Both fear and pain processing are altered in post-traumatic stress disorder (PTSD), as evidenced by functional neuroimaging studies showing increased amygdala responses to threats, and increased insula, putamen and caudate activity in response to heat pain. Using psychophysiology and functional magnetic resonance imaging, we studied conditioned and unconditioned autonomic and neuronal responses in subjects with PTSD versus trauma-exposed non-PTSD control (TENC) subjects. A design using an electric shock selected by subjects to be 'highly annoying but not painful' as an unconditioned stimulus (US) with partially reinforced cues allowed us to partly disentangle the expectancy- and prediction-error components from sensory components of the unconditioned response.Whereas responses to the conditioned stimulus (CS) were similar in PTSD and TENC, the former displayed higher putamen, insula, caudate and amygdala responses to the US. Reactivity to the US in the anterior insula correlated with PTSD symptom severity. Functional connectivity analyses using the putamen as a seed region indicated that TENC subjects had increased amygdala-putamen connectivity during US delivery; this connection was disengaged in PTSD.Our results indicate that although neural processing of fear learning in people with PTSD seems to be comparable with controls, neural responses to unconditioned aversive stimuli in PTSD seem to be increased. In classic fear conditioning, an initially neutral, and later conditioned stimulus (CS+) is paired with an aversive unconditioned stimulus (US) such as an electric shock. After pairing, the neutral stimulus is able to elicit fear on its own. Much has been learned about the neural responses induced by the CS+ presentation. In healthy people, conditioned stimuli activate the amygdala, brainstem, insula and parts of anterior cingulate cortex (ACC) . Less isUnderstanding the neural mechanisms that mediate aversive unconditioned responses may be clinically relevant. Brain regions involved in fear learning, such as the amygdala and the ACC, are implicated in the pathophysiology of several anxiety disorders such as post-traumatic stress disorder (PTSD). Other intrinsically aversive stimuli such as trauma reminders, aversive images and fearful faces also elicit increased responses in several brain regions in PTSD, such as the amygdala and the ACC . The uncpost hoc analysis, we explored inter-regional functional connectivity differences in US-elicited responses in the regions in which the subjects with PTSD were found to differ significantly from the TENC subjects.In the present study, we examined the neural correlates of responses to electrical shock stimuli in subjects with PTSD and in trauma-exposed non-PTSD (TENC) control subjects. Within an fMRI fear-conditioning paradigm, we examined neuronal and autonomic responses to CS+ and US presentations in a partial-reinforcement paradigm . The US The Partners Healthcare System Human Research Committee approved the study, and all subjects gave informed consent.Diagnostic and Statistical Manual, fourth edition (DSM-IV) [In total, 23 right-handed people with PTSD and 28 right-handed healthy TENC subjects were recruited from the community. All were administered the Structured Clinical Interview for the (DSM-IV)  to deterFor the TENC group, participants with any current mental disorder were excluded. Subjects with PTSD who had current substance dependence were also excluded, as were subjects who had used any psychotropic medication within 4 weeks before participation (1 year for neuroleptics). Data from four PTSD and four TENC subjects were excluded because of excessive head movement while in the scanner, leaving a total sample size of 19 PTSD and 24 TENC subjects. Data from a subset of these participants have already been reported in studies examining fear extinction ,16; howeThe Clinician-Administered PTSD Scale (CAPS) , NeurotiWe used a partial-reinforcement classic conditioning paradigm that has previously been described in detail . In the The US consisted of a 500 ms train of 1 ms spikes at 50 Hz delivered to the second and third fingers of the right hand, with currents ranging from 0.2 to 4.0 mA. Before the experiment, the shock current was individually adjusted so that it would be perceived as 'highly annoying but not painful' by the individual patient. Administration of the US immediately followed CS offset.Skin-conductance responses (SCRs) were measured using a skin-conductance coupler  with 8 mm (sensor diameter) radiolucent Ag/AgCl electrodes . Electrodes were filled with isotonic paste, and placed on the palm of the subject's left hand. The skin-conductance electrodes were separated by 14 mm. The SCR for each CS trial was calculated by subtracting the mean skin-conductance level 2 seconds before CS onset from the highest skin-conductance level recorded during the following CS duration of 6 seconds. SCR responses during the interval after the US, the omitted (non-delivered) US or the CS- offset were calculated by subtracting the mean skin-conductance level recorded during the first 2 seconds of this interval from the highest skin-conductance level during the ensuing 3 seconds. Because autonomic responses are delayed with respect to their antecedent neural activity , this 2-MRI data were collected using a 3.0 Tesla whole-body MRI system  equipped for echo planar imaging with a 12-channel head coil. Subjects were instructed to lie as still as possible, and head movement was restricted with foam cushions. After an automated scout image was obtained and automated shimming procedures performed, a high-resolution, T1-weighted, three-dimensional, magnetization-prepared rapid acquisition gradient echo (MPRAGE) volume was collected to facilitate spatial normalization and positioning of the subsequent scans. Functional MRI images, sensitive to blood oxygenation level-dependent (BOLD) contrast, were acquired with an interleaved gradient echo T2*-weighted sequence , collected in 45 coronal oblique slices tilted 30° down from the anterior-posterior commissural line. The voxel size was 3 ± 3 ± 3 mm.http://www.fil.ion.ucl.ac.uk) was used to process all MRI data. Structural images were segmented and spatially normalized to the Montreal Neurological Institute (MNI) 305 T1 template. Functional images were realigned, corrected for slice timing, coregistered with the structural volume, resampled to 2 ± 2 ± 2 mm, normalized into MNI space using parameters obtained from the structural normalization process, and finally smoothed with an 8 mm full width half-maximum Gaussian kernel to reduce spatial noise and to compensate for residual misregistration in the spatial normalization process. High-pass temporal filtering with a cutoff of 128 seconds was included in the first-level statistical model to remove the effects of low-frequency physiological noise. Serial correlations in the fMRI time series caused by aliased biorhythms were estimated using an autoregressive AR(1) model.SPM8 . However, because of multiple collinearity between regressors identifying cue-induced activations and activity immediately after the termination of the cue (r2 = 0.36), we did not examine this effect.We focused our analysis on the responses to three of the experimental events: 1) the CS+, 2) the US and 3) the omitted US. The CS- cue and the interval following its offset were used as control contrasts. This design allowed us to investigate not only fear-conditioned responses but to also directly contrast brain responses induced by the actual delivery of the US with brain responses induced by the immediate expectancy and then the unexpected omission of the US. Because both the US and omitted US intervals occurred immediately after the offset of the CS+, a combination of anticipatory- and prediction-error components influencing the shock response could be identified in the omitted US contrast, as limited by the temporal resolution of the present fMRI paradigm. Multi-collinearity between regressors may lead to instability of parameter estimation and a consequent reduction in sensitivity. In the present design, the inherent multiple collinearity of the classic conditioning design (a cue followed by a shock) was not problematic for these three contrasts, as the correlations between the regressors were very low , corrected for multiple comparisons, are reported.Recent studies of pain responses in PTSD have reported alterations in activity in the putamen, amygdala, hippocampus, cingulate, insula and caudate nucleus ,12,23. BP < 0.05 FWE are reported. Correlation models such as these have been criticized for overinflated correlation coefficients [r values.First-level contrast images representing the effects of CS+ and US were obtained for all subjects with PTSD, and modeled at the second level using a linear regression model with CAPS scores. Correlated clusters of more than 10 contiguous voxels adjusted for multiple comparisons at ficients , so we tP < 0.05 FWE are reported.To further understand alterations in US responses in PTSD, we performed a psychophysiological interaction (PPI) analysis, a technique allowing estimation of how inter-regional BOLD signal correlations vary with respect to an experimental psychological context. Based on the univariate results, we investigated whether the delivery of the US (versus omitted US) modulated the inter-regional functional connectivity between seed regions and targets elsewhere in the brain differently in PTSD and TENC subjects. Four seed regions  were chosen based on the peak observed hyperactivation to the US of these regions in the PTSD group. We contrasted inter-regional influences of the seeds during the US interval with those during the omitted US interval to elucidate how the delivery of the US modulated seed functional connectivity to other brain regions. The corresponding PPI design matrix had three columns: the interaction between the experimental manipulation and seed time series, the main effect of experimental manipulation (US or omitted US), and the physiological effect . In accordance with the task-related univariate analysis, we also included six estimated movement parameters to account for any residual effects due to inter-scan head motion. The voxel-wise regression of voxel time series from the entire brain on the three predictor variables resulted in a first-level PPI contrast for each seed region in every subject. Subsequently, these PPI contrast images were modeled in a second-level group analysis using a mixed-effects repeated-measures linear model with subjects, diagnosis and seed region as factors. This second-level analysis resulted in a statistical parametrical map of regions that displayed differences between TENC and PTSD subjects in alterations of seed functional connectivity depending on the presence or absence of the US. Clusters of more than 10 contiguous voxels adjusted for multiple comparisons at PTSD and TENC subjects chose similar shock levels and displayed similar levels of autonomic skin-conductance responses to the CS+ and the US. Subjects with PTSD displayed slightly higher skin-conductance responses to the non-reinforced CS- cue, but the magnitude of responses to the CS- was generally low (Table a priori regions of interest (ROI), subjects with PTSD displayed additional significant hyper-reactivity in the bilateral amygdala, left hippocampus, right dorsal ACC, bilateral posterior insula, left anterior insula, and bilateral caudate and putamen. No regions displayed lower reactivity in the PTSD group . Within the up Table .xyz = 28, 34, 0, cluster size = 329 voxels, Z = 7.93, PFWE corrected = 0.002)  and US-induced BOLD activation in the right inferior frontal gyrus/anterior insula , that is, connectivity, analysis.To further elucidate the findings from the univariate analysis, we performed post hoc to perform a functional connectivity analysis. The seed consisted of a 6 mm sphere centrered at the peak location . When comparing PPI effects of the left putamen between PTSD and TENC subjects, there were significant differences in functional connectivity shifts. The TENC group showed a a more positive PPI effect between the left putamen and the left temporal lobe including the amygdala. In other words, the shock delivery led to a larger increase in functional connectivity between the putamen and the amygdala in the TENC group as compared to connectivity shifts in the PTSD group . Contrasting PPI effects of the right putamen between PTSD and TENC subjects showed differences in functional connectivity shifts similar to the left putamen seed, with a more positive PPI effect in the TENC group between the right putamen and the left temporal lobe including the amygdala, that is, indicating a larger increase in functional connectivity between these two regions at the shock delivery in the TENC group compared with the PTSD group (Table The right putamen seed (a 6 mm sphere) was centred at the peak location . Contrasting PPI effects of the left middle frontal gyrus between PTSD and TENC subjects showed no significant differences in PPI effects.The left middle frontal gyrus seed (a 6 mm sphere) was centred at the peak location . Contrasting PPI effects of the left parahippocampal gyrus between PTSD and TENC subjects showed differences in functional connectivity shifts, with a more positive PPI effect in the TENC group between the left parahippocampal gyrus and right inferior parietal lobule, and the left medial frontal gyrus and the right precentral gyrus, that is, indicating a larger increase in functional connectivity between the seed and these three regions at the delivery of the shock in the TENC group compared with the PTSD group  was centred at the peak location (MNIup Table .The predictable and 'annoying' shock (the unconditioned stimuli (US)) elicited activation in several brain regions associated with pain processing , suggestThe CS+ led to comparable increases in skin conductance in both the PTSD and the TENC group, suggesting no differences in the fear induced by the conditioned cues. Moreover, there were no differences in the neural response to the CS+, replicating behavioral studies that suggested intact fear learning in PTSD -35, but Subjects with PTSD displayed increased putamen reactivity to the US, an effect that was not observed after the omitted US. Moreover, the putamen hyperactivation to the US in the PTSD group remained significant when controlling for responses to the omitted US, (PTSD (US vs. omitted US) vs. TENC (US vs. omitted US); Table With regard to the functional connectivity results, two major effects were seen when comparing connectivity at the omitted US and the delivered US: TENC subjects (compared with subjects with PTSD), displayed a larger increase in putamen to temporal lobe/amygdala connectivity at the delivered US compared with the omitted US. Moreover, TENC subjects (compared with subjects with PTSD) displayed an increase in parahippocampal gyrus connectivity to the inferior parietal lobule, the precentral gyrus and the medial frontal gyrus Figure . We specHigher right anterior insula US reactivity was correlated with higher PTSD symptom severity on the CAPS. This result is consistent with findings of Mickleborough and colleagues , who repThe putamen is best known as a motor output region , but botWe have interpreted the neuronal responses and connectivity modulations related to the omitted US as reflecting immediate expectancy ('I am going to get shocked right now'). It could be argued, however, that these results also represent a prediction error and relief signal . Our experimental design did not allow us to distinguish between these two responses, thus further studies with higher temporal resolution should be designed and conducted to further examine this point.Previous studies have implicated the putamen and insula in altered pain processing in PTSD. The present study extends to this literature by showing that the functional activation of these brain regions is also altered in response to highly annoying but not overtly painful stimuli, and that such alterations are likely to be driven by sensory aspects of the aversive stimulus rather than by expectancy- and prediction-error components.ACC: anterior cingulate cortex; BAI: Beck Anxiety Inventory; BDI: Beck Depression Inventory; CAPS: Clinician-Given PTSD Scale; CS conditioned stimulus; fMRI functional magnetic resonance imaging; FWE: family-wise error corrected for multiple comparisons; PPI: psychophysiological interaction; PTSD: post-traumatic stress disorder; SCR: skin-conductance response; STAI: Spielberger State Trait Anxiety Inventory; TENC: trauma-exposed non-PTSD controls; US: unconditioned stimulus.The authors declare that they have no competing interests.CL collected and analyzed the data, and wrote the manuscript. TAZ contributed to data analysis and to the final version of the manuscript. RKP participated in experimental design and the final version of the manuscript. MRM designed the study, collected data and contributed to the final version of the manuscript. All authors read and approved the final manuscript.Figure S1 Paradigm design and timing. Figure S2 Skin conductance responses within three different trials in one representative subject. Figure S3 Skin conductance responses to the conditioned stimulus. Figure S4 Skin conductance responses to the unconditioned stimulus. Figure S5 Skin conductance responses to the omitted unconditioned stimulus.Click here for file


Purpose of the study was to investigate alterations in midbrain serotonin transporter (SERT) binding in patients with epilepsy and symptoms of depression compared to patients with epilepsy with no symptoms of depression.123I-ADAM).We studied 12 patients with epilepsy . The presence of self-reported symptoms of depression was assessed using Beck Depression Inventory (BDI) and the Emotional State Questionnaire (EST-Q). The binding potential of the SERT was assessed by performing brain single photon emission tomography (SPET) using the SERT radioligand 2-((2-((dimethylamino)methyl)phenyl)thio)-5-(123)iodophenylamine (123I-ADAM binding was not significantly different between patients with epilepsy with and without symptoms of depression. In addition, 123I-ADAM binding did not show a significant correlation to either BDI or EST-Q depression subscale scores and did not differ between patients with focal vs. generalized epilepsy.Seven patients had BDI and EST-Q subscale scores greater than 11 points, which was interpreted as the presence of symptoms of depression. We found that The results of our study failed to demonstrate alterations of SERT binding properties in patients with epilepsy with or without symptoms of depression. The frequent coexistence of epilepsy and depression has encouraged researchers to explore potential shared mechanisms underlying these disorders. Depression occurs far more frequently in patients with epilepsy than in the general population, affecting approximately 30% of this patient population,2. At th1A receptor binding potential in various limbic and neocortical regions and the raphe nuclei[The dysfunction of the brain serotonin (5-hydroxytryptamine or 5-HT) system has been suspected to be the common denominator for the shared pathogenic mechanisms of epilepsy and depression. Alterations in serotonergic signaling are associated with the pathogenesis of depression in patients with major depressive disorder - findings that are clinically supported by the effect of selective serotonin reuptake inhibitors (SSRIs) in the treatment of depression. Several neuroimaging studies using different positron emission tomography (PET) or single photon emission tomography (SPET) tracers for various components of serotonergic system in the brain have supported the involvement of 5-HT in major depressive disorder. These alterations include increased serotonin transporter (SERT) binding in the thalamus and limbic regions, or decre nuclei.1A receptors to investigate the role of the serotonergic system in epilepsy and depression. These studies have concentrated on patients with both temporal lobe epilepsy (TLE) and depression and have shown reduced 5-HT1A receptor binding potential in the ipsilateral temporal lobe as well as in thalamic regions, hippocampus, anterior insula, anterior cingulate, and the raphe nuclei in the depressed patients[A growing body of evidence implicates the role of serotonin system in epilepsy. This includes studies in which the anticonvulsant effects of SSRIs have been confirmed, such as Favale et al.. In thispatients-20. Thus123I-ADAM) is a novel SPET tracer that has shown a high binding affinity for SERT as well as high selectivity for 5-HT transporter over those for norepinephrine and dopamine, and which has been proven to have excellent brain uptake in rats[2-((2-((dimethylamino)methyl)phenyl)thio)-5-(123)iodophenylamine  with epilepsy who were recruited from the outpatient clinic at the Department of Neurology and Neurosurgery in University of Tartu and West-Tallinn Central Hospital in Tallinn, Estonia. Patients were otherwise healthy, with no history of other neurological disorders except epilepsy, and did not use antidepressant medications prior to this study. All patients gave informed consent to participate in the study.123I-ADAM SPET study.In the current study, all patients with symptoms of depression were consulted regarding their affective disorder, and treatment with antidepressant medications was offered following the 123I-ADAM SPET imaging session. A cut-off score of > 11 points was used to define the presence of symptoms of depression on the BDI. EST-Q is a self-report questionnaire for depression and anxiety that uses the rating of 33 items on a five-point frequency scale. This questionnaire has five subscales: depression, anxiety, agoraphobia–panic, fatigue, and insomnia. In the current study, a cut-off score of > 11 points was used to define the presence of symptoms of depression on the EST-Q depression subscale.Patients were screened for self-reported symptoms of depression using the Beck Depression Inventory (BDI) and the 123I-ADAM). The subjects received a dose of 185 MBq 123I-ADAM  intravenously. To block the thyroid gland, potassium perchlorate  was given orally at least 20 min prior to the injection of 123I-ADAM. Brain SPET studies were acquired 4 hours after the injection of 123I-ADAM.We examined serotonin transporter (SERT) binding potential by performing brain SPET study with 2-((2-((dimethylamino)methyl)phenyl)thio)-5-(123)iodophenylamine  dual head gamma camera with low energy high-resolution collimators (Lehr collimators). The energy window was centered on 159 keV (+/-10%). SPET scans were acquired in a step and shoot mode with total angular range of 360 degrees thereby arc per detector being 180 degrees. View angle 3 degrees, 120 views, 30 sec per projection. Acquisition time was 30 min. Matrix size was 128 × 128, with a zoom of 1.0.123I-ADAM binding was assessed using MRI-guided ROIs in the midbrain and cerebellum. ROIs were placed on transaxial MRI slices over the midbrain and cerebellum and then transferred onto corresponding SPET slices. In addition, radio-uptake and the specific uptake ratios (SURs) of midbrain were assessed. As a measure of brain SERT availability, the ratio of specific-to-nonspecific 123I-ADAM binding for the midbrain compared to the cerebellum were calculated in mean counts/pixel using the following equation: SUR = specific binding/nonspecific binding = target- cerebellum/cerebellum.Data were reconstructed using the Xeleris Functional Imaging Workstation software . Transverse slices were reconstructed parallel to the canthomental plane. SPET data were reconstructed using a Butterworth filter , followed by the Chang attenuation correction . SPET and MRI data were automatically coregistered using MPI Tool software . To measure the individual SERT occupancy, irregular regions of interest (ROIs) were manually drawn over the midbrain and over the cerebellum as the reference area. The t-tests were used to compare variables between the two groups of patients (with symptoms of depression vs. without symptoms of depression). A correlation analysis was used to assess the relationship between depression scale scores, demographic, and clinical characteristics, and SERT binding potential.Statistical analysis was performed with STATISTICA 8.0. Student’s A summary of patient characteristics, including their demographic and clinical characteristics is included in Table Seven patients had BDI and EST-Q depression subscale scores greater than 11 points, which was interpreted as the presence of symptoms of depression. The mean BDI and EST-Q depression subscale scores for the whole patient group were 11.5 ± 6 and 14 ± 4.3, respectively. The maximal BDI score was 20; in all patients with symptoms of depression, the BDI score was in the range of mild to moderate severity.In the current study, patients with symptoms of depression, as a group, did not significantly differ from patients without symptoms of depression in either demographic or clinical variables Table . A compa123I-ADAM binding to SERT did not differ significantly between the patients with epilepsy who had symptoms of depression vs. those without. In addition, SERT binding potential of 123I-ADAM did not show any statistically significant correlation with either the BDI or the EST-Q depression subscale scores. SERT binding potential was also not correlated with any demographic or clinical characteristics, including age, duration of epilepsy, or age at disease onset. We also observed that the SERT binding potential did not differ between patients with focal vs. generalized epilepsy.Using SPET, we observed that 123I-ADAM imaging study.One patient with moderate symptoms of depression on BDI committed suicide shortly following the In the current study, we sought to study SERT binding properties in the midbrain region in patients with epilepsy, and to determine whether SERT binding differed between depressed vs. non-depressed patients with epilepsy. Our results did not indicate any difference in SERT binding potential between these patient groups.123I-ADAM SPET to measure SERT binding in major depressive disorder have also indicated decreased SERT binding in the midbrain, medial temporal lobe, and basal ganglia of depressed patients compared to controls[123I-ADAM in patients with depression compared to healthy controls have also been published[There could be several reasons for these negative results. Previous work with PET and SPET tracers for SERT in depressed patients has shown some conflicting results. The majority of reports show increased SERT binding in the thalamus and limbic regions of depressed patients compared to controls, but othcontrols,27. At tublished,32. It hSLC6A4 gene, which encodes the SERT protein, exhibit differences in SERT binding properties in neuroimaging studies[SLC6A4.Another contributing factor to these negative results may be the genetic variability that has been shown for SERT expression in the human brain. For example, individuals with polymorphisms in the promoter (5-HTTLPR) of the  studies-35. Gene studies. GeneticThere are several limitations to the current study. Perhaps the most important, and the one likely to be largely responsible for our negative results, is the relatively small study sample size. The statistical power of the comparison of SERT binding in groups of patients with and without depression was 0.755. We calculated that increasing the power to 0.8 under the same conditions would need 40 subjects, which considering the nature and cost of SPET imaging, would be unachievable.Since in all patients with symptoms of depression, the BDI score was in the range of mild to moderate severity, it could also be considered as a weakness contributing to the negative results of our study.1A receptor imaging studies, are all well-documented in cases of TLE. Little is known about the same aspects in case of focal extra-temporal epilepsies, and even less about generalized epilepsy syndromes. Although some findings have indicated that depression could be specifically related to TLE and mesial temporal sclerosis[vs. generalized epilepsy in terms of the prevalence of depression. One work did assess symptoms of anxiety, and found that patients with FLE have much higher anxiety scores than patients with generalized epilepsy[vs. generalized epilepsy were comparable in terms of presence of depressive symptoms.The heterogeneity of our study group, regarding the clinical characteristics of epilepsy, could also have led to the observed lack of differences in SERT binding properties. The characteristics of depression, depression-related treatment outcomes, and serotonergic system involvement, based on 5-HTclerosis, this haclerosis. It has epilepsy. In our These previous findings seem to indicate that the bidirectional relationship between epilepsy and depression is not specific to TLE. It has been shown that preoperative depressive symptoms predict postoperative seizure outcome in both TLE and FLE. TherefoThe results of our study failed to demonstrate alterations of SERT binding potential in patients with epilepsy with symptoms of depression compared to patients with epilepsy without symptoms of depression. Further studies are needed to clarify the role of SERT and, more generally, the serotonergic system in the common pathogenesis of epilepsy and depression.123I-ADAM: 2-((2-((dimethylamino)methyl)phenyl)thio)-5-(123)iodophenylamine; SPET: Single photon emission tomography; SERT: Serotonin transporter; BDI: Beck depression inventory; EST-Q: Emotional state questionnaire; 5-HT: Serotonin; SSRIs: Selective serotonin reuptake inhibitors; PET: Positron emission tomography; TLE: Temporal lobe epilepsy; ROIs: Regions of interest; MRI: Magnetic resonance imaging; FLE: Frontal lobe epilepsy; AEDs: Antiepileptic drugsThe authors declare that they have no competing interests.ML participated in the conception and design of the study, acquisition and analysis of data, and drafted the manuscript; MP participated in the design of the study, acquisition of SPET data, and revised the manuscript critically; LV participated in the conception of the study, acquisition of data, and revised the manuscript critically; KGP participated in the conception of the study, acquisition of data, and revised the manuscript critically; SH participated in the conception and design of the study, coordinated the study, and revised the manuscript critically. All listed authors have read the manuscript and have given their final approval of the version to be published.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/13/204/prepub


Previous studies suggested that the overproduction of TACE is causally related to the pathogenesis of inflammatory diseases and cancers. To test this hypothesis, we generated a transgenic line in which the transcription of exogenous Tace is driven by a CAG promoter. The Tace-transgenic mice were viable and exhibited no overt defects, and the quantitative RT-PCR and Western blot analyses confirmed that the transgenically introduced Tace gene was highly expressed in all of the tissues examined. The Tace-transgenic mice were further crossed with −/+Tace mice to abrogate the endogenous TACE expression, and the Tace-transgenic mice lacking endogenous Tace gene were also viable without any apparent defects. Furthermore, there was no difference in the serum TNFα levels after lipopolysaccharide injection between the transgenic mice and control littermates. These observations indicate that TACE activity is not necessarily dependent on transcriptional regulation and that excess TACE does not necessarily result in aberrant proteolytic activity in vivo.TNFα-converting enzyme (TACE/ADAM17) is a membrane-bound proteolytic enzyme with a diverse set of target molecules. Most importantly, TACE is indispensable for the release and activation of pro-TNFα and the ligands for epidermal growth factor receptor  This proteolytic activity, also referred to as “ectodomain shedding,” plays essential roles in the functional regulation of membrane-bound molecules. Previous studies revealed that the TNFα-converting enzyme (TACE), also known as A Disintegrin and Metalloprotease 17 (ADAM17), is one of the most critical proteolytic enzymes involved in ectodomain shedding in vivo.There is evidence suggesting that the overexpression of TACE is causally related to the pathogenesis of various disorders in vitro, including growth factors, phorbol esters, osmotic pressure, ultraviolet irradiation, and cholesterol deprivation, without changing the amount of its mature form TACE is initially produced as a proteolytically inactive pro-form and is processed by a furin protease in the secretory pathway to produce the mature enzyme Tace-transgene in any of the tissues examined. This study, therefore, shows that the overproduction of TACE does not necessarily result in the hyperactivation of shedding activity and suggests that the level of TACE activity cannot be explained solely by its level of transcription or the amount of the mature form of the protein.To address these issues, we generated a transgenic line of mice that express TACE under the control of a CAG promoter and found that the mutant mice were viable and exhibited no apparent defects. A higher amount of TACE transcripts and mature protein in the mutant mice tissues were confirmed by quantitative RT-PCR and Western blotting, respectively. The expression levels of the TIMP3 transcripts were not affected by the introduction of the Tace cDNA with an HA-epitope (YPYDVPDYA) sequence added to the 3′-teminus was generated using PCR-based methods and subcloned into the pCAGGS vector. HA-epitope tagged-TACE has been shown to exhibit comparable shedding activity towards its substrates to non-tagged wild-type TACE Tace cDNA, HA epitope, and polyadenylation signal sequence  and a BX50 microscope .RNA was extracted from the tissues or cultured cells using Sepasol RNA I Super G  and reverse-transcribed using ReverTra Ace . The PCR amplification and quantification were performed using SYBR premix ExTaqII  and LightCyclerII (Roche). The relative mRNA expression levels were normalized to the expression level of the β-Actin transcripts. The sequences of the oligonucleotides used in this study will be provided upon request.The tissues and cells were lysed in lysis buffer , 1 mM 1,10-phenanthroline, and protein inhibitor cocktail (Sigma-Aldrich)), and the lysed samples were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The anti-sera against the cytoplasmic domain of mouse TACE was produced as previously described Tace-Tg embryos were immortalized with the SV40 large T antigen, as previously described Mouse embryonic fibroblasts (mEFs) collected from E13.5 The cells were washed twice in ice-cold PBS, and the cell surface molecules were labeled using Pierce Cell Surface Protein Isolation Kit (Thermo) according to the manufacturer's instructions, with some modifications. The labeled samples were affinity precipitated with neutravidin beads overnight at 4°C, and the affinity precipitated material was separated by SDS-PAGE and analyzed by Western blotting.Septic shock was induced by the intraperitoneal injection of lipopolysaccharide  and D-galactosamine (20 mg). The mice were closely monitored until the mortality was 100%. For the analysis of the serum cytokines, the sera were collected at 3 h after the treatment in a separate experiment. The serum levels of TNFα, TNFR1, TNFR2, and CD62L were analyzed by sandwich ELISA  according to the manufacturer's instructions.t-test for two samples, assuming equal variances, was used to calculate the p values. The statistical analyses were performed using Prism 5 (GraphPad software), and p values smaller than 0.05 were considered statistically significant. All of the experiments were conducted in triplicate.The Student's Tace transcripts would affect the TACE activity in vivo, we generated transgenic mice that expressed an HA-tagged TACE driven by a CAG promoter (in vitroTace-transgenic mice (henceforth referred to as Tace-Tg mice) were viable and fertile and did not show any overt developmental defects .To understand how the enhanced expression of promoter . The HA- defects . MatingsTace transcripts in the Tace-Tg mice by quantitative RT-PCR. As shown in Tace was observed in all the tissues examined, including the liver, lung, skin, spleen, bone marrow (BM), and thymus. The relative increase was lower (approx. 4–6 times) in the immune organs  and higher (approx. 30–300 times) in the other tissues, indicating that the basal levels of Tace transcripts and/or CAG promoter activity differ across these tissues. The increased expression of the TACE protein in these tissues was also confirmed using an anti-HA antibody  and an anti-TACE antibody   . Necrotoal level . These oTace transcript levels in the mEFs derived from the Tace-Tg mice . As shown in Tace-Tg embryos. The shedding activity was similarly enhanced upon PMA stimulation and suppressed by a broad-range metalloprotease inhibitor, GM6001. These observations indicate that the excess TACE protein in the Tace-Tg-derived mEFs does not significantly affect the overall shedding activity, at least under the present experimental conditions.The apparent lack of developmental defects in the Tace-Tg mice were intraperitoneally injected with LPS to induce TNFα production; LPS is a major component of the cell membrane of Gram-negative bacteria, and it elicits a strong immune response in mammalian immune cells. Because LPS can stimulate the production of TNFα in immune cells, we hypothesized that the amount of soluble TNFα released into the supernatant would show a positive correlation with the amount of TACE expressed in the immune cells. However, contrary to our expectation, there was no difference in the serum levels of TNFα or any of the TACE substrates examined  between the control and Tace-Tg mice under unchallenged conditions or after LPS treatment , a proteolytically inactive member of the rhomboid protease family, are defective for this process and incapable of releasing soluble TNFα /− mice . Furtherve model .in vivo. The observation that the amount of cleaved molecules in the serum remained static, irrespective of the TACE expression levels (In summary, this study demonstrates the lack of a direct correlation between the TACE expression levels and the shedding of TACE substrates n levels , may sugin vivo does not lead to overt defects.During the revision of the manuscript, an independent study


Primary signet-ring cell carcinoma of the urinary bladder is extremely rare and patient survival is very poor. The disease usually presents at advanced stages because the cancer progresses rapidly. The only option for effective treatment is radical cystectomy, and no effective chemotherapy has been established for this variant. We report a case of signet-ring cell carcinoma of the urinary bladder with a long-term survival of 90 months owing to radical cystectomy and combination adjuvant chemotherapy with S-1 and cisplatin. To our knowledge, this is the first report to demonstrate the long-term therapeutic activity of combination S-1 and cisplatin adjuvant chemotherapy against invasive signet-ring cell carcinoma of the urinary bladder. Adenocarcinoma of the urinary bladder accounts for 0.5%–2% of all bladder cancers with signet-ring cell carcinoma being an aggressive phenotype characterized by signet-ring cells containing intracellular mucin-filled vacuole, which displaces the hyperchromatic nucleus to one side of the cell . Primary2, days 1–14) and cisplatin  for three cycles. This regimen was approved by the institutional chemotherapy review board at Nagoya City University Hospital and conducted in accordance with Declaration of Helsinki. As a result, no remarkable adverse event was associated with this chemotherapy. He is still alive 90 months with no evidence of tumor recurrence or metastasis.A 53-year-old Japanese man with a history of pain on urination and hematuria for several months was referred to our hospital. He had no past medical history and was treated with a course of antibiotics for urethritis, which did not resolve the complaint. Urinalysis revealed RBC 50–99/HPF and WBC 10–19/HPF and was negative for bacteria and for urinary cytology. Serum tumor markers including carcinoembryonic antigen were within normal limits. Cystoscopy revealed widely prevalent nonpapillary raised lesions and edematous mucosa on the posterior wall of the bladder. Pelvic computed tomography (CT) scan demonstrated marked diffuse bladder wall thickening without swelling of the pelvic lymph nodes. Magnetic resonance imaging (MRI) produced results similar to CT. Moreover, sagittal MRI showed bladder wall thickening from the anterior to posterior wall . TransurSRCCU is a rare histologic variant of poorly differentiated adenocarcinoma. Most cases of SRCCU occur in middle age, and common complaints are hematuria and irritation on voiding, both of which were seen in this patient. The duration of presenting symptoms was 3 months in the review by Naeim et al.  and 3.5 The histogenesis of SRCCU remains controversial. Many experts suggest that mesonephric remnants of the trigone of the bladder may be the source of this cancer. However, this would not explain signet-ring cell carcinoma at other locations. Another theory is metaplasia of urothelium, which may occur along the surface of or in areas of cystitis cystica within the bladder . In thisRadical cystectomy is the only therapy that offers the possibility of a cure when the tumor is localized. However, treatment options for advanced and invasive SRCCU have not been well defined because of the rarity of the tumor. Although combinations of methotrexate, vinblastine, doxorubicin, and cisplatin or gemcitabine and cisplatin are standard chemotherapy regimens for the treatment of advanced and invasive urothelial carcinoma, SRCCU is generally resistant to these therapies that have several limitations. Long-term follow-up has shown considerable toxicity, including myelosuppression and renal toxicity. To date, some patients fail to respond to standard platinum-based regimens but derive a significant clinical benefit from colon cancer type regimens such as 5-fluorouracil- (5-FU-) based regimens of capecitabine. In addition, some reports have shown the effectiveness of combination chemotherapy with 5-FU and cisplatin in SRCCU . In Japan, S-1 is the most widely used 5-FU-based drug for the treatment of gastric carcinoma including signet-ring cell carcinoma. It is an oral 5-FU derivative consisting of tegafur, gimeracil, and oteracil. Gimeracil  is a dihydropyrimidine dehydrogenase inhibitor that inhibits degradation of 5-FU. Oteracil  reduces gastrointestinal toxicity caused by phosphorylating 5-FU –17. AdmiWe consider the 5-FU-based chemotherapeutic regimen with cisplatin used in gastric cancer as an effective therapy for SRCCU. Our conclusion is limited by the fact that it is based on only one case; however, adjuvant chemotherapy with S-1 and cisplatin can be considered as an option for patients with invasive SRCCU.


Open surgical rerouting and proximal ligation of one or more supra-aortic vessels prior to endovascular stent-graft placement has become an alternative to major open thoracic surgery in the treatment of complex thoracic aortic disease. Complications owing to failed surgical ligation of the left subclavian artery are rare. In this report, 3 cases of failed ligation are presented. Diagnosis was made by CT-scan and treatment was performed by transcatheter coil and plug embolization, avoiding redo neck surgery. Endovascular repair has become a valuable alternative to open repair for the treatment of several thoracic aortic pathologies -4. HowevFrom 1999 to end of 2009, 172 thoracic stent-graft procedures in 160 patients were performed in the author's institution. In 49 patients (30%), supra-aortic rerouting was performed. In 41 out of these 49 patients 84%) perioperative surgical ligation of the left subclavian artery was performed in association with supra-aortic rerouting. All patients were followed up according to the EUROSTAR guidelines [4% periopA 65-year-old man presented with persistent thoracic pain since two weeks. Serial computed tomography (CT) scans revealed an aortic dissection, Stanford type II starting at the origin of the left subclavian artery Figure  and withControl CT-scan one year later showed a progressive increase in diameter of the distal thoracic aorta below the stent-graft. An extension stent-graft  was successfully placed landing at the tenth thoracic vertebra. The patient was discharged 3 days later. CT-scan at 1, 2 and 3 years follow-up after placement of the extention stent-graft revealed complete thrombosis of the false lumen and occlusion of the left subclavian artery with the occlusion-plug in place. The diameter of the thoracic aorta remained stable with a maximum diameter of 50 mm  after placing a carotidosubclavian bypass and ligation of the proximal left subclavian artery in order to minimize potential postoperative neurological symptoms related to myelum ischemia. The postoperative period was uneventful except for fever up to 38°C for 3 days; no signs of arm claudication were noted. Control CT-scan 6 months later revealed discrete increase of the aneurismal sac diameter up to 69 mm owing to a type II endoleak by retrograde sac perfusion through the incompletely ligated proximal left subclavian artery. It was decided to treat the endoleak. Under local anesthesia, the left brachial artery was punctured and a 45 cm long 8 F sheath  was inserted. Angiography revealed the retrograde opacification of the prevertebral segment of the left subclavian artery, resulting in a type II endoleak. A 16 mm nominal diameter vascular plug  was placed at the origin of the left subclavian artery, with complete disappearence of the endoleak. Control CT-scan at one and two years follow-up revealed absence of any residual type II endoleak and stable diameter of the thoracic aneurysm up to 68 mm.A 68-year-old man presented with an asymptomatic, focal atherosclerotic aneurysm of the aortic arch  and another, focal, thoraco-abdominal aneurysm with a diameter of 5.5 cm, ending at the level of the origin of the renal arteries. Eleven years ago, the patient also underwent an elective surgical repair for an infrarenal abdominal aortic aneurysm. It was decided to first treat the arch aneurysm with use of a hybrid vascular procedure: A carotid-carotid bypass with additional bypass to the left subclavian artery was performed using a Silver 8 mm vascular graft; concomitantly a surgical ligation of the left subclavian artery proximal to the origin of the left vertebral artery was performed. Afterwards a stent-graft  starting at the origin of the brachiocephalic trunk and ending in the descending thoracic aorta, was inserted and resulting in a complete exclusion of the aneurysm. Postoperative follow-up was uneventful and three months later, patient underwent a Crawford operation for his thoraco-abdominal aneurysm with reimplantation of all visceral arteries including celiac trunk, superior mesenteric artery and both renal arteries. Six months later, follow-up CT-scan revealed a growing thoracic arch aneurysm and a type II endoleak by retrograde perfusion of the aneurysmal sac through an incompletely ligated left subclavian artery Figure . ControlCombined open and endovascular surgical repair is recently propagated as a less invasive treatment option for the management of aortic arch pathologies like aneurysms, dissections or penetrating ulcers ,7,9-12. In summary, three cases of persistent flow through the left subclavian artery after combined open en endovascular surgery for thoracic aortic disease are presented. CT-scan clearly identified the persistent left subclavian artery opacification, despite previous surgical attempt of ligation; catheter-angiography confirmed these findings. Definitive occlusion of the prevertebral part of the left subclavian artery can be performed using plug or coils, resulting in disappearance of the endoleak and in cessation of the aneurismal or false lumen growth.In our institution no approval of the Ethical Committee is required for case reports.The authors declare that they have no competing interests.GM has taken care of the concept, design and the acquisition of data. SH as well as JV have taken care of the acquisition of data, the revision of the manuscript, and the final approval for the manuscript to be published. All contributing authors have read and approved the final manuscript.


Neu-glycolyl (NeuGc)-containing gangliosides are attractive targets for immunotherapy with anti-idiotype mAbs, because these glycolipids are not normal components of the cytoplasmic membrane in humans, but their expression has been demonstrated in several human malignant tumors. Racotumomab is an anti-idiotype mAb specific to P3 mAb, an antibody which reacts to NeuGc-containing gangliosides, sulfatides, and other antigens expressed in tumors. Preparations containing racotumomab were able to induce a strong anti-metastatic effect in tumor-bearing mice. Different Phase I clinical trials have been conducted in patients with advanced melanoma, breast cancer, and lung cancer. The results of these clinical trials demonstrated the low toxicity and the high immunogenicity of this vaccine. The induced antibodies recognized and directly killed tumor cells expressing NeuGcGM3. A Phase II/III multicenter, controlled, randomized, double blind clinical trial was conducted to evaluate the effect of aluminum hydroxide-precipitated racotumomab vaccine in overall survival in patients with advanced non-small cell lung cancer. The clinical results of this study showed a significant clinical benefit in the patients who were treated with the anti-idiotype vaccine. Actively induced immunotherapy is one of the most promising fields in cancer research and numerous approaches are being studied to design effective cancer vaccines. An approach to generate an effective immune response against tumor-associated antigens involves the use of an anti-idiotype monoclonal antibody (Ab2 mAb). The use of Ab2 as a vaccine is a consequence of Jerne’s idiotypic network theory, which postulates that, due to the large potential diversity of immunoglobulin variable regions, the idiotype repertoire can mimic the universe of self and foreign epitopes . Thus, pStudies in animal models have demonstrated the efficacy of these vaccines for triggering the immune system to induce specific and protective immunity against tumors of different origin . In the An anti-idiotype antibody that has shown to activate T cell responses against the nominal antigen is the 105AD7. This antibody has both amino acid and structural homology with the complement regulatory protein CD55, a glycoprotein that protects the cells from the attack by complement and which is overexpressed in osteosarcoma and colorectal cancer cells. Some of the osteosarcoma patients vaccinated with 105AD7 elicited an IFN-gamma T cell response against the vaccine, and TNF-alpha and GM-CSF responses not only to the vaccine, but also toward the native antigen. This antibody was also injected in colorectal cancer patients, where again it induced proliferative responses and TNF-alpha and GM-CSF secretion against the nominal antigen. These researchers have not seen correlation between the induced T cell responses and patient’s survival, probably due to the small number of patients included in the studies , 2008.Abagovomab, an anti-idiotype mAb directed against CA125, has shown to induce active immune response against this tumor-associated antigen in a Phase Ib/II clinical trial in advanced ovarian cancer patients. In this study, those patients who developed anti-anti-idiotype (Ab3) response had a significant longer overall survival . On the Also, anti-idiotype mAbs have been used to mimic non-protein antigens in patients with different tumors. One example of these kind of antigens are the gangliosides which are sialic acid-containing glycosphingolipids normally expressed in the membrane of eukaryotic cells , but theThe anti-idiotype mAb 1A7, which functional mimics the ganglioside GD2,  has beenAnother antibody used as an anti-idiotype vaccine in cancer patients is the mAb Bec2, which mimics GD3 ganglioside . This anIn particular the Neu-glycolyl (NeuGc)-neuraminic acid variant of gangliosides is widely expressed in most mammalian tissues, but is rarely found in normal human cells. The lack of expression of this type of sialic acid in humans is due to the inactivating mutation in the CMP-Neu5Ac hydroxylase gene, enzyme responsible for Neu5Gc biosynthesis of Neu5Gc-containing molecules . HoweverThe most accepted theory for this phenomenon is the incorporation of Neu5Gc from dietary sources. Free sialic acids from the medium can be taken up into cells via pinocytosis. The content of the resulting pinocytotic vesicles and endosomes would eventually be delivered to the lysosome, where a sialic acid transporter then delivers the molecules into the cytosol . The expWe first generated a mAb, named P3, which distinguish between NeuGc and NeuAc gangliosides . This mAOur hypothesis was that racotumomab could act as a surrogate of NeuGc-gangliosides generating autologous antibodies in the immunized species which react with the Ab2 idiotype and with the NeuGc-containing gangliosides.We tested the hypothesis using different animal models with a different NeuGc-sialic acid expression: mice and monkeys that express NeuGc-glycoconjugates  and chicAnimals received different doses of preparations containing racotumomab and when we evaluated the serological antibody response we found that all animals of the different species developed Ab3 response. It is noteworthy that in monkeys and chickens where racotumomab is a xenogenic immunoglobulin the serological response against racotumomab was stronger when compared with other isotype-matched mAb used as a control (immunodominance of the idiotype determinants). Besides, the Ab3 antibodies generated by immunization with racotumomab mAb were characterized to share idiotopes with P3 mAb (Ab1), as evidenced by their capacity of inhibiting racotumomab binding to P3 mAb Ab3 Id+; .In contrast, when the antibody response against NeuGc-containing gangliosides induced by racotumomab immunization was studied, a completely different pattern of response was detected. In mice and monkeys, no specific humoral response against NeuGc-containing gangliosides was detected in the sera of racotumomab immunized animals. In the case of mice and monkeys, since NeuGc-containing gangliosides are normal tissue components , toleranOn the other hand, most chickens in our study developed a specific Ab3 response against NeuGcGM3 and NeuGcGM2 gangliosides (Ab3 Ag+), due to the immunization with racotumomab . In addiAlthough these results, it is known that the induction of antigen-specific humoral immune response is not predictive of the biological effect induced by an anti-idiotype mAb. Thus, we studied the effect of different preparations containing racotumomab in different experimental tumor murine models.In BALB/c mice, vaccination with several intraperitoneal doses at 14-day intervals of racotumomab coupled to keyhole limpet hemocyanin in Freund’s adjuvant, significantly reduced subcutaneous tumor growth of F3II carcinoma cells and the formation of spontaneous lung metastases. The effect of racotumomab as a biological response modifier on lung colonization was evaluated in C57BL/6 mice injected with B16 melanoma cells. Interestingly, intravenous administration of uncoupled racotumomab antibody, 10–14 days after inoculation of tumor cells, dramatically reduced the number of experimental metastases in comparison with lungs from mice treated with an irrelevant IgG .Later, we evaluated the anti-metastatic effect of racotumomab in Alum adjuvant (racotumomab-Alum) in the 3LL-D122 Lewis lung carcinoma model. Racotumomab-Alum immunization was tested in two different settings distinguished by the frequency of the immunizations, the amount of vaccine and the initiation of the vaccine schedule related to the tumor challenge. Independently to the immunization schedule, racotumomab-Alum promoted a significant reduction of spontaneous lung metastases. The therapeutic effect was associated to the increment in the number of T cells infiltrating metastases, a reduction of new blood vessels formation, and the increment of apoptotic tumor cells in lung nodules. It is noteworthy that active immunization with the mAb in Alum does not induce measurable antibodies to racotumomab molecule, the NeuGcGM3 or tumor cells, which may suggest a different mechanism which has to be elucidated .More recently, preclinical studies were carried out with the main objective to determine the anti-tumor effect of racotumo-mab-Alum vaccine coadministered with low dose of cyclophosphamide in a murine mammary carcinoma model, based on their potentially shared antiangiogenic properties and/or a complementary proapoptotic effect by racotumomab-Alum vaccine. The results of the study showed that the combination enhanced the efficacy of chemotherapy or immunotherapy alone in the F3II carcinoma model, both when the mAb was obtained from mice ascites fluid or from bioreactor supernatant .Taking in account the preclinical results together with the results of the toxicology studies, and after the approval of the Ethical Committees of the hospitals and the Regulatory agencies, different Phase I clinical trials were performed in advanced melanoma , breast Racotumomab-Alum vaccine resulted very immunogenic, a strong, and specific response against NeuGc-containing gangliosides was detected in the sera of most of the immunized patients by ELISA and TLC-immunostaining . One intThe finding that in most of the patients treated with racot-umomab-Alum vaccine we detected a relatively high titer of anti-NeuGcGM3 antibodies of both IgM and IgG isotypes is a relevant result taking into account that it is difficult to obtain an IgG antibody response against these antigens . Even thAlthough these studies were not designed to evaluate the therapeutic efficacy of the vaccine, a prolonged survival was observed in several patients. In particular, the clinical study performed in NSCLC patients stage IIIb/IV showed encouraging clinical benefit. This clinical trial study included 34 stage IIIb and 37 stage IV NSCLC patients. These patients were treated with the anti-idiotype vaccine, after received standard chemotherapy and radiotherapy, in a compassionate-use basis study. Patients received five bi-weekly injections of 1 mg of racotumomab-Alum, other 10 doses at 28-day intervals and later the patients who maintained a good performance status continued to be immunized at this same time interval. No evidence of unexpected or serious adverse effects was reported, although the patients were repeatedly injected with racotumomab-Alum and even when some of them received more than 15 doses of this vaccine. The overall survival of the patients who entered the study was superior to the one reported  for a grImmunological studies performed in 20 of these patients suggested an association between the induction of antibodies against NeuGcGM3 and longer survival times . Then, oOur results from flow cytometry studies, where the sera of patients were incubated with a cell line expressing NeuGcGM3 and the effect on their viability was studied using the propidium-iodine exclusion assay, showed that sera of most of the patients not only recognize the cells, but also directly killed the cells, by a mechanism independent of complement activation. When the cells were examined by scanning electron microscopy it was shown that the cytotoxic hyperimmune patient’s sera produce large lesions on cell’s membranes . These rThen, a Phase II/III randomized, multicentric, double blind, clinical study was performed in advanced NSCLC patients looking to the proof of concept with significant clinical benefit in the patients who were treated with the anti-idiotype vaccine in comparison with the placebo group (manuscript in preparation). Now, a Phase III multinational clinical trial is ongoing to confirm our clinical results in advanced NSCLC patients.Racotumomab-Alum vaccination has proved to be safe and highly immunogenic in patients with advanced melanoma, breast and lung cancer. Racotumomab-Alum vaccination seems to be effective to prolong overall survival in patients with NSCLC and preliminary data suggested a correlation between the induction of antibodies against NeuGcGM3 and increased survival times of vaccinated patients. The current ongoing Phase III clinical trial will give a definitive answer to the potential clinical benefits offered by this vaccine. Furthermore, studies will be required to determine the more efficient combination with chemotherapy or other immune interventions to prevail over the tumor-induced immunosuppression.Dr Ana M. Vázquez is an inventor of patents related with P3 mAb and its anti-idiotypes, however, she has signed the assignment of her rights to the assignee Center ofMolecular Immunology. Dr Roberto E. Gómez is a full time employee at ELEA Laboratories.


Post-operative pulmonary complications add to the morbidity and mortality of surgical patients, in particular after general anesthesia >2 hours for abdominal surgery. Whether a protective mechanical ventilation strategy with higher levels of positive end-expiratory pressure (PEEP) and repeated recruitment maneuvers; the "open lung strategy", protects against post-operative pulmonary complications is uncertain. The present study aims at comparing a protective mechanical ventilation strategy with a conventional mechanical ventilation strategy during general anesthesia for abdominal non-laparoscopic surgery.2O with recruitment maneuvers (the lung-protective strategy) or mechanical ventilation with the level of PEEP at maximum 2 cmH2O without recruitment maneuvers . The primary endpoint is any post-operative pulmonary complication.The PROtective Ventilation using HIgh versus LOw positive end-expiratory pressure ("PROVHILO") trial is a worldwide investigator-initiated multicenter randomized controlled two-arm study. Nine hundred patients scheduled for non-laparoscopic abdominal surgery at high or intermediate risk for post-operative pulmonary complications are randomized to mechanical ventilation with the level of PEEP at 12 cmHThe PROVHILO trial is the first randomized controlled trial powered to investigate whether an open lung mechanical ventilation strategy in short-term mechanical ventilation prevents against postoperative pulmonary complications.ISRCTN70332574ISRCTN:  Mechanical ventilation is a life-saving strategy in patients with respiratory failure. There is unequivocal evidence that mechanical ventilation in critically ill patients has the potential to aggravate or even initiate lung injury ,2. PatieMechanical ventilation is frequently mandatory in patients who undergo surgery. The effects of short-term intra-operative mechanical ventilation on pulmonary integrity are less well defined . In addiPost-operative pulmonary complications, in particular after general anesthesia >2 hours for abdominal surgery, add to the morbidity and mortality of surgical patients ,13. We hPROVHILO aims at comparing the effects of such protective strategy and conventional mechanical ventilation in biomarkers of lung injury, post-operative pulmonary complications, extra-pulmonary complications and length of hospital stay in patients undergoing general anesthesia for open abdominal surgery.The PROtective Ventilation using HIgh versus LOw positive end-expiratory pressure ("PROVHILO") trial is a worldwide investigator-initiated multicenter randomized controlled two-arm trial.http://www.controlled-trials.com with trial identification number ISRCTN70332574.The Institutional Review Board of the Academic Medical Center - University of Amsterdam, Amsterdam, The Netherlands, approved the trial. The PROVHILO trial is conducted in accordance with the declaration of Helsinki and was registered on October 29 2010 at Figure Local investigators screen consecutive patients scheduled for non-laparoscopic abdominal surgery in participating centers worldwide. Demographic data on screened patients regardless of meeting enrolment criteria are recorded . A total of 900 patients are randomized to the 2 different mechanical ventilation strategies. In the participating centers at least 2 investigators are involved with the study. One researcher is involved with mechanical ventilation practice in the operation room, he/she will be blinded for the randomized intervention most closely to the time of tracheal intubation  - the second investigator, blinded for randomization arm, will score the primary and secondary post-operative endpoints.Patients with high or intermediate risk for post-operative pulmonary complications following non-laparoscopic abdominal surgery with general anesthesia are eligible for participation. To identify such patients the ARISCAT risk score , and patients who consented for another interventional study or decline to participate. In addition, patients who were on mechanical ventilation >30 minutes  within last 30 days, are excluded. Other important exclusion criteria include: any previous lung surgery, history of previous severe chronic obstructive pulmonary disease (COPD) with (non-invasive) ventilation and/or oxygen therapy at home and/or repeated systemic corticosteroid therapy for acute exacerbations of COPD, ALI or acute respiratory distress syndrome expected to require prolonged post-operative mechanical ventilation, persistent hemodynamic instability or intractable shock (considered hemodynamic unsuitable for the study by the patient's managing physician), severe cardiac disease , and recent immunosuppressive medication (receiving chemotherapy or radiation therapy within last 2 months).Patients planned for laparoscopic surgery are excluded from participation, as are non-adult patients (age <18 years), patients with a body mass index >40 kg/mAll patients are asked for signed informed consent, as required by the institutional review board in accordance with the Declaration of Helsinki.Randomization is performed using a dedicated, password protected, SSL-encrypted website. Randomization sequence is generated using random blocks and is stratified per center. No blocking is applied to other trial factors.2O with the use of recruitment maneuvers (the lung-protective strategy) or mechanical ventilation with levels of PEEP at maximum 2 cmH2O without recruitment maneuvers . The PEEP level in the protective strategy is chosen to be 12 cmH2O, to achieve maximal interventional effect without causing harm to participating patients and to make the intervention acceptable for the participating clinicians. The conventional strategy is chosen based on a national survey in France that showed >90% of responding anesthetists to use levels of PEEP of 0 - 4 cmH2O without recruitment maneuvers [2O, the level of PEEP is set at a maximum of 2 cmH2O with the conventional strategy. However the lowest possible level of PEEP is always chosen.Patients are randomly assigned to mechanical ventilation with levels of PEEP at 12 cmHaneuvers . Since nPatients are ventilated with a volume-controlled mechanical ventilation strategy. Although it is left to the discretion of the attending anesthesiologist to use different fractions of inspired oxygen, it is advised to use at least 0.4, with the lowest oxygen fraction to maintain oxygen saturation ≥ 92%. The inspiratory to expiratory time ratio (I:E) is set at 1:2, and the respiratory rate is adjusted to reach normocapnia . Tidal volumes of <8 mL/kg predicted body weight (PBW) are advised to be used . PBW is Recruitment maneuvers, as part of the lung-protective strategy, are performed directly after intubation, after any disconnection from the mechanical ventilator, and directly before tracheal extubation. Recruitment maneuvers should not be performed when patients are hemodynamic unstable, as judged by the attending physician.Recruitment maneuvers are not easily applied with available anesthesia ventilators since not all machines have an inspiratory hold function or other adequate facilities. To obtain standardization among centers, recruitment maneuvers are performed as follows:2O1. peak inspiratory pressure limit is set at 45 cmH2O2. tidal volume is set at 8 ml/kg PBW and respiratory rate at 6-8 breaths/min , while PEEP is set at 12 cmH3. inspiratory to expiratory ratio (I:E) is set at 1:22O4. tidal volumes are increased in steps of 4 ml/kg PBW until a plateau pressure of 30-35 cmH2O5. 3 breaths are administered with a plateau pressure of 30-35 cmH2O6. peak inspiratory pressure limit, respiratory rate, I:E, and tidal volume are set back to settings preceding each recruitment maneuver, while maintaining PEEP at 12 cmHAnesthesiologists are allowed to change the ventilation protocol at any time point upon the surgeon's request, or if there is any concern about patient's safety. The level of PEEP can be modified according to the anesthesiologist in charge if the systolic arterial pressure drops <90 mmHg for more than 3 minutes despite intravenous fluid infusion and/or start of vasopressors, if dosages of vasopressors are at the highest level tolerated, if new arrhythmias develop which are unresponsive to treatment suggested by the Advanced Cardiac Life Support Guidelines , if ther2 <90%), after excluding airway problems, severe hemodynamic impairment and ventilator malfunction, a rescue strategy is proposed, which improves oxygenation with respectively a decreasing level of PEEP with increasing FiO2 in the lung-protective strategy group, and increasing levels of PEEP and FiO2 in the conventional group  pain score <3, to use regional or neuroaxial analgesia if indicated, to use physiotherapy by early mobilization, deep breathing exercises with and without incentive spirometry, and stimulation of cough in the post-operative period, to avoid fluid overload (according to the discretion of the responsible physicians) and to use appropriate prophylactic antibiotics when indicated. Data on the applied procedures will be collected and analysed.Baseline variables are collected pre-operative at the pre-anesthetic visit or before induction of general anesthesia. The following variables are collected; gender, age, height, weight, functional status , physical status (according to the American Society of Anesthesiologists (ASA), cardiac status , acute coronary syndrome, or persistent ventricular tachyarrhythmia's),COPD and use of inhalation therapy and/or steroids, respiratory infection in the last month, smoking status, alcohol status in the past 2 weeks, history of active cancer, weight loss >10% in the last 6 months, history of diabetes mellitus, use of oral anti-diabetics, use of antibiotics in the last 3 months, use of statins, type of scheduled surgery , transfusion of blood products in the preceding 6 hours, vital parameters , blood pressure, heart rate), airway secretion score , VAS-scores for dyspnea and pain, blood samples  and a chest X-ray .During the intra-operative period variables are recorded hourly after induction of anesthesia during the recruitment maneuver. These variables include duration and type of both anesthesia and surgical procedures, all administered drugs during anesthesia , ventilator settings, vital parameters, fluid- and transfusion requirements, need of rescue therapy for hypoxemia and intra-operative complications possibly related to recruitment maneuvers .Patients are assessed at the first five post-operative days and at the last day before discharge from the hospital. On day 90 hospital free-days are recorded; if the patient is still admitted to the hospital on day 90, this day will be recorded as last day of follow-up. Clinical data and the presence of pulmonary and extra-pulmonary postoperative complications are scored; the day of development of any complication is indicated. A chest X-ray will be taken on the first post-operative day, blood samples for laboratory tests  will be taken on day 1, 3 and 5 and blood samples for biomarkers are collected directly after surgery and on day 5. As mentioned above, one local investigator, blinded for randomization group will score the primary and secondary post-operative endpoints.2 <60 mmHg or SpO2 <90% in room air but responding to supplemental oxygen, (b) severe respiratory failure , (c) development of ALI/ARDS  mild respiratory failure (PaOidelines ), (d) suSecondary clinical endpoints - include (a) intra-operative ventilation strategy related complications , (b) unexpected need for ICU admission or ICU readmission, (c) hospital-free days at follow-up day 90, (d) post-operative wound healing and (e) post-operative extra-pulmonary complications. Extra-pulmonary complications include SIRS, sepsis, severe sepsis, septic shock , extra-Other study parameters - Blood samples will be collected and analyzed for systemic markers of lung injury (including but not limited to soluble Receptor for Advanced Glycation Endproducts (sRAGE), Clara Cell protein-16 (CC-16), surfactant proteins A and D and levels of proinflammatory and procoagulant/antifibrinolytic mediators (including but not limited to interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α, and thrombin-antithrombin (TAT), protein C, and plasminogen activator inhibitor (PAI)-1). The abovementioned biomarkers of lung injury, acute inflammation and coagulopathy have been shown to correlate with poor clinical outcome in patients with ALI/ARDS . NotablyThe injury induced by mechanical ventilation originates in the lung, but may also affect distal organs by release of mediators from the lung into the systemic circulation ,29. Ther2 test with a 0.05 two-sided significance level will have 80% power to detect the difference (in post-operative pulmonary complications) between conventional mechanical ventilation (24%) and open lung mechanical ventilation (16.5%) (Odds ratio of 0.626) when the sample size in each group is 450.Sample size calculation - the required sample size is calculated from an estimated effect size derived from data collected in the ARISCAT study  and prevp-value < 0.0005 is found at 300 patients or <0.014 at 600 patients, termination of the study is considered. The third and final analysis is performed at 900 patients with a p-value of 0.045 for significance. When post-operative pulmonary complications occur significantly more frequent in the intervention group, terminating the study due to harm will be considered when p ≤ 0.022 for each interim analysis.Interim analysis - one main concern is not to withhold positive effects of the open lung mechanical ventilation strategy to the control group. Therefore, interim analyses are performed after 300 and 600 patients. The first interim analysis is performed when 300 patients have successfully been included and followed-up. If the intervention has a strong trend for improving post-operative pulmonary complications (as defined above) with a t-test will be used. Likewise if continuous data are not normally distributed the Mann-Whitney U test will be used. Categorical variables will be compared with the Chi-square test or Fisher's exact tests or when appropriate as relative risks. Where appropriate statistical uncertainty will be expressed by 95% confidence levels.Normally distributed variables will be expressed by their mean and standard deviation; not normally distributed variables will be expressed by their medians and interquartile ranges; categorical variables will be expressed as n (%). In test groups of continuous normally distributed variables, Student's t-test will be used or when not normally distributed the Mann-Whitney U test will be used.Primary outcome is the total occurrence of pulmonary complications within the first 5 post-operative days, presented as a percentage. The percentage will be analyzed as continuous data. If the data is normally distributed, Student's As this is a randomized controlled trial, we expect that randomization in this large study population will sufficiently balance the baseline characteristics. Baseline balance is tested and imbalance compensated in all pre-operative variables and on ARISCAT scores  (as mentTime to event variables (primary and secondary outcomes) are analyzed using a proportional hazard model adjusted for possible imbalances of patients' baseline characteristics. Time course variables  are analyzed by a linear mixed model. The linear mixed models procedure expands the GLM so that the data are permitted to exhibit correlated and non-constant variability. The model includes two factors: 1) study group , each level of the study group factor can have a different linear effect on the value of the dependent variable; 2) time as a covariate, time is considered to be a random sample from a larger population of values, the effect is not limited to the chosen times.The Executive Committee is constituted of the study principal investigator and the principal investigators of the investigating centers that approved the final trial design and protocol issued to the clinical sites and to the Data and Safety Monitoring Board (DSMB).The independent DSMB watches over the ethics of conducting the study in accordance with the Declaration of Helsinki, monitors patient safety and reviews safety issues as the study progresses. All serious adverse events, and all unexpected and related or possibly-related adverse events will be reported blinded to the appointed international SAE-manager, who assesses the events and reports this information to the DSMB within 24 hours of that event in the case of a serious adverse event or within one week in the case of an adverse event.The Steering Committee is composed of the principal investigators of the principle participating centers who contribute to the design and revisions of the study protocol.The National Coordinators are responsible for administrative management and communication with the local principle investigator and provide assistance to the participating clinical sites in trial management, record keeping and data management.It has become clear that mechanical ventilation can attenuate lung damage and may even be the primary factor in lung injury ,3. ALI/AProtective mechanical ventilation using lower tidal volumes could reduce ventilator-associated lung injury. Indeed, the use of lower tidal volumes has been found beneficial in patients who needed long-term mechanical ventilation for ALI/ARDS ,15. Two During general anesthesia reductions in end-expiratory lung volume and increases in airway closure is commonly seen . Both coVarious studies showed mechanical ventilation according to an open lung concept to improve ventilatory efficacy of the lungs in patients with healthy lungs undergoing general anesthesia ,37. StudThe PROVHILO trial is the first randomized controlled study powered to investigate whether protective mechanical ventilation using higher levels of PEEP complemented by recruitment maneuvers attenuates post-operative pulmonary complications. The two ventilation strategies used in the PROVHILO trial are composed to match as many clinically applied anesthesia ventilators as possible. With these standardized ventilation strategies, we aim to minimize variation between ventilation strategies used in the participating centers.The primary endpoint of this trial is a composed endpoint (post-operative pulmonary complications). This could be seen as a shortcoming, since the effect of the intervention on one post-operative pulmonary complication could be diluted if other post-operative pulmonary complications are not affected, or affected to a lesser content. However, since we collect and report on all post-operative pulmonary complications, it may still be possible to determine the effects on separate complications.The main concern in the statistical interim analysis is not to withhold positive effects of the treatment to the control group. However, to achieve maximal protection for patients and to have a lower chance of achieving positive effects of the intervention on post-operative pulmonary complications if they were not really present, different stopping rules are defined for a strong beneficial effect on post-operative pulmonary complications of the intervention versus a worse effect on post-operative pulmonary complications.The spectrum of ventilator-associated lung injury does not only include pulmonary inflammation, but also an increase in systemic inflammatory mediators ,42-44. TSeveral confounding factors can be suggested. Post-operative pain is a commonly acknowledged contributor to post-operative atelectasis ,47. RespIn conclusion, the PROVHILO trial is a worldwide investigator-initiated randomized controlled trial powered to test the hypothesis that an open lung mechanical ventilation strategy using higher levels of PEEP and recruitment maneuvers during short-term intra-operative mechanical ventilation prevents against post-operative pulmonary complications. The PROVHILO trial also determines the effect of an open lung approach on post-operative extra-pulmonary complications. Finally, in the PROVHILO trial the effect of lung-protective mechanical ventilation is monitored by highly specific biomarkers of lung injury.AE: Adverse Event; ALI: Acute Lung Injury; AR: Adverse Reaction; ARDS: Acute Respiratory Distress Syndrome; COPD: Chronic Obstructive Pulmonary Disease; EU: European Union; ICU: Intensive Care Unit; I:E: Inspiratory to Expiratory ratio; PBW: Predicted Body Weight; PEEP: Positive end-expiratory pressure; PPC: Post-operative pulmonary complications; RM: Recruitment maneuver; SpO2: Oxyhemoglobin saturation by pulse-oximetry breathing air in supine position; VALI: Ventilator-associated lung injury; WBC: White Blood CellsThe authors declare that they have no competing interests.SH: preparation of the initial drafts of the manuscript and preparation of the final version. All: review of the initial drafts of the manuscript. JB: planned the statistical analysis and revised the manuscript. MS, MGdA, PP designed the study, reviewed the initial drafts of the manuscript. All authors approved the final version of the manuscript.


The advent of novel and high-throughput sequencing (next generation) technologies allowed for the sequencing of the genome at an unprecedented depth. The majority of transcribed RNAs have been classified as non-coding RNAs. Among them, long non-coding RNAs (lncRNAs) are emerging as important regulators in many biological processes. Here, we discuss the role of those lncRNAs which are under the control of p53 or that are able to regulate its activity, due to the central role of p53 pathway in many conditions. We also briefly discussed the emerging need of having novel strategies and computational tools to completely unravel the multifaceted roles of lncRNAs and to pave the way to the development of novel diagnostic and therapeutic applications based on these peculiar molecules. In biology, the classical view of gene expression and regulation has been focused around protein-coding genes via the central dogma of DNA → mRNA → protein. However, over the past decade, evidence from numerous next generation sequencing (NGS) experiments suggested that the evolution of developmental processes, which regulate the complexity of the organism, is mainly due to the expansion of the regulatory potential of the non-coding portions of the genome . In compi.e., the p53 pathway). Huarte et al. have demonstrated that numerous lncRNAs are key constituents in the p53-dependent transcriptional pathway [bona fide p53 transcriptional targets.In eukaryotes, lncRNAs have been implicated in many biological processes with different functional roles such as X chromosome inactivation ,13, geno pathway . MoreoveIn this review, we focused the interest on those lncRNAs related to p53 regulation (induced or activating), owing to the central role of p53 in many processes. We therefore considered and discussed LincRNA-p21, PANDA, H19, MEG3 lncRNA and LincRNA-EPS.et al. gained also mechanistic clues into how LincRNA-p21 functions to repress such a large subset of genes in the p53 transcriptional response. Biochemical experiments such as RNA-pulldown, native RIP, cross-linked RIP, and deletion-mapping experiments, allowed the authors to identify a specific interaction between the LincRNA-p21 and the heterogeneous nuclear ribonucleoprotein K (hnRNP-K)  .Moreover, they identified a highly conserved 780 nt 5′ region in the sequence of LincRNA-p21 which is required for hnRNP-K binding and subsequent induction of apoptosis. In fact, the two major phenotypic consequences of p53 pathway activation are the growth arrest and the induction of apoptosis . In theiThe long non-coding RNA PANDA (a ~1.5-kb transcript) is located ~5 kb upstream of the CDKN1A (p21) transcription start site, is evolutionarily conserved, specifically induced by DNA damage in a p53-dependent manner and mediates anti-apoptotic functions . HoweverPromoter regions of p53-dependent cell death genes are characterized by the presence of a binding site for the transcription factor NF-YA, which interacts with PANDA in a highly specific manner . MoreoveDuring erythropoiesis more than 400 putative lncRNAs are differentially expressed. One of these lncRNAs, named long intergenic non-coding RNA erythroid prosurvival (LincRNA-EPS), is induced during the terminal differentiation of murine erythroid cells and has been recently investigated . LincRNAet al. employed microarrays technology to assess the gene expression of lineage-negative (Lin-) fetal liver cells, highly enriched for erythroid progenitors [To study the functional link between LincRNA-EPS and Pycard and how this lncRNA can inhibit apoptosis, Hu genitors  and ectoThe nuclear localization of LincRNA-EPS suggested that the gene expression regulation might occur through nuclear events such as epigenetic modifications, transcription, or mRNA splicing, or simply by its association with chromatin modifiers which repress the transcription of Pycard and the other genes promoting cell apoptosis. Finally, further studies aimed at discovering this lncRNA and other potentially associated factors in humans and novel and more powerful computational methods and prediction algorithms will surely help to identify all the actors involved in this mechanism.The lncRNA H19 has been recently investigated and found upregulated in many tumors ,34. Moreet al. demonstrated that the H19 promoter is efficiently repressed by p53 [et al. demonstrated the association of H19 and p53, and by transfecting H19 in AGS cells they observed a significant decrease of p53 activity, suppressing also the protein level of the p53 target Bax. Therefore, these data emphasize the fact that the upregulation of the lncRNA H19 contributes to tumorigenesis through p53 activity regulation, at least in gastric cancers.In fact, several groups have studied the link between H19 and p53 ,37. Dugii.e., human pituitary, including normal gonadotroph cells) but absent in a continuously increasing list of primary human tumors and tumor cell lines [The transcript of the maternally expressed gene 3 (MEG3) is reported to have several isoforms originating from the human MEG3 gene which contains 10 exons . The lenll lines ,40. MEG3ll lines . Gene dell lines . In concll lines .et al. pointed out for the first time that the folding structure of MEG3 lncRNA is more important to its biological function than its primary sequence [et al. assessed that p53-mediated transactivation varies between these different isoforms and that M2 domain has a critical role. As a direct consequence of their results, the most fascinating observation is that also the folding structures of lncRNAs are important factors to consider as they are intimately linked to their functions. We can imagine that, like proteins, also lncRNAs can fold into tertiary structures determining different outcomes and functions. Further crystallographic and RNA-protein studies will surely shed light on these interesting, but still unanswered, questions.Interestingly, Zhang sequence . In factMany studies have recently identified and discussed the role of numerous lncRNAs which are involved in p53 regulation or have a role in carcinogenesis or cancer growth ,44–48. TThe advent of next-generation sequencing techniques has paved the way to important discoveries also in the field of non-coding RNAs, but the exact biological role of this regulatory molecules has still to be clarified. Notwithstanding, many studies are beginning to shed light on lncRNAs and their mechanisms and more information are expected to arise in the next few years.i.e., the threshold) required to a lncRNA to function properly, or the relative abundance in various tissues. To partially answer these questions, a novel integrative approach consisting in a computational approach integrating RNA-seq data with available annotation resources has been recently reported [In this brief review, we emphasized only the role of a selection of these lncRNAs, in particular of those molecules involved in p53 pathway and in its regulation owing to the central importance of this pathway in many conditions. We are aware that many other lncRNAs may play a role in this pathway, although this role is not completely unraveled so far. Moreover, little is known about the expression level (reported . The resreported  of 8195 Finally, the bioinformatics tools that will be developed in the near future will surely help researchers to fill the knowledge gaps and to face the present challenges . We beli