and an increase in IL-10 levels after SIC compared to baseline values   . We foun20 before SIC in the OA and NoOA Groups . We did not found any correlation between cell changes and the other pulmonary function test parameters.We found a significant negative correlation between the percentage of eosinophils and the PC4 levels were observed in patients with OA due to exposure to HMW agents, but not in those exposed to LMW agents. An increase in sputum neutrophils and IL-8 was also observed in subjects exposed to LMW agents who were ultimately diagnosed as having other respiratory diseases.To our knowledge, this is the first study comparing inflammatory cell percentages and inflammatory markers in sputum samples of subjects with and without OA, distinguishing between HMW-induced and LMW-induced asthma. Increases in sputum eosinophils, neutrophils, and IL-10 concentration and decrease in LTBSeveral studies have evaluated the differences in cell counts in induced sputum before and after SIC . In fourThis is the first study in which no significant differences in cell types in induced sputum after SIC were observed in patients with OA due to LMW agents. Some authors have reported an increase in sputum eosinophils after SIC in these patients Another interesting finding of the present study is that subjects testing negative on SIC and diagnosed with other respiratory diseases experienced an important increase in sputum neutrophils after SIC. This finding has been reported by other authors, who have suggested that these patients may have had false-negative responses to SIC 4 levels were measured because previous studies have reported that it could be an important marker of disease in patients with OA 4 levels after SIC in induced sputum 4 in OA induced by HMW agents. Interestingly, in the present study a decrease in LTB4 levels after SIC was found in all patients except one with OA due to HMW agents. It has been demonstrated in multiple studies that in IgE-mediated asthmatic reactions caused by allergens there is an increase in cysteinyl leukotriene (Cys LT) 4 and Cys LT occurs through alternative pathways 4 production. Although this hypothesis has not been demonstrated in relation to SIC, it has been observed in asthma exacerbation In the present study, LTBTo our knowledge, only two studies have evaluated IL-10 in patients with OA The study has some limitations. Although no significant differences were found in the differential cell count of the groups studied, it cannot be excluded that there may have been some influence due to smoking habit. In any case, this effect would be observed before SIC, but would likely be independent of the response after SIC, considering that during the test the patients did not smoke. Another aspect that may have influenced the observed results is the fact that approximately 50% of patients in both groups received treatment with inhaled corticosteroids. Although treatment with B-2 agonists was withdrawn in all patients prior to initiation of the SIC, inhaled corticosteroids treatment was not changed to avoid destabilization of asthma, which would invalidate the test. Lastly, the low yield in obtaining sputum samples from patients in the NoAO group might seem to be a limitation, but we believe the study illustrates the true situation of our laboratory and does not show selection bias.In conclusion, the results of this study show that it is necessary to differentiate between patients with OA due to HMW agents and LMW agents when investigating airway inflammation. In OA patients exposed to HMW agents, an increase in the number of neutrophils can be found in parallel to the increase of eosinophils, although this does not contradict an IgE-mediated mechanism. Exposure to LMW agents can result in increased neutrophilic inflammation in patients with airway diseases unrelated to OA. Moreover, we found variability in the responses observed in patients with OA exposed to LMW agents. Future studies should examine if more than one mechanism may be involved in the genesis of the disease.


An advantage of the Intensity Modulated Radiotherapy (IMRT) technique is the feasibility to deliver different therapeutic dose levels to PTVs in a single treatment session using the Simultaneous Integrated Boost (SIB) technique. The paper aims to describe an automated tool to calculate the dose to be delivered with the SIB-IMRT technique in different anatomical regions that have the same Biological Equivalent Dose (BED), i.e. IsoBED, compared to the standard fractionation.Based on the Linear Quadratic Model (LQM), we developed software that allows treatment schedules, biologically equivalent to standard fractionations, to be calculated. The main radiobiological parameters from literature are included in a database inside the software, which can be updated according to the clinical experience of each Institute. In particular, the BED to each target volume will be computed based on the alpha/beta ratio, total dose and the dose per fraction . Then, after selecting the reference target, i.e. the PTV that controls the fractionation, a new total dose and dose per fraction providing the same isoBED will be calculated for each target volume.The IsoBED Software developed allows: 1) the calculation of new IsoBED treatment schedules derived from standard prescriptions and based on LQM, 2) the conversion of the dose-volume histograms (DVHs) for each Target and OAR to a nominal standard dose at 2Gy per fraction in order to be shown together with the DV-constraints from literature, based on the LQM and radiobiological parameters, and 3) the calculation of Tumor Control Probability (TCP) and Normal Tissue Complication Probability (NTCP) curve versus the prescribed dose to the reference target. Irradiation techniques with Intensity Modulated Radiotherapy (IMRT) allow doses to be delivered to the target with a high conformation of prescribed isodose, sparing Organs at Risk (OARs), compared to conventional 3D-CRT techniques. Another advantage of the IMRT technique is the possibility to achieve the so-called Simultaneous Integrated Boost (SIB), which provides different levels of therapeutic doses to different target volumes during the same treatment session, once the fraction number has been set -5.Historically, to obtain the desired tumor control, the doses were determined using a conventional fractionation that ranged between 50 to 70 Gy at 2 Gy per fraction.Whereas, in order to obtain Tumor Control Probability (TCP), equivalent to that of a conventional fractionation, the total dose simultaneously delivered to the targets have to be determined according to the Linear Quadratic Model (LQM) to be used with the SIB technique . Thus, tBased on the Biological Equivalent Dose (BED) formalism, a new total dose and the fraction dose can be calculated in order to obtain the same biological effect, named IsoBED herein ,8.2-VH) and compare these amongst themselves and with the Dose-Volume constraints (DV- constraints), 3) calculate and compare the TCPs and the Normal Tissue Complication Probabilities (NTCPs) obtained from different DVHs.The paper aims to: 1) describe home-made software, based on the IsoBED formula, able to calculate the total dose and the dose per fraction with the same TCP as the conventional fractionation, that will be used with the SIB technique, 2) import the DVHs from different TPSs or different plans, convert them into a normalized 2 Gy-fraction-Volume Histogram (NTDsf) of cells in the tissues exposed to a total radiation dose D (expressed in Gy) and to a dose per fraction d(expressed in Gy). The logarithm of the surviving fraction, in the absence of any concurrent re-population, can be expressed as:This approach was based on the LQM, widely used for fractionated external beam-RT, to describe the surviving fraction  ratio is a parameter which takes into account the radiobiological effect of fractionation in tumor or OARs.and the (Equation (2) is the basis on which a comparison of different treatment strategies is performed.1 and D2) and dose per fraction (d1 and d2), the following equation can be invoked:In order to obtain the same cell survival with two fractionations having a total dose  we obtain:If we have a fractionation schedule with i.e.and thenThe solution of which is:2 is the new dose per fraction delivered in n fractions, resulting in a new total dose D2 = d2 n,Where dEquation (7) is valid for both PTVs and OARs (following the LQM).The software has been developed using the Microsoft Visual Basic 6.0. The main form - the IsoBED Calculator- gives a choice between IsoBED calculation and DVHs analysis modules.α/β) ratio of investigated tumor must be inserted to calculate the corresponding BED.The software allows the anatomical district to be selected. The user has to introduce the total dose, dose per fraction  for each target (up to 3) and, the (Then the software requires the selection of the reference target (which determines the fractions number in the SIB treatment), in order to calculate the new fractionation for the remaining targets, based on equation (7). Furthermore, the software permits a comparison of the biologically equivalent schedules using hyper/hypo-fractionated as well as conventional regimes. It also includes a database with the main DV- constraints at 2 Gy per fraction for different OARs derived from literature and clinical experience in the radiotherapy department of our Institute -20 whichThe DV-constraints are converted to those of the new schedule (i.e. hypo or hyper-fractionated) calculated by IsoBED.Then the converted constraints for OARs can be printed and used as constraints for IMRT optimization.After the IMRT optimization using commercial TPSs , the obtained DVHs can be imported to our software and can be used to compare techniques and/or dose distributions from the same or different TPSs.The software automatically recognizes the DVH file format exported from each TPS source and imports it into the patient directory without any changes. In particular, import procedures consist of copying DVH files into a subfolder with the patient's name, contained in a directory where the IsoBED.exe file is held.Then, a specific window permits the analysis of DVHs to be carried-out. Cumulative or differential DVHs can be visualized after setting dose per fraction and fraction number. In this window up to five plans imported from BrainScan, Eclipse and Pinnacle can be compared. The volumes and the minimum, mean, median, modal and maximum doses can be visualized for OARs and PTVs.2VH (Appendix 1 equation 1.6) by using the appropriate (α/β)ratio, which may be changed by the user.For each volume the software calculates NTDFinally, the TCP, NTCP and Therapeutic Gain (P+) curves can be calculated from the DVHs based on radiobiological parameter sets, derived from literature but upgraded by the user, according to the formulas reported in Appendix 1 -27.To illustrate this user friendly IsoBED software some case examples are shown.The following test cases were considered in order to illustrate the usefulness of the home made software for comparing sequential versus SIB plans for three clinical treatments in this paper.The first case regards irradiation using IMRT of prostate and pelvic lymph nodes.The comparison was made between the sum of 2 sequential IMRT plans  and an SIB IMRT plan .α/β)ratio = 1.5 Gy for both targets .-12.10-12The second case regards the treatment of a rinopharynx cancer patient.The prescribed dose was 53 Gy at 2.12 Gy per fraction to the Planning Elective Tumor Volume , 59.36 Gy at 2.12 Gy per fraction to the Planning Clinical Target Volume  and 69.96 Gy at 2.12 Gy per fraction to the Planning Gross Target Volume .The first plan, the sequential treatment, was calculated to deliver 53 Gy in 25 fractions to PETV followed by 6.36 Gy in 3 fractions to the PCTV and another 10.6 Gy in 5 fractions to the PGTV, for a total of 33 fractions.For the SIB plan, the IsoBED doses derived from prescription and the calculated doses from our software were considered in order to deliver 69.96 Gy in 33 fractions to the PGTV.The setup of the IMRT plan was calculated with Pinnacle 8.0 m TPS  and based on seven 6 MV photon beam techniques  -17.In a lung cancer patient two volumes had to be irradiated in a hypofractionaction regime . The preIn both cases, the plans were performed by the Pinnacle TPS using 6 MV photon energy and 3 coplanar fields . The acceptance criteria for the primary plan had to meet treatment goals (prescribed dose to >95% of the volumes) for all target while keeping the maximum dose of the healthy lung, spinal cord, esophagus and heart under DV-constrains of sequential and SIB plans value are inserted. For the BED calculation it is necessary, as previously described, to select the target, named reference target, that will determine the fraction number. Thus, BED values are calculated by clicking on the button "BED and Fractionaction Calculation".Figure Then the SIB schedule is calculated by selecting the control box "IsoBED Calculation". The results of such evaluations are visualized in the "IsoBED DOSES" area. The dose limits are visualized in the "OAR CONSTRAINTS" area.Import procedures consist of copying DVH files, exported from TPS, in a folder with the patient's name contained in a directory where an IsoBED.exe file is installed. DVH files are different depending on the TPS source. IsoBED can import DHV data files from Eclipse, Pinnacle and Brainscan.Figures Figures α/β)values can be set for each structure by choosing a dropdown menu in which the list of parameters incorporated in a dedicated database appears. These values are derived from literature data and from experience at our Institute [2VH value while designing the fractionation scheme might aid the prediction of different effects (such as acute and late effect) related to clinical trials.Furthermore, the possibility of changing the  model was used to calculate the NTCP. For uniform irradiation of a fraction where s is defined as:TD50 (veff) are the slope of the NTCP curve versus the dose and the tolerance dose at 2 Gy per fraction to a fraction veff of the organ, respectively.where m and In order to generalize the LBK method each DVH has been converted into a single value using a DVH reduction method.veff) method was chosen as a histogram reduction scheme for non-uniform organ irradiation:The effective volume . By Eq. (1.4), an inhomogeneous dose distribution is converted into an equivalent uniform irradiation of a fraction veff of the organ treated at the maximum dose (Dmax).where TD50 (veff) can be calculated using the following equation:The TD50(1) is the tolerance dose to the whole organ, leading to a 50% complication probability.where i = Di/N and d = Dmax/N, where N is the number of fractions), both Di(received by the volume fraction vi) and the maximum dose Dmax are converted to the nominal standard dose , applying the following equations:In order to take into account the new dose per fraction (dandrespectively.Equation (1.4) becomes:By using this formula, each dose step in the DVHs was corrected separately. This formalism presumes complete cellular repair between treatment fractions and neglects the role of cellular re-population. The latter assumption is valid for late-responding normal tissues but is inaccurate for acute-responding tissues and tumors. This limitation may be important when using the LQM to compare treatment schedules differing in overall treatment times in terms of their acute effects (for which time-dependent repopulation may be important). For late effects, time factors are generally thought to be of minor importance.Therapeutic gain is used to compare optimization outcomes in treatment plans calculated with different modalities taking into account both tumor control and normal tissue complications. The following expression is used:


The Mre11/Rad50/Nbs1 (MRN) complex is a regulator of cell cycle checkpoints and DNA repair. Defects in MRN can lead to defective S-phase arrest when cells are damaged. Such defects may elicit sensitivity to selected drugs providing a chemical synthetic lethal interaction that could be used to target therapy to tumors with these defects. The goal of this study was to identify these defects in the NCI60 panel of cell lines and identify compounds that might elicit selective cytotoxicity.We screened the NCI60 panel in search of cell lines that express low levels of MRN proteins, or that fail to arrest in S-phase in response to the topisomerase I inhibitor SN38. The NCI COMPARE program was used to discover compounds that preferentially target cells with these phenotypes.HCT116 cells were initially identified as defective in MRN and S phase arrest. Transfection with Mre11 also elevated Rad50 and Nbs1, and rescued the defective S-phase arrest. Cells of the NCI60 panel exhibited a large range of protein expression but a strong correlation existed between Mre11, Rad50 and Nbs1 consistent with complex formation determining protein stability. Mre11 mRNA correlated best with protein level suggesting it was the primary determinant of the overall level of the complex. Three other cell lines failed to arrest in response to SN38, two of which also had low MRN. However, other cell lines with low MRN still arrested suggesting low MRN does not predict an inability to arrest. Many compounds, including a family of benzothiazoles, correlated with the failure to arrest in S phase. The activity of benzothiazoles has been attributed to metabolic activation and DNA alkylation, but we note several cell lines in which sensitivity does not correlate with metabolism. We propose that the checkpoint defect imposes an additional mechanism of sensitivity on cells.We have identified cells with possible defects in the MRN complex and S phase arrest, and a series of compounds that may preferentially target S phase-defective cells. We discuss limitations of the COMPARE program when attempting to identify compounds that selectively inhibit only a few cell lines. Many anticancer agents kill cells as a consequence of damaging their DNA. While this approach targets rapidly proliferating cells, it is only somewhat selective for tumors and can lead to undesirable toxicity to the patient. Differences exist between normal and tumor cells with respect to the cellular response to DNA damage and it may be possible to exploit these differences to selectively target tumor cells. One such difference is in cell cycle checkpoint regulation. In response to DNA damage, cell cycle checkpoints are activated causing the arrest of cell cycle progression . This alThe S-phase checkpoint is activated in response to DNA damage that occurs during replication. For example, topoisomerase I inhibitors such as SN38 produce a single-strand break in DNA which is converted to a double-strand break upon collision with the replication fork . ProcessMutations in the MRN complex have been identified in lymphoid, breast and colorectal tumors ,9. A defStudies in our laboratory have confirmed that HCT116 cells have very low levels of Mre11 and also that they arrest poorly in S phase in response to SN38. By transfecting HCT116 cells with a vector expressing Mre11, we show here that we can reconstitute the MRN complex. Our data indicate that Mre11 is necessary for the stability of Rad50 and Nbs1 and rescues the S-phase arrest in HCT116.http://dtp.nci.nih.gov). We then correlated the MRN protein levels to sensitivity to the compounds in the NCI database using the COMPARE program [MRN defects if present in some tumors could render these cells sensitive to DNA-damaging agents and thereby provide a tumor selective therapy. The primary goal of these studies was to find compounds that preferentially target cells with defects in the proteins of the MRN complex. We have examined the frequency of MRN defects across the NCI60 cell line panel which is derived from many different cancer types  . These c program ,17. Sinc2.NCI60 cancer cell lines were obtained from the Developmental Therapeutics Program at the National Cancer Institute and were cultured in RPMI 1640 media (Hyclone) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone) and Antibiotic-antimycotic (Gibco). Cells were cultured at 37°C with 5% COElevated expression of Mre11 was achieved by cloning Mre11 from pDB20  into pCR3.1 (Invitrogen) . This plSN38, the active metabolite of irinotecan, was obtained from Pfizer, and 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole  was obtained from the Developmental Therapeutics Program at the National Cancer Institute . Both drugs were dissolved in DMSO and stored at -20°C.For cell cycle analysis, cells were rinsed with phosphate-buffered saline (PBS), harvested with 0.05% trypsin, and fixed in 70% ethanol overnight. Cells were stained with 100 μg/ml propidium iodide in 1 mg/ml ribonuclease A and analyzed on a FACScan flow cytometer (Becton Dickinson).Following treatment, cells were rinsed with phosphate buffered saline, lysed by direct addition of Laemmli buffer to the wells, boiled for 5 min, and stored at -80°C. Protein levels were quantified using EZQ Protein Quantitation Kit (Molecular Probes). 15 μg of each lysate was separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes. Membranes were blocked and incubated overnight at 4°C with primary antibodies specific for Nbs1 , Rad50 , Mre11 or actin . Membranes were washed and incubated for 30 min at room temperature with anti-rabbit or anti-mouse secondary antibody conjugated to horseradish peroxidase. Proteins were visualized with enhanced chemiluminescence. Densitometry scans were quantified using Scion Image software (Scion Corporation) and normalized to a serial dilution of the same protein from MDA-MB-231 present on each blot. Several exposures of each western blot were scanned to avoid over-exposure and to ensure that the samples were within the range of the standards.http://dtp.nci.nih.gov). In their screen, all cell lines were incubated with a range of drug concentrations, usually for 48 h, and then fixed and stained with sulforhodamine B.Cells were seeded at low density (500-1000 cells) in 96-well plates and then incubated with drug for 24 h. Following treatments, cells were washed and grown in fresh media for 5-7 days at 37°C. Prior to attaining confluence, cells were washed, lysed, and stained with Hoechst 33258, as previously described . FluoresDNA damage, such as that caused by the topoisomerase I inhibitor SN38, triggers checkpoints that arrest cell cycle progression to allow for DNA repair. We have previously compared seven cell lines for their ability to arrest in S phase . In geneThe National Cancer Institute has screened more than 100,000 compounds in a panel of 60 cell lines and this data is publicly available. The database also has information on the gene expression patterns in each cell line. However, our data in Figure While this analysis was designed to identify cell lines with low levels of the MRN proteins, we also identified several lines in which the ratio of these proteins far exceeded the expected values. For example, SW620 cells had about 10 fold more Mre11 than Rad50, whereas Nbs1 was intermediate between these two levels. On the other hand, HCC2998, K562 and EKVX had about 2 fold more Rad50 than Mre11. For these latter cell lines, Mre11 and Nbs1 were relatively similar suggesting that it is the elevated level of Rad50 that maybe abnormal. Whether these cell lines contain functionally defective MRN remains to be determined.To extend this analysis further, we compared the protein levels to mRNA levels obtained from the NCI database Figure . The cor-4 M). Hence, sensitivity to these latter compounds does not correlate with expression of the MRN proteins. This reflects a limitation of the COMPARE program that is discussed below.Considering that defective MRN can cause chemo- and radio-sensitivity, we hypothesized that other drugs in the NCI database would correlate with the low levels of MRN observed. Accordingly, we used the NCI COMPARE program to find correlations between MRN proteins and compound activity. We set the correlation coefficient to 0.5 to restrict the number of compounds identified. The search recovers both positive and negative correlations, that is, a positive correlation represents high protein level correlating with sensitivity whereas a negative correlation represents a cell line with low protein but high sensitivity to a compound. Mre11 and Nbs1 protein levels correlated with the activity of only a few compounds, and approximately equally distributed between positive and negative correlations Table . The othWhile the MRN complex has a critical role in the S-phase checkpoint, there are many other proteins that are also involved. In search of other possible S phase checkpoint defects, we screened all of the cell lines in the NCI60 panel for their ability to S-phase arrest in response to the topoisomerase I inhibitor SN38 Figure . SeveralUsing the COMPARE program to analyze the values in Figure The sensitivity of cells to benzothiazoles is reported to be due to the ability of cells to metabolically activate these compounds which then go on to damage DNA ,23. MCF7The overall goal of this study was to identify molecular defects in cell cycle checkpoint regulation using the NCI60 panel of cell lines, and then to identify compounds that might target cells with these defects. Eventually, such compounds might provide leads for the development of drugs for personalized cancer therapy. We began by screening the cell panel for potential defects in MRN proteins and the S-phase checkpoint. We uncovered a number of interesting relationships with respect to MRN proteins and their mRNA that impact the level of this complex. In addition, using the COMPARE program, we have identified several compounds of interest which warrant further study.Our results implicate protein stability in the regulation of MRN protein levels. Transfection with Mre11 not only increased Mre11 levels but also Rad50 and Nbs1 levels. In comparing the protein levels in each cell line across the NCI60 panel, we also found a strong correlation between the levels of these three proteins. The overall level of the complex appeared to be primarily regulated by transcription of Mre11 as there was much lower correlation between mRNA and protein levels for Rad50 and Nbs1. These results suggest that the protein levels of Rad50 and Nbs1 are primarily regulated by stability of the complex while transcription of Mre11 contributes more to the overall level of the complex.While Mre11 expression may be more highly regulated by transcription, among the cell lines with low levels of Mre11 protein, only the IGROV1 cells had a very low level of mRNA. HCT116 had a relatively high level of Mre11 transcript but low protein, and this has been shown to be due to a splicing defect in its mRNA . This deThis screen identified a small number of compounds which correlated with either Mre11, Rad50, or Nbs1. However, there were three compounds identified which correlated with both Mre11 and Rad50. The activity of one of these, NSC 734406, was significant at the concentrations tested and correlates with low levels of Mre11 and Rad50 , we may have missed many potential drug candidates. Some compounds may correlate with only some of the checkpoint defects responsible for the lack of SN38-induced S-phase arrest. The fact that each defect may be rare in the NCI60 panel makes it a challenge to use this approach to identify novel drug candidates. For example, a compound that has the same activity in 60 cell lines would exhibit a very high correlation with a dataset in which 56 cell lines arrested and 4 did not, the correlation coefficient being driven by the 56 cell lines rather than the 4 interesting queries. Accordingly, each compound discovered in the screen had to be individually analyzed and validated. Many compounds elicited no obvious correlation with the query set. In some cases , three of the four cell lines that failed to arrest in S phase had not even been screened against this compound. These limitations are unfortunate because we had hoped to use this approach to identify compounds that killed only a few cell lines, and might therefore be candidates for selective therapy for tumors with that particular phenotype . Given the limitations of this screen, the fact that most of the cells defective for S phase arrest were indeed sensitive to compounds in the benzothiazole cluster gives strong support for further investigation into this class of drugs.Genomic instability is one of the characteristics of cancer and is frequently attributable to defects in DNA damage response genes involved in either DNA repair or checkpoint regulation. This provides a therapeutic opportunity as these defective cells may be hypersensitive to various drugs. We have identified cells with potential defects in the MRN complex and in S phase arrest, and a series of compounds that may preferentially target S phase arrest-defective cells. The in silico screen used has limitations when seeking compounds that are cytotoxic to only a few cell lines. But considering this limitation, the identification of benzothiazoles as compounds that preferentially target cells with a defect in S phase arrest warrants further investigation. Compounds such as these that kill only a few cell lines likely represent the future of individualized cancer therapeutics.The authors declare that they have no conflicting interestsKMG and AE conceived the project and shared in its design. KMG performed most of the experiments. KMG and AE performed data analysis and wrote the manuscript. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/206/prepubTable S1: Correlations between Mre11, Rad50 and Nbs1 protein and mRNA.Click here for file


Objective: To evaluate the effects of different hemostatic agents upon the outcome of periapical surgery. Design: A retrospective study was made of patients subjected to periapical surgery between 2006-2009 with the ultrasound technique and using MTA as retrograde filler material. We included patients with a minimum follow-up of 12 months, divided into two groups according to the hemostatic agent used: A) dressings impregnated in anesthetic solution with adrenalin; or B) aluminum chloride paste (Expasyl™). Radiological controls were made after 6 and 12 months, and on the last visit. The global evolution scale proposed by von Arx and Kurt (1999) was used to establish the outcome of periapical surgery.Results: A total of 96 patients  with a mean age of 40.7 years were included. There were 50 patients in the aluminum chloride group and 46 patients in the anesthetic solution with vasoconstrictor group. No significant differences were observed between the two groups in terms of outcome after 12 months - the success rate being 58.6% and 61.7% in the anesthetic solution with vasoconstrictor and aluminum chloride groups, respectively (p>0.05).Conclusion: The outcome after 12 months of follow-up was better in the aluminum chloride group than in the anesthetic solution with vasoconstrictor group, though the difference was not significant. Key words:Aluminum chloride, bleeding control, hemostasis, periapical surgery, outcome. Many factors can influence the outcome of periapical surgery. In this context, adequate bleeding control is essential for the success of periapical surgery, since it improves visualization of the surgical site, minimizes the operating time, and is a requirement for the insertion of most retrograde filling materials ,2.Different agents and techniques have been used to secure hemostasis in periapical surgery. Bone wax has been used for many years and is easy to handle, though remaining traces of this material can cause adverse tissue reactions . SimilarVon Arx et al.  introducA number of studies have been published involving different materials used to secure hemostasis in periapical surgery, though none have correlated the outcome of periapical surgery to the hemostatic agent used.The present study was carried out to compare the outcome of periapical surgery when using two different hemostatic agents: dressings impregnated with anesthetic solution containing a vasoconstrictor , and aluminum chloride paste (Expasyl™).-Sample selectionA retrospective study was carried out in the Oral Surgery Unit  covering the period between October 2006 and March 2009 and including the patients subjected to periapical surgery with the ultrasound technique, using MTA  as retrograde filling material. The study was approved by the local Ethics Committee, and all patients gave informed consent to participation in the study.In all cases dressings impregnated in anesthetic solution with vasoconstrictor, or aluminum chloride paste, were used for securing hemostasis, and the minimum follow-up period was 12 months. Specifically, we used dressings impregnated in anesthetic solution with vasoconstrictor  in the patients treated between October 2006 and December 2007, and aluminum chloride  in the patients treated between January 2008 to March 2009.In the patients with periapical lesions measuring over 20 mm in diameter, with tunnel lesions rupturing the vestibular and lingual cortical layers, or with apicomarginal defects with complete vestibular cortical loss, hemostasis was secured with dressings impregnated in anesthetic solution with vasoconstrictor, due to the difficulty of eliminating the remaining traces of Expasyl™ in such situations. In these cases we always used bone regeneration techniques, and decided to exclude the patients from the study, since they all belonged to the adrenalin with vasoconstrictor group.We likewise excluded those patients in which no hemostasis material had been used (9 patients). The resulting initial study sample consisted of 123 patients, divided into two groups according to the hemostatic agent used: A) dressings impregnated in anesthetic solution with adrenalin (55 patients); or B) aluminum chloride paste (Expasyl™)(68 patients).Eight patients were excluded due to incomplete study protocols (one in the dressings group and 7 in the Expasyl™ group), 14 because of a lack of follow-up (3 in the dressings group and 11 in the Expasyl™ group), and 5 patients in the dressings group in which bone regeneration techniques had been used .The final study sample thus consisted of 96 patients: 46 belonging to the dressings with anesthetic and vasoconstrictor group and 50 to the Expasyl™ group.-Surgical techniqueAll the operations were made by the same surgeon (MPD). Locoregional and infiltrating anesthetic techniques were used with 4% articaine and adrenaline 1:100,000 . Intrasulcular incisions were made with one or two vertical releasing incisions for raising triangular or trapezoidal mucoperiosteal flaps. Ostectomy was performed using a rounded 0.27 mm tungsten carbide drill  under abundant irrigation with saline solution. The minimum apical resection needed to access the apexes was made, followed by apical curettage. The cavity was prepared for retrograde filling using a Piezon Master® 400 ultrasound instrument  at full power with diamond-surfaced ultrasound tips for periapical surgery. To facilitate the procedure, use was made of a Medi Pack Pal endoscope  and Moeller® Dental 300 surgical microscope . Bone cavity drying and bleeding control was carried out in group A by means of small sterile dressings impregnated with anesthetic solution with adrenaline as vasoconstrictor at a concentration of 1:100,000 , placed within the bone crypt. Dry dressings were then placed and pressure was applied for two minutes, after which all the dressings except the first impregnated dressing were removed, and the surgical procedure was continued – removing the impregnated dressing before saline irrigation and suturing , together with digital panoramic X-rays .Radiographic controls were made immediately after surgery, after 6 and 12 months, and on occasion of the subsequent annual control visits. The months elapsed from surgery to final control (counting to the last visit of each patient) or until failure were recorded for calculating mean patient follow-up.-Scales of successThe outcome of periapical surgery was assessed based on the global evolution scale proposed by von Arx and Kurt , which c-Data collection and statistical analysisA clinical history was compiled for all patients, following a previously established protocol - recording the personal antecedents of interest, collecting all the clinical and radiographic data of the patients in detail, and registering the pre-, intra- and postoperative characteristics.The SSPS version 15 statistical package for MS Windows was used for analyzing the data. In each study group we performed a descriptive analysis of all the variables , checking that there were no differences between the two groups for any of these variables. The outcomes of periapical surgery after 6 and 12 months and at the final control, in each group, were compared using the chi-squared test, with a statistical significance level of p<0.05. In all cases the necessary tests for confirming the applicability of the different statistical techniques were carried out.The study comprised a total of 96 patients  with a mean age of 40.7 years (range 12-67). The Expasyl™ group consisted of 50 patients , while the anesthetic solution with vasoconstrictor group comprised 46 patients . The mean duration of follow-up was 17.1 months (range 1-41). In the anesthetic solution with vasoconstrictor group the mean duration of follow-up was 18.6 months (range 1-41), versus 16.6 months (range 1-26 months) in the Expasyl™ group. There were no significant differences in the duration of follow-up between the two groups of patients .The global outcome in each group after 6 and 12 months and on occasion of the final control, as established from the global evolution scale of von Arx and Kurt , is repoThe outcome of periapical surgery can differ considerably from one study to another, depending on the patient inclusion criteria used, the scales employed to score success, and the duration of follow-up . In the On occasion of the control visit after 12 months, 60.4% of the teeth showed treatment success, 33.8% improvement, and 5.8% failure. At the final control (after an average of 17.4 months of follow-up), 60.9% of the teeth showed treatment success, 29.7% improvement, and 9.4% failure. The success rates reported by other authors also using ultrasound and MTA, independently of the hemostatic material used, range from 74-92% -24, thouOnly one study to date  has corrIn our study we found no significant differences in the outcome of periapical surgery between the two groups. In the control performed after 6 months, the outcome was seen to be poorer in the Expasyl™ group, though not significantly so, while after 12 months the opposite situation was observed . The evolution between 6 and 12 months after surgery was more favorable in the Expasyl™ group than in the group treated with dressings impregnated with anesthetic solution and vasoconstrictor. This could be explained by the presence of remaining traces of Expasyl™ - despite having favored bleeding within the bone cavity - which could have delayed bone formation during the first months. However, the more positive course seen in this same group over the subsequent months was probably due to better quality retrograde fillings resulting from the excellent field dryness and visibility conditions afforded by Expasyl™, and which would minimize bacterial filtration between the periapex and root canal system. Nevertheless, since patient assignment to each group was not randomized (this being a retrospective study), the comparatively more favorable results obtained in the Expasyl™ group may have been a mere coincidence or could have been influenced by the surgeon learning curve. Randomized, controlled prospective studies involving large sample sizes are therefore needed to more firmly establish the effects of Expasyl™ upon the outcome of periapical surgery. The healing percentages (summing success and improvement) after 12 months were 96.3% in the Expasyl™ group and 91.4% in the group treated with dressings impregnated with anesthetic solution and vasoconstrictor. These figures are slightly higher than the 90.2% healing rate mentioned above, corresponding to the study of von Arx et al.  using ExIt has been seen that aluminum chloride produces inflammatory reactions in the soft tissues when used for gingival retraction ,29. In tThe outcome after 12 months of follow-up was better in the Expasyl™ group than in the anesthetic solution with vasoconstrictor group, though the difference was not significant.


Considering the major progresses made in interventional cardiology since the advent of BA, it would be reasonable to question what relevance these data have in the era of third- and fourth-generation drug-eluting stents (DESs) that have biodegradable polymers or are polymer free or have a completely bioabsorbable platform. This report comes at an opportune moment in interventional cardiology because of the emergence and availability of the ABSORB everolimus-eluting bioresorbable vascular scaffold . These data might be our “crystal ball” into predicting late and very late events after nonpermanent stent alternatives such as drug-eluting balloons (DEBs) or BVS (the BA group) or after implantation of a DES with biodegradable or no polymers (the BMS group).In this issue, Yamaji et al1 report the very long-term outcomes of patients who were free from early restenosis. The main findings were the following:In the current study, Yamaji et alThe cumulative incidence of all-cause death (44.4% versus 45.4%), cardiac death (19.5% versus 20.6%), or composite of death or myocardial infarction (52% versus 51.6%) at 15 years was similar for the BA and BMS groupsP<0.001). The difference in TLR rates was most marked within the first year after the index procedure (31.5% versus 16.1% at 14 months), with a plateau phase between 14 months and 4 years (33.8% versus 18.5% at 4 years).The cumulative incidence of target lesion revascularization (TLR) was higher in the BA group  occurred less frequently after BA (16.3% versus 21.4%).In the BA group, less residual stenosis at early follow-up angiography was associated with a lower incidence of late TLR.Target lesion thrombosis after 1 year was similar for the BA and BMS groups (1.5% versus 0.7%).In applying these data, we must realize that there are a number of limitations that may make the vision through our crystal ball hazy and limit the scope of our predictions. The authors have clearly outlined these limitations and the large selection bias of the patients in this study, which limits the clinical applicability of the conclusions. Furthermore, regarding generalizability, these data do not apply to patients undergoing percutaneous coronary intervention for acute myocardial infarction or in vessels <3 mm in diameter. Keeping these limitations in mind, let's attempt to use this study to make predictions about late events with current-generation devices.5 The first-generation ABSORB BVS was not associated with repeat revascularization at 4 years,6 and the second iteration of ABSORB BVS 1.1 was associated with a 3.6% TLR rate at 12 months.5 However, we do not know what plaque progression will be in a scaffolded segment when the artery is no longer caged. Furthermore, after the initial phase of drug elution, we do not know if BVSs will have a plateau phase between 1 and 4 years like the BA and BMS groups in this study or a progressive, albeit slow, increase in TLR as seen with both first- and second-generation DESs with durable polymers.7 However, after 4 years, it reasonable to assume that BVSs will have a TLR pattern like that in the BA group. We could even assume that BVSs will perform better, concerning very late events, because of the passivation action of everolimus without the trigger of the foreign body: the metal.The available data have confirmed the efficacy of the drug-elution profile of BVSs.The low very late TLR rate after BA is very encouraging; nevertheless, these findings can be interpreted in 2 ways: (1) the lack of a foreign body, as with a BMS, allows a better “restitutio” of the vascular integrity with all the advantages of unaltered physiology and (2) the BA group is “cleaned” by excluding patients who underwent early TLR (of whom there were more in the BA group than in the BMS group). Indeed, a major limitation of this study with extended follow-up is that early events tend to be more frequent with BA, which results in a selected population of patients. This fact gives the BA group a clear advantage.8 might be about 21% at 15 years .The findings reported by these authors can also be used to predict the long-term outcome after a successful angioplasty with a DEB. If this were true, then looking through our crystal ball, the cumulative incidence of TLR after a DEB procedure, given the 6-month TLR rate of 4.4% in the recently completed BELLO  trial,4 Furthermore, as experience with postdilating BVS increases, postprocedural and early follow-up diameter stenoses should become even lower, thus resulting in an even lower late TLR rate. Another theoretical advantage of BVSs that could further contribute to lower late TLR is that degradation of struts might remove the ongoing inflammatory stimulus for intimal hyperplasia and the absence of a vessel cage would not prevent late positive remodeling.9However, we would expect the results with BVSs to be superior to that of the BA group solely on the basis that the BVS will result in a better angiographic result and less residual stenosis. Lower residual stenosis at the end of the procedure and at early follow-up angiography is known to be associated with less TLR during follow-up. Analogously, patients in the present study with a smaller percentage diameter stenosis at early follow-up angiography had a lower late TLR rate than those with a larger diameter stenosis (14.5% versus 28.0%). The average diameter stenosis in this group (26.7±7.1%) is similar to that seen at 6 months with a second-generation BVS (24.0±9.6%).11 However, unstable lesions characterized as thin-cap fibroatheromas or plaque rupture are more frequent in BMSs probably because of the longer implant duration. In all cases, however, there is usually no communication between the lesion within the stent and the underlying native atherosclerotic plaque.11 In rare cases, neoatherosclerosis may contribute to very late revascularization and thrombotic events in both BMSs and DESs. However, pathological data regarding neoatherosclerosis in DESs are limited to first-generation DESs with durable polymers.Although DESs are extremely efficacious in inhibiting intimal hyperplasia while the drug is being eluted, they clearly change the underlying plaque and function of neointima in a way that is different than that of BMSs. Indeed, we do not know if second-/third-generation biodegradable polymer or polymer-free DESs will behave as BMSs after the drug has eluted, with regard to late events and neoatherosclerosis. It is now accepted that neoatherosclerosis is more frequent and occurs significantly earlier in DESs than in BMSs.12 If we look through our crystal ball again, can we predict that these DESs will behave like BMSs after the polymer has degraded? If so, then these stents will have a late TLR rate of 1.6%/y, like the BMS group in the present study . However, the cumulative incidence curves will probably be very different and continue to progress slowly without showing a plateau phase. It is not known if this is related to neoatherosclerosis or just a more protracted neointimal response than occurs with BMSs.In the LEADERS (Limus Eluted from A Durable versus ERodable Stent coating) trial, the TLR rate at 4 years of a biodegradable polymer DES was 9%.13 All these adverse reactions can be potentially prevented by the implantation of a fully biodegradable stent.13 Similarly, in the LEADERS trial, the 4-year rates of very late definite stent thrombosis were significantly lower with biodegradable polymer DESs than with durable polymer DESs .12The causes of late stent thrombosis have been described as related to delayed endothelialization, chronic inflammatory response, and localized hypersensitivity reactions.1 Indeed, late target lesion thrombosis (after 4 years) was present in 7 of 10 patients with BA who underwent late TLR for acute myocardial infarction. Does this suggest that the absence of a permanent vascular scaffold does not protect from late thrombotic events? This would be a very controversial statement as the enthusiasm for BVSs is based on the fact that after degradation, the treated segment of the vessel will return to normal function, thus eliminating the dependence on long-term dual antiplatelet therapy and the risk of late thrombotic events.14 This concept is also circumstantially supported by data demonstrating normal vasomotion after BVS implantation.15The most important and challenging prediction to make is whether BVSs remove the risk of very late stent thrombosis from the clinical arena. To predict whether late events after implantation of a BVS will be similar to those after a BA procedure, we need to know if the mechanisms of healing and late events are similar. In this regard, an intriguing and important finding in the present study is that target lesion thrombosis was similar in the 2 groups.16 provided some preliminary data in this direction with their observation that BVSs result in a symmetrical and circumferential thick fibrous cap. The premise is that this healing process of BVSs might be used to stabilize vulnerable plaques before the scaffold disappears and leaves the vessel uncaged.17However, there is a paucity of long-term histopathological data after BA or BVS. It is not known if late events are due to further expansion of the underlying plaque or rupture/erosion of neoatherosclerotic plaques. One hypothesis is that BVSs may seal plaque, resulting in more “normal healing” and thus altering neoatherosclerosis. Brugaletta et alIn conclusion, BA and current stenting do not seem to fully protect the vessel from neoatherosclerosis or plaque progression. It may be possible that a fully bioresorbable scaffold, by eliminating the presence of a permanent vascular prosthesis, may passivate the vessel, thus resulting in better healing and a vessel less prone to atherosclerosis. Presently, there are insufficient scientific and clinical data to support the dream that sealing or passivation of critical and selected noncritical coronary plaques with nonpermanent scaffolds may prevent future events.