The resulting practice guideline report has been reviewed and approved by the gi dsg, which comprises medical and radiation oncologists, surgeons, a pathologist, and patient representatives.Evidence was selected and reviewed by two members of the Gastrointestinal Cancer Disease Site Group  to be superior to bolus 5-fu/fa/irinotecan (ifl) for rates of median survival and tumour response, with lower incidences of most adverse effects except peripheral neuropathy. For second-line treatment after fluoropyrimidine monotherapy, folfox is a reasonable alternative for patients with contraindications to second-line irinotecan. After progression on infusional 5-fu/fa/irinotecan (folfiri), folfox is the preferred therapy. Evidence from a single randomized trial suggests that additional benefits can be expected with the addition of bevacizumab to the folfox regimen in second-line treatment.The literature review found twenty-one randomized controlled trials and two meta-analyses. Evidence on first-line treatment found infusional 5-These recommendations apply to adult patients with advanced colorectal cancer who have high performance status (Eastern Cooperative Oncology Group score 0–2).Refer to folfox regimen is an important component of therapy for advanced colorectal cancer.The folfox was shown to be superior to ifl. The folfox regimen has superior rates of median survival and tumour response. Compared with ifl, folfox has lower incidences of severe nausea, vomiting, diarrhea, and febrile neutropenia, but a higher incidence of peripheral neuropathy.In one trial, fu/fa in combination with either oxaliplatin (folfox) or irinotecan (folfiri) are both acceptable alternatives for fit patients when combination therapy is the preferred treatment. Choice of first-line therapy may rely on patient factors and preferences—for example, less neuropathy with irinotecan versus less alopecia with oxaliplatin.Short-term infusional 5-fu/fa, capecitabine), irinotecan is standard second-line therapy. The folfox regimen is a reasonable alternative for patients with contraindications to the use of second-line irinotecan.After progression on first-line anti-thymidylate synthase monotherapy (for example, 5-folfox is the preferred therapy. Recent trials suggest that, as compared with folfox alone, folfox combined with bevacizumab provides additional survival benefits.After progression on both irinotecan and an anti-thymidylate synthase agent, The role of radiation therapy, either alone or in combination with chemotherapy, for locally advanced unresectable colorectal cancer is not addressed in this guideline.fu combinations, particularly chronomodulation of 5-fu in these combinations. Chronomodulation of oxaliplatin has not been extensively studied, and the topic of chronomodulation is beyond the scope of this guideline and is not addressed.Use of chronomodulated regimens is a topic that intersects with the use of oxaliplatin/5-folfox in second-line treatment, no first-line treatment data are available on which to make a recommendation.Although data exist to support the use of bevacizumab in combination with  For patients unable to tolerate a combination-therapy regimen, an alternative to folfiri is monotherapy with a first-line thymidylate synthase (ts) inhibitor such as 5-fu/lv, raltitrexed Currently, the standard first-line treatment for metastatic colorectal cancer  groupdach side groups inhibit dna base excision by mismatching the repair enzymes fu/fa with other combinations, a systematic review of the evidence and clinical practice guideline were warranted.Once patients are no longer responding to the combined use of a gi dsg) of Cancer Care Ontario’s Program in Evidence-Based Care (pebc), using the methods of the practice guidelines development cycle gi dsg and by a methodologist.This systematic review was developed by the Gastrointestinal Cancer Disease Site Group (rcts). The systematic review and clinical practice guideline are intended to promote evidence-based practice in Ontario, Canada. The pebc is editorially independent of Cancer Care Ontario and the Ontario Ministry of Health and Long-Term Care.The body of evidence in this review is primarily composed of data from mature randomized controlled trials (medline [1966 to June (week 1) 2006], cancerlit (1975 to October 2002), embase , Guidelines International Network, and Cochrane Library  databases was conducted in June 2006. The Medical Subject Heading search terms “colonic neoplasms,” “rectal neoplasms,” and “colorectal neoplasms” were combined with the text words “oxaliplatin”, “l-ohp,” “lohp,” and “folfox”. The results from that search were then combined with the terms “random” and “clinical trial” describing specific study designs. Results were limited to the English language. The conference proceedings of the 1999 to 2006 annual meetings of the American Society of Clinical Oncology, including the 2004 through 2006 gastrointestinal cancer symposia, were also searched for reports of new or ongoing trials. The reference lists from retrieved papers were searched for additional trials.A systematic search of the Articles were selected for inclusion in this systematic review of the evidence if they wereiii rcts of oxaliplatin (l-ohp) combined with 5-fu/fa as first-line or second-line therapy for advanced colorectal cancer.phase abstract reports of trials.English-language published reports.gi dsg decided not to pool the results of the trials found in the literature search:For the following reasons, the Treatments described were too heterogeneous to allow for pooling.Evidence from the studies obtained provided a clear indication of benefit or harm.Published meta-analyses of individual patient data were available. rcts) of first-line treatment rcts The literature search found thirty-three reports Sixteen of the included trial reports disclosed funding, either wholly or in part, from pharmaceutical companies rcts on first-line treatment, representing seventeen individual trials Twenty-seven reports fu, fa, and oxaliplatin: one in a standard infusion, and the other in a chronomodulated regimen. Both arms reported similar 1-year survival rates . Of the other six trials, only two detected statistically significant 1-year survival differences: one for folfox4 over ifl  oxafafu over irifafu  Only seven of the seventeen trials reported 1-year survival data p < 0.05) in response rates between treatment arms: three for chronomodulated regimens of 5-fu, fa, and oxaliplatin over standard infusion regimens of the same agents lv5fu2 and oxaliplatin over lv5fu2 alone fufox over fufa folfox4 over ifl and for irox over ifl oxafafu over irifafu folfoxiri over folfiri oxafafu over fafup value folfox and folfiri folfox4 and irox et al. folfiri→folfox6 and folfox6→folfiri.Only two of the seventeen trials did not report data on response rates rcts reported data on qol qol scores between the trial arms: lv5fu2 plus oxaliplatin versus lv5fu2 oxafafu versus fafu Only two of the seventeen rcts located by the literature search, first-line treatment with oxaliplatin was associated with significantly more peripheral and sensory neuropathy and neutropenia beyond the adverse effects expected with the other drugs given in the regimen.As reported in the et al. rcts involving 3186 patients. It detected a significant 3.5-month increase in median survival (p = 0.0083) in patients who received a first-line combination therapy  as compared with patients who received monotherapy. The results of the meta-analysis indicated that, for maximum overall survival benefit, patients on a first-line combination therapy containing oxaliplatin should be offered combination therapy with irinotecan as second-line treatment, and vice versa. It appears that second-line treatment may compensate for a less active first-line treatment, as evidenced by the fact that patients who had access to all three active drugs  showed the longest overall survival. However, this conclusion is confounded by the fact that patients who lived longer were more likely to have been treated with all three drugs. In addition, patients with lower performance status may have been excluded from second-line treatments using oxaliplatin and irinotecan. Additionally, oxaliplatin was not available to all patients, especially in older trials. The meta-analysis detected longer median survival times in more recent trials—trials more likely to use oxaliplatin in first- and second-line treatment.The meta-analysis reported by Grothey et al. cm) infusion to flat infusion in the first-line treatment of mcc. Significant benefits were detected in overall response rate , complete surgical resection , and median progression-free survival  favouring cm therapy; however, no difference was detected in median survival . Pooling the data from the two trials did not detect any difference in survival at either 5 or 7 years . The results may have been confounded by an imbalance between the studies with regard to recurrent metastatic disease following surgery for liver metastases , and in addition, by significant treatment crossover from the flat infusion to the cm infusion arm (26% of patients), which affected median survival . The pooled results from those two trials confirmed that, as compared with flat infusion, cm infusion significantly improved the orr% and pfs.An abstract report by Lévi rcts of second-line treatment were obtained Three of the four reports qol.None of these abstract reports provided data on As indicated under the heading “Quality of Life” in subsection 4.2.1, treatment with oxaliplatin was associated with significantly more peripheral and sensory neuropathy and neutropenia beyond the adverse effects expected from other drugs given in the regimen.fu (folfox or folfiri) both represent acceptable treatment alternatives for first-line therapy in fit patients.The combination regimens using infusional 5-fu/fa (folfox). Oxaliplatin without 5-fu/fa does not appear to have meaningful activity. The folfox regimen has definite advantages over bolus ifl in terms of toxicity, objective response rate (45% vs. 31%), median ttp (8.7 months vs. 6.9 months), median survival (19.5 months vs. 15.0 months), and 1-year survival (71% vs. 58%) as demonstrated in the N9741 study Oxaliplatin is active in colorectal cancer, and the evidence supports its use in combination with infusional 5-The superior 1-year survival seen in the N9741 study may have two possible explanations:fu was given as an infusion in the folfox arm, but as a bolus in the ifl arm, and infusional 5-fu has demonstrated superiority over bolus 5-fu in terms of toxicity and tumour response rate. This fact alone may therefore account for the differences seen in the two regimens. It may also account for the lack of difference seen between folfox and folfiri in the gercor study, because both regimens used infusional 5-fu The 5-folfox arm may relate to the high rate of second-line irinotecan use. In the folfox arm, 53% of the patients received second-line irinotecan, but only 17% of patients on the ifl arm received second-line oxaliplatin. It is becoming increasingly clear that subsequent therapy can have a substantial effect on survival. From the evidence reviewed, it appears that the number of active drugs available to a study arm may positively affect survival, because when more drugs are made available to patients, median survival is increased. This finding does not rule out other factors, such as variations in study population and other variations in treatment over time, but it is highly supportive of the conclusion that access to all three active drugs  is important to optimize patient outcomes fu should be used. This recommendation to use 5-fu in an infusional schedule is now abundantly clear from single-agent studies, from combination studies in advanced disease, and from the adjuvant setting in early colorectal cancer. The role of bolus 5-fu in the management of colorectal cancer is becoming increasingly limited.Superior survival in the folfox does, and therefore no advantages The combination of oxaliplatin and irinotecan is also active, but it has lower tumour response rates and 1-year survival rates than fu pump programs, in combination with the drug’s unavailability in certain regions, has led to interest in oral capecitabine as a possible replacement for infusional 5-fu in oxaliplatin and irinotecan combinations. The few phase ii trials reported thus far have demonstrated response rates and toxicity that appear comparable to those seen with infusional 5-fu combinations. In the future, capecitabine may supersede the 5-fu pump and the need for central venous access devices; however, this development will depend on the results of ongoing phase iii trials. Until the results of those trials are available, infusional 5-fu regimens—either alone or in combination—are standard therapy.The inconvenience of infusional 5-cm regimen of infusional 5-fu is a compelling area of study, but no such regimen has been widely evaluated outside of a few specialized centres. This question extends beyond a simple review of 5-fu/oxaliplatin combinations. The pooled analysis of the two underpowered studies by Lévi et al. cm could both reduce toxicity and positively affect endpoints such as orr% and ttp. The intervention is worthy of further study, although the complexity of the therapy may put it beyond feasibility in many locales.The question of whether to use a ttp , and symptom control with second-line folfox as compared with oxaliplatin alone or infusional 5-fu/fa alone in patients who progressed on the ilf regimen. No overall survival analysis was performed. For patients who have progressed on both an anti-ts agent and irinotecan, folfox is the preferred therapy.The ECF4584 trial fu/fa, folfox is active, with an orr% that appears to compare favourably with that of folfiri, the standard regimen. Currently, more evidence supports second-line irinotecan than supports folfox or oxaliplatin alone, but for patients considered poor candidates for second-line irinotecan, folfox is a reasonable alternative. Further clarification of the role of oxaliplatin after progression on first-line 5-fu awaits more-mature data from the Sanofi-sponsored ECF4585 trial.In patients with tumour progression on first-line 5-folfox regimen. An abstract report by Hochster et al. tree-2 study—a first-line cohort study comparing folfox plus bevacizumab, oxafafu plus bevacizumab, and capecitabine and oxaliplatin (capox) plus bevacizumab—found that folfox plus bevacizumab resulted in the longest median survival with acceptable adverse events. In the tree-2 study, the median survivals for the treatment arms without bevacizumab were 19.2 months (folfox), 17.9 months (oxafafu), and 17.3 months (capox) as compared with 26 months (folfox + bevacizumab), 20.7 months (oxafafu + bevacizumab), and 27.0 months (capox + bevacizumab) tree-1 trial (no bevacizumab) were compared with the tree-2 trial data (bevacizumab added), the results were 12 months, 67.5% versus 79.1%; 18 months, 50.1% versus 64.7%; 24 months, 35.8% versus 50.7%—all in favour of the treatments including bevacizumab As treatment regimens for advanced colorectal cancer continue to evolve, recent trials rct reported by Giantonio et al. folfox4 with and without bevacizumab, found that the addition of bevacizumab to the folfox regimen resulted in significant gains in both median survival  and pfs . Based on these two trials, we conclude that the addition of bevacizumab to an infusional 5-fu, fa, and oxaliplatin regimen may provide benefits beyond those that would be possible with infusional 5-fu, fa, and oxaliplatin without bevacizumab.The second-line gi dsg consensus process was based on an earlier draft of the present document. That draft did not contain any of the evidence regarding the addition of bevacizumab to regimens of infusional 5-fu/fa plus oxaliplatin. References The fu/fa/ oxaliplatin (folfox) to be superior to bolus 5-fu/fa/ irinotecan (ifl), with more favourable rates of median survival and tumour response. Compared with ifl, folfox has lower incidences of severe nausea, vomiting, diarrhea, and febrile neutropenia, but a higher incidence of peripheral neuropathy. Therefore, for first-line treatment, short-term infusional 5-fu/fa in combination with either oxaliplatin (folfox) or irinotecan (folfiri) is acceptable for fit patients when combination therapy is the preferred treatment. Choice of first-line therapy may rely on patient factors and preferences—for example, less neuropathy with irinotecan versus less alopecia with oxaliplatin.The systematic review found one first-line therapy trial ts monotherapy , irinotecan is standard therapy. For patients with contraindications to the use of second-line irinotecan, folfox is a reasonable alternative. After progression on both irinotecan and an anti-ts agent, folfox is the preferred therapy.For second-line treatment after progression on first-line anti-cm regimens is a topic that intersects with the use of oxaliplatin/5-fu combinations, particularly cm 5-fu in those combinations. Chronomodulation of oxaliplatin has not been extensively studied and was not addressed, because the topic is beyond the scope of this guideline.The role of radiation therapy, either alone or in combination with chemotherapy, for locally advanced unresectable colorectal cancer was not addressed in this guideline. In addition, the use of gi dsg acknowledges that the combination of oxaliplatin with short-term infusional 5-fu and fa (folfox) is an important component of first- and second-line treatment of advanced colon cancer, and the dsg recommends that oxaliplatin be made available for the treatment of advanced colorectal cancer.In conclusion, the fu/fa plus oxaliplatin. References The practitioner feedback survey was based on an earlier draft of the present document. That draft did not contain any of the evidence regarding the addition of bevacizumab to infusional regimens of 5-gi dsg consensus, feedback was sought from Ontario clinicians.Based on the evidence and the draft recommendations presented at the time of gi dsg reviewed the results of the survey.Practitioner feedback was obtained through a mailed survey of 63 practitioners in Ontario . The survey consisted of items evaluating the methods, results, and interpretive summary used to inform the draft recommendations and asking whether the draft recommendations should be approved as a practice guideline. Written comments were invited. The practitioner feedback survey was mailed September 15, 2004. Follow-up reminders were sent at 2 weeks (post card) and 4 weeks (complete package mailed again). The From among the 63 surveys distributed, 29 responses were received (46% response rate). “Responses” include returned completed surveys and telephone, fax, and e-mail responses. Of the practitioners who responded, 18 indicated that the report was relevant to their clinical practice, and they completed the survey. Four respondents (22%) provided written comments. The main points were these:Access issues with oxaliplatin are ongoing.The guidelines seem to be directed to the medical oncologists who provide the treatments. For other caregivers in the cancer system, this key message needs to be delivered: Cancer treatment is in a state of continuous development with increasing efficacy, therefore assessment by a medical oncologist is important for all patients.Some of the stated recommendations are currently in use and are being accepted with enthusiasm by both clinicians and patients.This guideline has not included any information regarding the role of radiation in the local management of rectal tumours. However, the guideline makes good sense with respect to the recommended chemotherapy regimens. Could a recommendation, or at least a comment, be added somewhere in the document regarding radiation timing, and radiation in combination with the recommended chemotherapy regimens?gi dsg made the following modifications to the clinical practice guideline in response to the comments obtained during practitioner feedback:The gi dsg acknowledges the major barrier that access represents to putting its recommendations into practice. The gi dsg hopes that their recommendation of oxaliplatin will raise awareness of the issue and facilitate the process of making this drug available to patients.With respect to the ongoing access issues with oxaliplatin, the gi dsg added a qualifying statement to both the abstract and the main document: “The role of radiation therapy, either alone or in combination with chemotherapy, for locally advanced unresectable colorectal cancer is not addressed in this guideline.”With respect to the issue of radiation therapy, the


These observations suggest that HFS-STN not only alters action potential conduction on the way towards the SNr but also modifies synaptic transmission within the SNr. A classical conditioning-test paradigm was then designed to better separate the causes from the indicators of synaptic depression. A bipolar platinum-iridium macroelectrode delivered conditioning HFS trains to a larger group of fibers in the STN, while a separate high-ohmic glass micropipette in the rostral SNr provided test stimuli at minimal intensity to single fibers. The conditioning-test interval was set to 100 ms, i.e. the time required to recover the excitability of subthalamo-nigral axons after HFS-STN. The continuity of STN axons passing from the conditioning to the test sites was examined by an action potential occlusion test. About two thirds of the subthalamo-nigral afferents were occlusion-negative, i.e. they were not among the fibers directly activated by the conditioning STN stimulation. Nonetheless, occlusion-negative afferents exhibited signs of presynaptic depression that could be eliminated by blocking GABA(B) receptors with CGP55845 (1 µM). Further analysis of single fiber-activated responses supported the proposal that the heterosynaptic depression of synaptic glutamate release during and after HFS-STN is mainly caused by the tonic release of GABA from co-activated striato-nigral afferents to the SNr. This mechanism would be consistent with a gain-of-function hypothesis of DBS.Patients with akinesia benefit from chronic high frequency stimulation (HFS) of the subthalamic nucleus (STN). Among the mechanisms contributing to the therapeutic success of HFS-STN might be a suppression of activity in the output region of the basal ganglia. Indeed, recordings in the  These afferents are likely to be co-activated. In rodents, GABAergic axons originating in the To better understand the cellular mechanisms of HFS-STN, it is desirable to perform experiments where the effects of multi-unit HFS are analyzed along with those of single-unit HFS in functionally defined axons impinging onto single SNr neurons. This can be done by applying the method of extracellular minimal stimulation We shall present evidence suggesting that the artificial electrical stimulation of the STN enables a mechanism of cellular interaction that may not exist under condition of physiological signal transmission in the basal ganglia. In support of this gain-of-function hypothesis HFS-STN was found to tonically activate the release of GABA, presumably from striato-nigral afferents. Activation of GABA(B) receptors then enabled a form of heterosynaptic depression at the glutamatergic subthalamo-nigral terminals. This depression lasts longer than the depression of axonal excitability and is therefore relevant for subthalamo-nigral afferents with partially preserved/recovered transmitter release function.2010/63/EU for animal experiments and comply with the requirements for manuscripts submitted to Biomedical journals. The study was registered at the Office of Health Protection and Technical Safety of the regional government Berlin  and specifically approved by the Institutional Animal Care and Use Committee of the University Medicine (Charité).The present experiments were performed in fully adult (315 to 565 days) C57Bl6 mice either provided by the Charles River GmbH Germany or the Jackson Labs . Every precaution was taken to minimize stress for and the number of animals used in each series of experiments. The work described here has been carried out in accordance with the EU Directive 2 and 5% CO2). The isoflurane concentration was adjusted to prevent spinal reflexes. The mice were then transcardially perfused with 60 ml of ice-cold (∼4°C) saline containing (in mM): 125 choline chloride, 2 KCl, 1.25 NaH2PO4, 25 NaHCO3, 10 glucose, 0.5 CaCl2, 7 MgCl2, 0.001 MK-801, 0.5 pyruvic acid, 0.005 glutathione, 2.8 ascorbic acid . The brain was removed quickly (∼1 min), separated into two hemispheres and transferred into ice-cold oxygenated saline. To preserve the basal ganglia circuits 2PO4, 25 NaHCO3, 10 glucose, 2 CaCl2, and 1 MgCl2 .For slice preparation the mice were anesthetized by inhalation of a mixture of isoflurane and carbogen (95% O−1) and regularly inspected using a movable Zeiss microscope  equipped with a 20×1.3 NA water immersion objective  and a camera for infrared videomicroscopy  showed that under the given conditions slices from animals >1 year were best preserved at this temperature, the quality criterion being the level of neuronal membrane potentials at break-in, in addition to the appearance of the slices.For electrophysiological tests, the slices were placed into a recording chamber, maintained under constant flow of oxygenated ACSF  were recorded with electrodes pulled from thick-walled borosilicate glass capillaries  and filled with ACSF (resistance 0.8–1 MOhm). The pipettes were placed in the SNr and occasionally also in the STN as shown in 2, 5 EGTA, 25 HEPES, 2 MgATP, 0.3 GTP, pH was set to 7.2 with KOH. The pipette resistance ranged from 3 to 6 MOhm. The experimentally determined reversal potential of GABA(A)-induced chloride currents (ECl) was = −100 mV. The holding potential was set to −60 mV (glutamatergic currents) or to −50 mV (GABAergic currents). The signals were acquired at a sampling rate of 30 kHz using an EPC-8 amplifier connected to the 16-bit AD/DA board. Liquid junction potentials were not corrected. Only recordings with a series resistance below 30 MOhm were accepted . Series resistance compensation was not applied. Access resistance was controlled by regularly (1/6 s) applying hyperpolarizing pulses of 10 mV. Cells exhibiting more than 20% changes in the access resistance during an experiment were discarded. Whole cell capacitance and input resistance values were determined by fitting a mono-exponential function to the currents induced by 10 mV hyperpolarizing pulses or single fiber stimulation. The time constants of uIPSC decay were determined by an automatized evaluation routine fitting a monoexponential function to the IPSC decay.Postsynaptic currents and action potentials were recorded in the whole cell voltage clamp mode. If not mentioned otherwise, the intra-pipette solution contained (in mM): 150 potassium gluconate, 5 NaCl, 0.5 CaClThe arrangement of the pipettes for stimulation and recording are given for each type of experiment. Details of the pulse pattern are illustrated in The PSCs elicited by single-fiber stimulation are referred to as unitary PSCs . For the purpose of single-unit activation in the SNr (referred to as “Microstim”) we used ACSF-filled glass micropipettes with a resistance of about 10 MOhm. For Microstim purposes rectangular pulses with 0.5 ms duration were delivered through an optically isolated constant current source . As single fiber activation was performed blindly, it was necessary to explore at any given site a wide range of current intensities. The criteria used to accept a Microstim site are laid down in The principal stimulation pattern used for the pairing of multi-unit HFS-STN with single-unit stimulation in the SNr is illustrated in S)-3-(1S)-1-ethyl[amino-2-hydroxypropyl](phenylmethyl)phosphinic acid (CGP55845) was applied at a concentration of 1 µM. All these compounds were obtained from Tocris . Other chemicals were purchased from Sigma-Aldrich .The glutamatergic nature of ion currents was verified by applying the AMPA receptor blocker 6,7-dinitroquinoxaline-2,3-dione  and the NMDA receptor blocker DL-2-amino-5-phosphonopentanoic acid . Similarly, the GABA(A) receptor blocker 2-(3-Carboxypropyl)-3-amino-6-(4 methoxyphenyl)pyridazinium bromide  was used to block GABAergic currents. To test for involvement of GABA(B) receptors, , Origin 4.10  and SPSS 12.0 . EPSC amplitudes are given as absolute values or normalized to the control average obtained for each particular test. The success rate of the unitary EPSC (s.r.) was defined as s.r. = 100%-failure rate of the responses to the first in a pair of test pulses. The analysis of fluctuating EPSCs was performed as described before t-test or ANOVA, unless otherwise stated. The asterisks indicate the following: * - p<0.05, ** - p<0.01, and *** - p<0.001.The results are presented as mean ± S.E.M. In all figures the error bars indicate S.E.M. Differences between means were tested for significance using Student's LFPs were elicited by multi-unit activation via bipolar platinum-iridium electrodes in the STN (“Macrostim”) and recorded in the STN and SNr using ACSF-filled glass pipettes as described in Globus pallidus, the size of the FV proved to be a useful means to predict the preservation of glutamatergic afferents originating in the STN. We requested a FV in the SNr of at least 0.5 mV to proceed with a given slice. In most cases amplitudes were larger than that  may not be part of the multi-unit pool activated by Macrostim in the STN. A fiber activated in the STN may not reach the site of microstimulation in the SNr and, A classical occlusion test, as used in collision experiments to study peripheral nerve branching e.g. , served The examination of occlusion-negative afferents offers an opportunity to address non-intrinsic modulatory actions on release, e. g. effects derived from other HFS-STN-activated fibers that terminate in the neighbourhood of the tested axon. The plot obtained from the occlusion-negative afferent of Assuming that the exocytosis of single vesicles is a probabilistic process, unitary test responses must exhibit a certain fraction of failures even though the active zone of transmitter release is successfully invaded by an AP. The presence of a postsynaptic response can be regarded as the best proof that the AP invaded the terminals. In multiquantal responses , a presynaptic depression should then reveal itself by a decrease in amplitude and an increase in the PPR, which was the case . In uEPSAt this point we can conclude that the glutamatergic synaptic afferents to the SNr experience a strong and lasting depression at their presynaptic terminals. The depression is not entirely intrinsic. Additional afferents are likely to be involved.With the amount of presynaptic HFS-STN-induced depression already quantified for unitary glutamatergic afferents, we are still left with numerous possibilities of how this suppression is implemented. We turned to one of the most obvious possible causes of depression of the subthalamo-nigral pathway, i.e. modification of synaptic glutamate release by GABA acting at presynaptic GABA(B) receptors The sample traces of In the experiments described so far we have evaluated single-unit-induced uEPSCs as an indicator of changes produced by HFS-STN-conditioning. In the following series of experiments we refrained from single-unit activation, but instead recorded the compound current responses induced by HFS-STN itself . GlutamaThese experiments demonstrate that a source of GABA is available at any moment during HFS-STN, due to synaptic release from GABAergic afferents.Decay).Next we asked whether GABAergic afferents of different origin contributed equally to the persistent chloride conductance during and after HFS-STN. Again, the respective currents were pharmacologically isolated and kept at minimal intensity by threshold-toggling. Decay , and the mean PPR was 1.22±0.07 (n = 14). These values are not different from the means obtained from the “slow” group of afferents in Decay>11 ms and a PPR>1.2 as striato-nigral afferents.Two previous studies on juvenile mice Thus, a subclass of GABAergic afferents to the SNr retains release during single unit HFS and, most likely, these afferents originate in the striatum.Finally, single-unit HFS was also applied to glutamatergic afferents. This offered an opportunity to clarify the intrinsic depressant mechanisms in glutamatergic afferents . Fig. 7AIn the experiment of Comparing the amount of depression obtained with single-unit and multi-unit HFS in occlusion-positive afferents showed an apparent difference of the mean reduction of about 20%. However, with the relatively small number of tested afferents in each case this difference did not reach significance. We conclude that intrinsic depressant mechanisms are also present in the glutamatergic afferents.Globus pallidusAs already revealed by other studies in rodents Deniau and colleagues We suppose that the relative robustness of the presumed striato-nigral transmission is due to the lower, in comparison with pallidal afferents, exhaustibility of the vesicle pool. Based on these results one can assume that the most effective way to facilitate a dominance of the direct as opposed to the indirect pathway would be to use intermediate current intensities that stimulate not only the axons of the subthalamo-nigral and the pallido-nigral neurons in the STN itself, but also the striato-nigral afferents travelling next to the STN This gain of function hypothesis stands somewhat in contrast with the fact that the pharmacologically isolated FVs recorded in our experiments and by Our present study focused on the mechanisms available to reduce the excitatory synaptic input towards the SNr. Therefore we have used FV recordings mainly to establish a checkpoint for evaluation of synaptic activity independently on suppression in the main axon. Another approach was the demonstration of HFS-STN-induced depression in occlusion-negative unitary STN afferents with proven AP invasion into synaptic terminals . This allowed us to reveal two types of mechanisms, i) an intrinsic depression present in any unit that experiences single fiber HFS and ii) a mechanism that requires participation of additional elements (heterosynaptic depression).nd response. With the present day information on the dynamics of transmitter release in mind While a reduction of PSC amplitude can reflect pre- and postsynaptic parameters, the paired pulse ratio is often used to evaluate the state of the release machinery The abundance of GABA(B) receptors in the SNr is well documented Immunogold labelling for GABAB1 and GABAB2 was demonstrated on the presynaptic membrane of asymmetric, putative glutamatergic synapses. Presynaptic GABA(B) receptors reduce the Ca influx through voltage-activated channels Decay values apply to recordings with zero chloride in the pipette, a condition known to prolong the decay As for the source of GABA responsible for the HFS-STN-induced depression of GABA release, our data in combination with the results of A question deserving further attention concerns the possible role of postsynaptic receptor desensitization in the depression of glutamatergic afferents. The work of Windels and colleagues suggested that the glutamate elevation in the extracellular space outlasts the HFS-STN As already mentioned in the introduction, animal studies on akinesia and its treatment with HFS-STN pursue three main directions In any system where HFS co-activates two or more types of afferents, the final outcome of repetitive fiber activation will depend i) on the relative robustness of release from a given unit and ii) on the number of afferents reaching the target area. Our slice preparation was optimized to preserve a maximum of the basal ganglia network. In any given slice the intactness of the BG connectome was verified by recording nigral evoked LFPs in response to cortical, striatal and pallidal stimulation. We believe that about 15 to 20% of the respective rostro-caudal connections are retained in 400 µm off-sagittal slices, if compared to the whole brain preparation . This estimate is based on the fraction of cells that exhibit disynaptic EPSCs due to the activation of the recurrent trans-STN pathway and on the fraction of single fiber responses exhibiting occlusion by a preceding pulse through the subthalamic macro-electrode. The fact that among the striatal, pallidal and subthalamic afferents those of the most distal origin dominate the transmitter release in the SNr argues against fiber loss as a main cause of the shift of the glutamate/GABA balance towards lower levels of glutamate release.The reductionist approach taken in the present study naturally favors the isolation of particular connections or mechanisms. Further studies will show to what extent the present conclusions derived from the analysis of single fiber responses will be applicable to the more complex situation in the intact brain. It should also be noted that there might be species-dependent differences in the structural organization of the BG in general and the STN-SNr pathway in particular. Specifically, there is no final clarity with regard to the contribution that local recurrent collaterals of STN neurons can make to the compound response to STN stimulation in primates substantia nigra pars compacta (SNc). A low level of ambient dopamine may affect the glutamate/GABA balance in the SNr. Therefore, the present results should not be generalized to the situation in Parkinsonian mice. The animals studied here were WT siblings of z-Q175-KI homozygotes, a new mouse model of Huntington's disease. At the age of >1 year of life these mice exhibit a marked hypokinesia globus pallidus or STN and whether they generate enhanced oscillatory beta band activity, as it has been described for rodent toxin models of IPS , i.e. degeneration of dopaminergic neurons in the Despite of these caveats, the identification of heterosynaptic GABA(B)-dependent depression in the SNr is an essential step towards a better understanding of the highly complex cellular interactions associated with HFS-STN.


Oncolytic viruses (OVs) replicate selectively in tumor cells and exert anti-tumor cytotoxic activity. Among them, Newcastle Disease Virus (NDV), a bird RNA virus of the paramyxovirus family, appears outstanding. Its anti-tumor effect is based on: (i) oncolytic activity and (ii) immunostimulation. Together these activities facilitate the induction of post-oncolytic adaptive immunity. We will present milestones during the last 60 years of clinical evaluation of this virus. Two main strategies of clinical application were followed using the virus (i) as a virotherapeutic agent, which is applied systemically or (ii) as an immunostimulatory agent combined with tumor cells for vaccination of cancer patients. More recently, a third strategy evolved. It combines the strategies (i) and (ii) and includes also dendritic cells (DCs). The first step involves systemic application of NDV to condition the patient. The second step involves intradermal application of a special DC vaccine pulsed with viral oncolysate. This strategy, called NDV/DC, combines anti-cancer activity (oncolytic virotherapy) and immune-stimulatory properties (oncolytic immunotherapy) with the high potential of DCs (DC therapy) to prime naive T cells. The aim of such treatment is to first prepare the cancer-bearing host for immunocompetence and then to instruct the patient’s immune system with information about tumor-associated antigens (TAAs) of its own tumor together with danger signals derived from virus infection. This multimodal concept should optimize the generation of strong polyclonal T cell reactivity targeted against the patient’s TAAs and lead to the establishment of a long-lasting memory T cell repertoire. The resistance of cancers to conventional therapies necessitates the search for new treatment strategies. The idea of using replication-competent viruses to destroy cancers is attractive to this end but is not new. Already in the mid-1950s, Newcastle Disease Virus (NDV) was reported as having oncolytic activity [The concept of using NDV for immunotherapy has been pioneered by William Cassel with NDV oncolysate. The approach was further followed by the senior author of this review who developed the live cell vaccine ATV-NDV derived form the patient’s tumor cells by infecting them with a non-lytic strain of NDV. The discovery of dendritic cells (DCs), in 1973, revolutionalized the field of vaccination only recently with the approval in 2010 by the FDA of the first DC-based cancer vaccine [NDV got its name in 1926 from an outbreak among chickens at a farm near Newcastle-upon-Tyne in England ,4. This (i)i.e., α2-3 and α2-6-N-linked sialic acids [Binding, fusion, transduction of the viral genome and transcription of viral genes: This first step involves the binding of the virus—via a lectin-like cell binding domain of the HN molecule—to ubiquitously expressed host cell surface receptors expressing distinct carbohydrate side chains (ic acids . This me(ii)Viral replication (second step): The anti-genome is then used as a template for amplification of the viral genome. Very interestingly, NDV can trigger, shortly after infection, autophagy to enhance virus replication . The M pInfection of cells by NDV can be schematically divided into two sequential steps b.(i)BinAmong all the OVs, which have been investigated as anticancer reagents, NDV has emerged as a particularly promising agent for virus therapy of cancer . This inIntegral to the live cycle of all RNA viruses is the formation of double-stranded RNA (dsRNA), which activates a spectrum of cellular defence mechanisms involving interferon (IFN)-α and -β. Tumors provide a relatively permissive substrate for the propagation of RNA viruses such as NDV because mutations in tumor cells often cripple the IFN system to allow un-inhibited proliferation and to provide resistance to apoptosis . The firThrough all the studies performed, NDV showed selective replication in nearly all the tested human/murine tumor cells and transformed cells ,13. NDV,The feature of selective replication in tumor cells appears to be directly related to the lower capacity of tumor cells, in comparison to normal cells, to mount a strong type 1 IFN response. In normal cells, NDV establishes first an early anti-viral state through a functional IFN signalling cascade of transcription factors that leads to prevention of viral genome amplification and consequently to inhibition of viral replication. Tumor cells have a weaker type 1 IFN response and a weaker sensitivity to type 1 IFN-receptor (IFNR) signaling. The impairment of interferon pathways appears to be a rather common feature during tumorigenesis. That makes tumor cells highly susceptible to NDV infection and to the ensuing oncolytic events. An inverse correlation exists between the expression of four anti-viral genes and the susceptibility of cells [normal cells and tumor cells) to infection by NDV: (i) Retinoic acid-Inducible Gene (RIG)-I, (ii) Interferon Regulatory Factor (IRF)-3, (iii) IRF-7 and (iv) IFN-β . In addiIt is of special significance that the activation of a rapid and strong type 1 IFN response by NDV in normal cells prevents virus replication, cytotoxic effects and pathology in normal tissues. This explains the high safety profile of this biologic agent. The anti-viral response of normal cells is initiated through the recognition of viral products such as dsRNA by the two types of pathogen recognition receptors (PRR): (i) Toll-like receptors (TLRs), especially TLR3 and (ii) RIG-like receptors (RLRs). Among the RLRs, RIG-I was demonstrated to be a cytoplasmic viral RNA receptor , this avian paramyxovirus, is not a human pathogen. NDV, when applied to humans, usually induces only mild fever for a day or conjunctivitis. No severe adverse effects have been reported despite applications in several thousands of people during 2 decades in Europe and the USA . 9 infectious particles when the virus is applied by the intravenous route and 4.3 × 1012 infectious particles by the intra-tumoral route [No mechanism of NDV to evade immune responses in mammalian normal cells has been observed and described. At present, there exists an extensive safety database for NDV . This vial route . In concin vivo mechanisms, such as cell fusion and syncytium formation, which allow virus escape from neutralizing antibodies. The general human population is seronegative when tested for antibodies against NDV antigens [These observations can be combined with other advantageous characteristics related to the biology of the virus. In particular, the modular nature of gene transcription, the undetectable rate of recombination, and the lack of a DNA phase in the replication cycle make NDV a suitable candidate for the rational design of a safe attenuated vaccine and gene therapy vector . There mantigens ,25. The in vitro and in animals when applied intra-tumorally or peri-tumorally [in vitro. In vivo, such oncolytic effects seem to be limited because of the interferon (IFN) response, which blocks NDV replication quickly after virus administration. Another interesting property of NDV, which is expected to play an important role during the systemic application of the virus, is its potential to induce tumor lysis after infection. Naturally occurring NDV strains have been reported to be effective oncolytic agents in a variety of animal tumor models . NDV canumorally ,29. The et al. to mediate its oncolytic effects by both intrinsic and extrinsic caspase-dependent pathways of cell death [Like many viruses, NDV induces apoptosis by activating the mitochondrial pathway. And this is responsible for the cytopathic effect caused in cells, which are infected by NDV . NDV hasll death . In anotll death . This call death . This sell death . It limill death . NDV coull death .in vitro to the death of approximately 10,000 cancer cells in 2–3 days.NDV has a wide host range with at least 27 of 50 orders of birds susceptible to infection. It is categorized into three pathotypes depending on the severity of the disease that it causes in birds: (i) lentogenic (avirulent), (ii) mesogenic (intermediate) and (iii) velogenic (virulent) . LentogeOne major difference between lytic and non-lytic strains is that lytic strains are able to produce infectious progeny virus particles in human neoplastic cells, whereas non-lytic strains are not . ConsequNDV has been shown over the last 2 decades to possess interesting immunostimulatory properties. One associated important feature is its capacity to induce large amounts of type 1 IFN when in contact with human peripheral blood cells. This is linked to the nature of the dsRNA structures, which are produced within the cytoplasm during the viral replication, thereby provoking a strong cellular IFN response. The viral surface HN protein has also been shown to be an important element for the immunostimulatory properties of this virus. NDV infection of tumor cells modifies the tumor cell surface . ThrougAll the danger signals, which derive from the infection by NDV  ,46, alloin vitro [NDV, as a strong inducer of type 1 IFN, is expected to have a strong effect on the immune response, given the recently appreciated role of type 1 IFNs in anti-glioma immunosurveillance in mouse studies . The proin vitro .In contrast, other viruses, which have adapted to the mammalian immune system, have developed virally encoded inhibitors of immunity such as TAP-inhibitors, cytokine decoys, micro-RNAs and viral proteins that antagonize type 1 IFN induction .In conclusion, NDV presents three key features—tumor selective replication, oncolytic capacity and immunostimulatory properties—which make it a promising agent to be developed for cancer therapy in man .The concept of using viruses in tumor therapy dates back to the early 1900s when observations were made of remissions of human cancers following natural viral infections ,57  conditions) was administrated weekly for 6 months by the inhalation route to patients with advanced cancers [Csatary’s concept was to use this strain as inhalation vaccine for systemic treatment and non-specific immune stimulation based on the strong capacities of the virus to activate host immune responses in terms of cytokine production. It was entered for clinical evaluation in Hungary, where an attenuated variant, designated as  cancers . Laszlo  cancers . Fifty-fIn the 1980s, the development of chemotherapeutic reagents brought new hope for cancer treatment. And virus treatment was not “en vogue” anymore during these years. But the continuous problems with the development of tumor resistance to treatment with chemotherapeutic agents, which resulted in cancer relapses, eventually led to a regain of interest from clinicians for OVs as tumor cell killers during the past two decades .73T and MTH-68, stimulated further evaluations of their anti-cancer properties. To this end, genetically immunosuppressed SCID mice carrying xenografted human neuroblastoma or fibrosarcoma were used. These mice responded well to a single intra-tumoral injection of the NDV 73T virus strain and showed complete or partial remission [PV701, which was derived via plaque purification from the naturally attenuated vaccine strain MK107 [The interesting clinical observations, which had been made in the past with the NDV strains emission ,70. Baseemission ,70, Lorein MK107 . PV701. It showed for 79 patients with various advanced solid cancers [10 to 1.2 × 1011 pfu were well tolerated with only grade 1 and 2 toxicity . Several dose regimens were evaluated. It was also observed that initially giving lower virus doses permitted escalation of doses later (desensitization procedure). This allowed a reduction in some cell toxicity, especially at the tumor site. Of the 79 patients, one partial remission was observed and 14 patients showed no tumor progression between 4 and 30 months after therapy. The most common adverse events were fever and other flu-like symptoms.A first Phase I clinical trial investigated the safety aspects of  cancers  that intIn total, the obtained results after systemic application of NDV were quite disappointing ,73,74,75MTH-68/H as a treatment modality for patients with various cancers. They reported on a case series of 4 patients and on a phase II trial. There was some benefit to some patients with glioblastoma after repeated i.v. applications of this oncolytic NDV strain [HUJ were performed at the Hadassah Medical Center . From the involved patients suffering from glioblastoma multiforme (GBM) and receiving systemic application of the HUJ virus strain, one patient has been reported to have achieved a complete response [A Hungarian group studied the use of the NDV strain V strain ,77. For response . From all these clinical studies, it appears that an effect of NDV as anti-cancer agent can be observed, even when applied to patients suffering from advanced cancers. But the data obtained with oncolytic NDV were not as good as one had hoped for. This concept is based on using NDV as biologic adjuvant for an effective tumor vaccine knowing that virus-induced augmentation of the antigenicity of tumor antigens has been observed in several model systems ,80. It iTwo main strategies for active specific immunotherapy (ASI) including NDV—respectively as lytic or non-lytic virus—as adjuvant were evaluated in clinical trials .et al. developed already in 1977 a second direction based on tumor oncolysates. These were obtained from melanoma upon infection with the oncolytic NDV strain 73-T [To stimulate the anti-tumor immune responses, William Cassel ain 73-T  to inducain 73-T ). The raet al. [The trial gave an unprecedented over-60% survival rate after 10-years follow-up compared to only 33% of comparable non-vaccinated patients . From alet al. ,83,84, tUlster) of NDV and γ-irradiation [Ulster virus shows an abortive monocyclic replication cycle in tumor cells [6–107 cells. NDV infection was initiated by one hour adsorption of the virus to the tumor cells and then the cells were inactivated by 200 Gy-irradiation. This treatment inhibits the tumor cells proliferation capacity via DNA cross-linking while it does not inhibit the RNA virus from replicating in the cells’ cytoplasm. The virus modified tumor vaccine was injected intradermally. In vivo viral replication within the tumor cells at the site of vaccine application takes approximately 6–40 hours [The concept of exploiting the immunostimulatory properties of NDV to augment the immunogenicity of tumor cells was further developed by the senior co-author of this review An autologous live cell tumor vaccine, called ATV-NDV, was obtained upon modification of patient’s tumor cells via infection with a lentogenic strain  and also allowed for activation of multiple innate immune cells  ,87. In 1986, it was reported for the first time that post-operative immunotherapy with ATV-NDV prevented metastatic spread in a mouse tumor model . The suret al. described for the first time that the induction of type 1 IFN by NDV potentiates tumor-specific CTL activity [in vitro MLT-CTL cultures, they reduced the generation of CTL activity.In 1990, von Hoegen activity . The appThe introduction of danger signals into tumor cells via NDV infection appeared able to break T cell tolerance to tumor antigens. For example, it could be demonstrated in an autologous human anti-melanoma CD4+ T helper clone that infection by NDV of autologous melanoma cells caused a T cell co-stimulatory activity . In contThe first description of clinical results following post-operative vaccination with ATV-NDV modified tumor cells was reported for primary operated breast cancer patients in 1996 . In thisData from 5 years’ observations are also available for colorectal carcinoma patients. OS in vaccinated colorectal carcinoma  was increased by ATV-NDV: 61% of the patients were still alive after 5 years, which compares favorably with the expected 39% under standard therapy . OS at 5p = 0.042) and with respect to metastases-free survival (p = 0.047). In contrast, in rectal carcinoma patients, the same treatment with ATV-NDV did not influence OS, recurrence free survival (RFS) or metastases-free survival. This difference in responsiveness to ASI between patients with two different tumor entities is of biological relevance but not yet understood. That this difference came out so clearly and that the follow-up period was much longer than in other published studies underlines the high quality of the trial. For colon cancer patients, there is thus a good future prospect with the ATV-NDV vaccine. The trial indicates the value and potential of cancer vaccines using NDV-infected cells. Results from a randomized-controlled Phase II/III study were as follows. Patients with histologically confirmed liver metastases from colon cancer or rectum cancer were randomized to an ATV-NDV vaccination or to a control arm . After cA further study with higher numbers of patients is warranted and further investigations into the implied mechanism, efficacy and long-term immunity are required.In conclusion, it can be stated that clinical efficacy in terms of survival has been observed when NDV was used as adjuvant in strategies of active specific immunotherapy—that is when NDV and tumor cells were combined together before application to cancer patients. This vaccine has been evaluated from 1989 to 2008 in at least ten Phase II trials and one randomized, controlled Phase II/III study. Positive effects on long-term survival of cancer patients upon post-operative anti-tumor vaccination with ATV-NDV have been summarized and discussed ). The reex vivo large quantities of DCs.DCs expressing tumor material of the patient’s own cancer are known to be capable of inducing a Th1 tumor-specific immune response. But it took a long time from DC discovery to their clinical application. One reason is that the number of circulating DCs in the blood is extremely low. That hindered clinical application of a DC therapy for a long time. Within the last 2 decades, new insights into the biology of DCs together with new developments in biotechnology have opened the possibility for generation of DCs outside the body. Starting from circulating white blood cells and employing speciﬁc culture conditions, it is now possible to generate DCs were first described by Ralph Steinman in 1973 when observing cells from mouse spleen that adhered to glass or plastic surfaces. In addition to mononuclear phagocytes, granulocytes, and lymphocytes, a subpopulation of cells with striking dendritic shape was noticed . Nearly in vivo in an immunogenic or tolerogenic fashion. They take up antigens and efficiently process them for their presentation as peptides in association with MHC molecules (either class-I or class-II) to respectively CD8 and CD4 T cells. MHC class-I molecules present self- or pathogen-derived antigens that are synthesized within the cells, whereas exogenous antigens derived via endocytic uptake are loaded onto MHC class-II molecules for presentation to CD4+ T cells. DCs are also specialized to process exogenous antigens into the MHC class-I pathway for presentation to CD8+ T cells. This process, known as cross-presentation and shown for the first time in 1976 [et al. [All the characteristics exhibited by DCs make these cells unique candidates for immunotherapy aiming at inducing effective T cell-mediated anti-tumor immunity. As first reported by Finkelman and colleagues in 1996 , DCs can in 1976 , provide in 1976 . DCs are [et al. . In ordeThe design of an efficient anti-tumor vaccine is influenced by an important paradigm shift in the field of immunology regarding the regulation of immunity. Indeed, a new concept has emerged that proposes that the regulation of immunity and tolerance is not only determined by the specificity of immune T cells as previously thought, but also by the context in which the antigens are presented to the immune system. This new hypothesis originally presented by CA Janeway Jr.  suggestein vitro generation of mature DCs and for their genetic modification, both through viral and nonviral approaches, to provide the three signals , which are required for T cell activation [The danger signal hypothesis by Polly Matzinger  allowed tivation . Signal tivation ,108. In tivation ,110. TheWhen all the necessary signals are present, the interactions between DCs and T cells in tumor-draining lymph nodes and in bone marrow lead to T cell activation, clonal expansion, and differentiation into effector and memory cells. To initiate such immune responses, cells from the innate immune system  play an important role, although the exact role of these immune effector mechanisms in protecting individuals from tumors is not well defined. An alternative mechanism, referred to as “cross-dressing”, has been shown to exist in mice in a context of viral infection, by which an Antigen Presenting Cell (APC) can transfer antigen via direct contact from its surface MHC-peptide complexes to another APC without the need of further processing .And this was shown to boost the memory  but alsoet al. demonstrated that isolated DCs pulsed ex vivo with antigen could, after injection into mice, elicit an immune response against cells bearing the same antigen [in vitro [et al. published the ﬁrst clinical trial on DC vaccines in B cell lymphoma patients. In this study, all the four patients developed measurable anti-tumor immune responses [Very quickly after the discoveries of the DCs, Steinman and colleagues demonstrated that DCs are able to mature and that they acquire novel functions following microbe encounters (reviewed in ). But th antigen . Two yeain vitro . This presponses . Two yeaesponses . Based oesponses ,122. Howex vivo generated DCs. These were employing diverse methodologies for DC propagation, antigenic loading, and administration [Thereafter, DC-based tumor vaccines have been extensively explored, and have become attractive tools for cancer immunotherapy. They have been tested for the treatment of more than 20 malignancies, most commonly melanoma, renal cell carcinoma, prostate cancer, and colorectal carcinoma ,124. Meastration . From the plethora of DC-based vaccine trials, several generalizations can be made. The first concerns safety and feasibility of this approach. The second concerns the efficacy of DC-based vaccines. Data in humans showed that such therapies were able to induce T cell responses, although not in all the vaccinated patients. However, objective clinical responses were anecdotal and concerned a minority of the vaccinated cancer patients. Although the vaccination may delay tumor progression and improve survival for some patients with advanced cancer, its capacity in inducing tumor regression was limited in general when considering the whole population of vaccinees. And at that time, DC-based vaccines largely failed the expectation of being an effective means of treating cancer. The limited success may be attributed to a variety of factors regarding the preparation and administration of the vaccine, the disease stage of participants in the trials, the heterogeneous nature of most tumors rendering the clinical analysis difficult, the limited number of tumor target antigens present within the DC vaccine and the context of immuno-suppression. Most of the clinical studies have been performed in immunocompromised patients with advanced disease, which are known to have dysfunctional DCs ,127. In addition, the existence of immuno-suppressive factors in the tumor micro-environment including the production of inhibitory cytokines , the activation of STAT3, the expansion of Treg cells, and the significant suppressive effect of myeloid derived suppressor cells (MDSCs) have been shown to create a tolerogenic milieu ,129,130.ex vivo antigen-loaded DC do not directly prime T cells but they shuttle their antigen to the draining LN where the antigen is acquired by resident DCs that subsequently prime T cells [One other explanation why only sub-optimal immune activation was achieved could be linked to some observations which suggest that only a small proportion of the injected antigen-loaded DC ever reach the LNs ,132 wher T cells . The depAmong all the clinical studies performed with DCs, two major results marked the development of the DC field as explained in the two following paragraphs. In 2006, DC vaccines suffered a major setback in a phase III clinical trial, which was performed in advanced melanoma patients . It was ex vivo with a recombinant fusion protein construct consisting of full-length Prostatic Acid Phosphatase (PAP) linked to a full-length GM-CSF. This cytokine is included to target the fusion protein to APCs through the GM-CSF receptor (CD164), where it stimulates T cell immunity to PAP. Of all the antigen platforms currently available in late-stage development, PAP is the only defined target that may be considered organ-specific, or even tumor-specific, as it has no evidence of expression outside of the tumor and the tumor-bearing organ. Although this system targets a single antigen, the immune response engendered may be polyvalent if it recognizes more than a single PAP epitope [The breakthrough came in 2010. The approval by the US FDA of the ﬁrst DC vaccine, named Provenge (or Sipuleucel-T), for the treatment of prostate cancer [ epitope . p = 0.01). The D9902a trial was designed like the D9901 trial but enrolled 98 patients. The median survival time for patients treated with Provenge was 19.0 months compared to 15.3 months for placebo-treated patients, but did not reach statistical significance. The IMPACT trial served as the basis for licensing approval of Provenge by the FDA [p = 0.032). The prolongation of the median OS of vaccinated patients by 4.1-month is numerically the greatest survival benefit that has been seen in the treatment of HRPC. Sipuleucel showed no effect on time to progression in asymptomatic or minimally symptomatic metastatic castrate-resistant patients. Provenge showed OS benefit to patients in three double-blind randomized phase III clinical trials: D9901 ,136; D99The FDA approval of Provenge  has beenFurther encouraging proof of principle results came from a Phase I trial on glioblastoma, in which patients were treated with DCs loaded with autologous acid-eluted tumor peptides, leading to increased time to tumor progression and OS . DespiteIt has been shown that DCs, when isolated from cancer patients, can have major dysfunctions. They can have a reduced capacity to stimulate an immune response than do DCs isolated from healthy donors ,139. DCsMultiple stroma- and tumor-derived factors have been recognized to have the potential to alter the DCs at tumor sites or at a systemic level. All of these mechanisms result in a lack of T cell stimulation. A major challenge for successful DC-based cancer therapy consists then in overcoming the immune suppression induced by the tumor micro-environment. To this end, it appears of importance to identify and optimize the appropriate combination of molecular signals that results in the induction of potent and clinically significant anti-tumor immunity. Determining parameters of optimal DC loading with TAAs and of their activation is expected to be of major significance for improvement of clinical efficacy.ex vivo DC activation in order to maximize the quantity and quality of effector T cells and to minimize the development of Treg that undermines anti-tumor responses . It combines NDV and DCs in 2 sequential steps: (i) first, NDV is injected systemically; (ii) thereafter, vaccinations are performed via intradermal application of a DC vaccine loaded with NDV infected autologous tumor oncolysate. This second step is based on The NDV-DC therapy combines immune conditioning via oncolytic NDV with DC-oncolysate immunization. It represents a new immunotherapeutic concept by its potential to increase innate immunity mechanisms and adaptive anti-tumor immune responses through priming of naive T cells and through re-stimulation of anti-tumor memory T cells. de novo tumor specific T cell responses from the pool of naive T cells.The studies with ATV-NDV revealed the importance of tumor cell viability and vaccine tumor cell number for clinical effectivity . In the MTH-68 is based on the effects described above: (i) the selective replication of the MTH-68 virus in tumor cells, (ii) the oncolytic properties of NDVand (iii) the expected post-oncolytic immune effects to reinforce pre-existing anti-tumor immune memory of the cancer patient. It is also based on indirect effects: (i) the NDV-induced production of high levels of type 1 IFNs,  thFNs, see  and . Upon rPlasmacytoid DCs (pDCs) have been identified as a minor cell population, which is responsible for the production of large quantities of type 1 IFNs upon NDV triggering through direct contact with these cells. The existence of these different pathways using autocrine and paracrine mechanisms ,141 mighin vitro more effectively [Type 1 IFNs are considered to be major players for linking innate to adaptive immunity. They were first discovered for their innate anti-viral activity ,145. Theectively . Such DCectively . In addiectively . in vitro, although they induce STAT4 activation. The co-presence of IL-12 appears important for a strong CD4+ T cell response able to drive Th1 development [in vivo is expected to help the induction of an anti-tumor response during the vaccination steps of the NDV-DC therapy. Type 1 IFNs also have an effect on the regulation of effector and memory cells by inhibiting their death . Type 1 elopment ,153. TheThere are other considerations, which corroborate the new concept, as for instance, the inhibitory effect of type 1 IFN on Th2 and Th17 differentiation of T cells. Cancer patients have often an immune system which is polarized towards a Th2 cytokine profile. Some subsets of leukocytes certainly exhibit antitumor activity, including CTLs and NK cells , other lType 1 IFNs play a role in this balance since they inhibit secretion of Th2 cytokines (IL-4 and IL-5) and stimulate IFN-γ production ,158. CanWe conclude that NDV as OVs (i) upon systemic administration can modify the immune context and (ii) consequently can facilitate the generation of anti-tumor immunity. The systemic administration of NDV before DC vaccination is a new strategy for the treatment of cancer patients.de novo anti-tumor immune responses (from naive T cells) and to reactivate anti-tumor reactive memory T cells from the repertoire of pre-existing memory T cells, including partially anergized cells. Strong stimulatory signals are required since vaccine efficacy may be blunted by the immuno-suppressive milieu, which is characteristic for cancer patients. This is linked to numerous mechanisms such as the increased presence of Tregs, and inhibitory pathways such as the PD-1/PDL-1. The rationale for the vaccination with NDV-DC is based on the use of autologous DC, autologous tumor cells and NDV having oncolytic and immunostimulatory properties.The objective of this vaccination step is, via injecting activated and TA-presenting DCs, to generate ex vivo generated DCs is expected to side-step the cancer-associated dysfunction of endogenous DCs and to deliver the key instructive signals needed for effective anti-tumor responses. Such information through DCs as APCs is important for the immune system to identify, via TAAs, the target  and for the T cells to associate such target antigens with danger signals derived from the virus. The use of Candidate tumor antigens for vaccination include shared self-antigens including cancer/testis antigens and tissue differentiation antigens and unique (mutated) antigens ,161. TheHowever, the enthusiasm for these antigens might be dampened by the 2 following reasons: (i) the repertoire of T cells might be deleted of high avidity clones through negative selection by shared antigens in the thymus  and (ii)Unique (or mutated) antigens are postulated to be superior to shared antigens ,161: theThe use of autologous tumor cells enables a close match between the TAAs of the vaccine and those of the patient’s tumor and includes common and individually unique TAAs . Unique The dsRNA replicating forms, which are synthesized in the cytoplasm during NDV replication, are recognized by TLR3 and RIG-I in a cell type and pathogen type-specific manner ,43. It hIt was shown that dsRNA, which is present in the apoptotic bodies of virus-infected dead cells, is recognized by CD8α+ DCs that have high expression of TLR3 . This prMaturation of DCs will be accomplished by NDV infection via activation of pathogen sensing pathways through PRRs, including the TLR- and RLR-pathways, which are mentioned above. Very interestingly, a recent study shows that NDV favors development of type 1 DCs, also called «DC1» . These uin vivo inoculation, their ability to migrate and survive was enhanced compared with control transduced DCs in human clinical trials [When DCs were transduced with IFN-α prior to l trials . TogetheIt is now clear that IFN-α has an important adjuvant function in the immune response. This effect could explain why most live viruses elicit strong immune responses whereas viral peptides are poorly immunogenic or tolerogenic unless supplemented with exogenous adjuvant. NDV, via induction of IFN-α, has been shown to induce in NK cells and monocytes TNF-related apoptosis-inducing ligand (TRAIL) and cell-mediated cytotoxicity . InteresType 1 IFN has been also reported to induce the IL-12 receptor-α chain in T cells . TogetheThe role of type 1 IFN in the induction of the immune response is reinforced by its function as link between innate and adaptive immunity . The innThe first published danger model of immunity  proposedIn conclusion, the NDV-DC vaccine, which combines autologous DCs with oncolysate from NDV infected autologous tumor, shows excellent prerequisites for superior activity. In comparison to classical DC therapies, the combined NDV plus NDV-DC approach is also new. “Classical” DC therapies have mainly employed DCs pulsed with TAs and matured by exposure to cytokine cocktails. These DCs are sufficient for T cell activation, but they may not be adequate to provide continuous co-stimulation to mount and maintain a pro-inflammatory immune environment and to recruit additional effector components. Since an existing tumor creates an immuno-suppressive environment, successful DC-based cancer vaccines should be prepared to be able, via immunization, to prime a strong and persistent primary immune response against tumor cells and to re-stimulate tumor-specific memory T cells. ex vivo antigen-loaded DCs would not directly activate naive T cells as efficiently as DCs that naturally acquire antigen in vivo, the combination of NDV and tumor antigens with DCs make these cells as carriers of TAs  and of danger signals  resistant to tolerogenic effects of the microenvironment. That appears to be a way to target tumor antigens and danger signals also to endogenous DCs. Even if the in vivo. Numerous investigations have demonstrated that DCs can be simultaneously modified with multiple genes and/or immune factors [The combination of NDV with DCs can allow for a continuous supply of processed tumor antigen and immune-stimulating molecules with redundant and pleiotropic activities. It is therefore expected to provide more robust and persistent anti-cancer immunity  factors ,178. HowRecently, NDV has been suggested in tumor therapy to help not only to stimulate T effector cells but also to block Treg cells, thereby alleviating a brake to anti-tumor activity . InactivClinical trials of DC-based cancer vaccines have revealed that activated NK cells are better predictive of vaccine efficacy than CTL responses ,182. AccThe strategy based on the use of oncolytic NDV, autologous tumor material and DCs appears to be sufficient for delivery of TAs, co-stimulatory molecules, and environmental signals. The use of oncolysate provides TAs directly to the cytosol of DCs after pinocytosis. This is expected to result in the induction of T cells with desirable effector functions and tumor-relevant homing properties. Combining DC vaccination with NDV oncolytic virotherapy might paves the way for a new strategy of cancer immunotherapy. This includes patients with tumors having poorly-defined rejection antigens, after primary or secondary tumor resection . This group of patients is most likely to benefit from vaccination in an adjuvant situation.in vivo (oncolysis in vivo) or ex vivo (by generating oncolysates). The observations made over this long period demonstrate the safety and potential of NDV as a new biologic agent against cancer (During the last 6 decades, NDV has been tested as an agent against cancer. From the beginning, the experimental studies with NDV were performed in animals but also in humans. Results were published as case-reports and as prospective non-randomized clinical studies. These trials were based on the oncolytic properties of the virus, either directly t cancer a,b.The selectivity of NDV for replication and cytolytic activity only in tumor cells has been attributed primarily to a defective type 1 IFN response which is a typical feature of tumor cells. However, in the recent years evidence has accumulated that also other features are of importance to explain the selective oncolytic activity of NDV ,187,188.The development of the field of tumor immunology over the same time period changed the focus of the development of tumor treatment with virus, from virological considerations to more immunological ones, as shown by the pioneering work of William Cassel with vaccination with tumor lysates. The Heidelberg vaccine ATV-NDV and the associated basic and clinically applied research revealed new insights into the mechanism of antitumor immune properties of NDV and paved the way for further exploitation of the potential of NDV for anti-tumor vaccination. DC-based cancer vaccines have shown a parallel development, although being more recent. Their further development faces new challenges for the future but also new hopes for success. Data from recent clinical studies suggest that DC-based vaccines have the potential to induce strong anti-tumor responses and to prolong the survival of cancer patients.New data reveal that NDV is a biologic agent to be combined with DC therapy to improve the efficacy of anti-tumor vaccination and to bring it to clinical efficacy . In an aThe scientific data presented in this review provide new insights into the design and mechanism of function of NDV-DC. This vaccine works as a potent Th1 response mediator, favoring the induction of DC maturation, the release of pro-inflammatory cytokines and the improvement of antigen cross-presentation. All these steps are essential for the priming and activation of a CD8+ T cell-mediated tumor-protective immune response.Many milestones in nearly 50 years have shaped the scientific basis and clinical evaluation of the NDV-DC therapy see . It restThe early clinical evaluations of the anti-tumor properties of NDV, which started in the 1960s and which were presented as case reports, were essentially experimental and observational. The vaccination studies, which were performed by William Cassel in melanoma patients with oncolysate, gave rise to interesting results, although their validity cannot be warranted ,82,83,84


Abutilon mosaic virus, AbMV, Geminiviridae) in intra- and intercellular molecule exchange. The chaperone cpHSC70-1 was shown to interact with the AbMV movement protein (MP). Bimolecular fluorescence complementation confirmed the interaction of cpHSC70-1 and MP, and showed a homo-oligomerization of either protein in planta. The complexes were detected at the cellular margin and co-localized with plastids. In healthy plant tissues cpHSC70-1-oligomers occurred in distinct spots at chloroplasts and in small filaments extending from plastids to the cell periphery. AbMV-infection induced a cpHSC70-1-containing stromule network that exhibits elliptical dilations and transverses whole cells. Silencing of the cpHSC70 gene revealed an impact of cpHSC70 on chloroplast stability and restricted AbMV movement, but not viral DNA accumulation. Based on these data, a model is suggested in which these stromules function in molecule exchange between plastids and other organelles and perhaps other cells. AbMV may utilize cpHSC70-1 for trafficking along plastids and stromules into a neighboring cell or from plastids into the nucleus. Experimental approaches to investigate this hypothesis are discussed.Stromules are dynamic thin protrusions of membrane envelope from plant cell plastids. Despite considerable progress in understanding the importance of certain cytoskeleton elements and motor proteins for stromule maintenance, their function within the cell has yet to be unraveled. Several viruses cause a remodulation of plastid structures and stromule biogenesis within their host plants. For RNA-viruses these interactions were demonstrated to be relevant to the infection process. An involvement of plastids and stromules is assumed in the DNA-virus life cycle as well, but their functional role needs to be determined. Recent findings support a participation of heat shock cognate 70 kDa protein (cpHSC70-1)-containing stromules induced by a DNA-virus infection ( Tobamovirus [turnip vein-clearing virus (TVCV) and tobacco mosaic virus (TMV); Potexvirus [Alternanthera mosaic virus (AltMV) and potato virus X (PVX); In plants, transport of endogenous macromolecules such as proteins and nucleic acids over cellular boundaries occurs in a highly selective and regulated manner . These cGeminiviridae) replicate within the nucleus, and systemic infection requires the crossing of two cellular barriers, the nuclear envelope via pores and the cell wall via plasmodesmata (Begomovirus) consists of two DNA molecules: DNA A and DNA B. The two DNA B-encoded proteins, nuclear-shuttle protein (NSP) and MP, mediate the viral transport processes , and tomato crinkle leaf yellows virus (TCrLYV) were found to interact with two classes of receptor-like kinases from thaliana . The furthaliana . Additiothaliana  and withthaliana ,b. AtNSIthaliana ,b suggesin vitro and in vivo co-immunoprecipitation (Co-IP) experiments showed an interaction of H3 with NSP and MP of BDMV as well as with CPs of different geminiviruses  family is induced in response to developmental signals and various abiotic and biotic stress stimuli . Some faa maxima  and by cN. benthamiana . It is hypothesized that cpHSC70-1 is present in the same type of “packet” structure as GFP in the preceding experiments, probably associated with vesicles and actively transported.participate in plastid motility and in facilitating transport of various molecules, e.g., proteins, metabolites and signaling components, into and out of a plastid, among plastids and even between plastids and other organelles , 2011. Cbidopsis . Moreovebidopsis . A simplbidopsis , 2011. TFigures 2).The data obtained by By contrast, in non-infected tissues only short cpHSC70-1-containing filaments were found which extended solely from cortex positioned plastids to the cell periphery. However, molecular transfer from the plastids to the nucleus or vice versa with the aid of stromules remains to be confirmed. That a retrograde protein exchange between plastids and the nucleus can occur was demonstrated recently. Plastid-encoded HA-tagged Whirly1 protein was translocated to the nucleus in transplastomic tobacco plants, where it stimulated pathogen-related gene expression . The chlN. benthamiana and Arabidopsis) were comprehensively analyzed for plastid morphology and marker protein transfer. Although the differently colored plastids and stromules were in very close proximity, the labeled organelles remained separate as indicated by the absence of color mixing. That the method applied in these studies is suitable to detect an exchange of material between organelles upon a fusion was confirmed by analogous experiments using a mitochondria-targeted version of the fluorescent protein. In contrast to our observations from geminivirus-infected plants, the results of Figure 3).In addition to the induction of stromule biogenesis, AbMV-infection influences the localization of a plasmodesmata-associated protein. The plasmodesmata callose binding protein 1 (PDCB1) fused to mCherry was investigated as a marker for plasmodesmata by Figures 3), but no punctate structures were detected anymore. Thus, the protein seems to have lost the plasmodesmata localization. We hypothesize this as an AbMV-induced remodeling of the plasmodesmata aperture by callose depletion in the neck region.Surprisingly, upon AbMV-infection PDCB1:mCherry signals were still distributed at the cell periphery, most likely the cell wall  might be the fusion of the outer and inner envelope membrane with the target membrane. AbMV-infection might create a cellular environment that allows such a fusion event of stromules and the consequent transposition of quantities of stromal components. Alternatively, a transfer process might be initiated, which does not comprise a fusion of the inner envelope membrane or the envelope at all. This might consist of transport along the cytoplasmic leaflet of the outer envelope membrane, through the intermembrane space after fusion of the outer envelope with the target membrane or via envelope-coated vesicles released from stromules . Irrespe of AbMV , suggestFigure 4) can be proposed in which MP/cpHSC70 interaction and stromule induction facilitate intra- and intercellular macromolecular trafficking along plastids and stromules into the neighboring cell or in the other direction from plastids into the nucleus. Whether this represents an accidental event or is of major significance for AbMV propagation and/or symptom development remains to be investigated.In summary, a model  To resolve the geometric relation of the different cellular structures high resolution confocal imaging and time lapse imaging is required. (ii) Test protein expression should be done in combination with a set of fluorescent marker proteins, probably photoconvertible, for different compartments to monitor the subcellular distribution, temporal activities, and macromolecular trafficking events. A cytoplasmic marker would be needed to investigate if AbMV-induced stromules actively associate with the nucleus or are just pressed by other organelles, like a large vacuole, toward the nucleus. (iii) Careful controls should be included to account for stresses (including agro-infiltration) that are known to influence stromule formation. (iv) To minimize the influence of agro-infiltration, transgenic plants expressing required fluorescent test proteins and alternate methods to introduce AbMV (biolistic bombardment of vDNA) should be applied. To investigate the precise molecular function of cpHSC70-1 on AbMV infection and presumably macromolecular trafficking, the impact of the following scenarios on AbMV spread could be investigated: (i) transgenic plants overexpressing wild-type cpHSC70-1 or a non-MP-interacting, dominant-negative cpHSC70-1 variant and (ii) infection with AbMV DNA encoding a non-cpHSC70-1-interacting MP mutant. Finally, the relevance of the findings should be tested for additional geminiviruses other than AbMV. This knowledge gained will contribute significantly to the elucidation of the geminiviral intra- and intercellular movement process.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Medulloblastoma, the most frequent brain tumor in childhood, also occurs with a wide range of characteristics in adult patients. Late relapse is common in adult medulloblastoma, and the overall survival of relapsed patients usually ranges from 12 to 15 months. Treatment at recurrence is still debated and after reoperation includes stereotactic or normofractionated radiotherapy, and high-dose chemotherapy with autologous bone marrow transplantation.We report on the case of a 31-year-old Caucasian woman who underwent re-irradiation for a recurrence of medulloblastoma at nine years after first irradiation (56Gy), focusing on the radiobiological background and a review of previous studies involving re-irradiation of recurrent medulloblastoma. After surgical excision of the relapsed tumor and medical multi-agent treatment, the site of recurrence was treated using three-dimensional conformal radiotherapy to a total dose of 52.8Gy (1.2Gy/fraction/twice daily). A total biological equivalent dose of 224.6Gy (α:β = 2 Gy) was delivered to the posterior fossa (first and second treatments). No radionecrosis or local recurrence was evident at 18 months after re-irradiation.Re-irradiation can be considered a possible and safe treatment in selected cases of recurrent medulloblastoma in adults. The reported radiobiological considerations could be useful in other cases involving re-irradiation of brain tumors. Medulloblastoma is a primitive neuro-ectodermal tumor that is the most frequent primary brain neoplasm in childhood. About 50% of these tumors occur in children aged less than five years, whereas they are rare in adolescents and young adults. Medulloblastoma behaves differently in adults than in children, and is identified as a different biological and clinical entity. It exhiThe outcome for patients with recurrent medulloblastoma has historically been poor, with most patients dying of disseminated disease: usually within 12 to 15 months after retreatment with either chemotherapy or radiotherapy. The optimal treatment for local relapse in non-metastatic patients remains controversial. A number of chemotherapy regimens (with or without high-dose chemotherapy and bone marrow autologous transplantation) have been proposed, along with temozolomide-6 and siThe patient, a 31-year-old woman, complained of headache and dizziness and underwent magnetic resonance imaging (MRI) of the brain; this showed a contrast-enhancing lesion in the posterior fossa. Radical removal of the lesion was achieved by means of right posterior fossa craniectomy and laminectomy of the first cervical vertebra; histological examination confirmed the diagnosis of T2  M0 (no gross subarachnoid or hematogenous metastasis) (Chang’s classification), desmoplastic medulloblastoma. At one month after surgery, cranio-spinal irradiation was carried out. The entire cranio–spinal axis was treated. The treatment consisted of two parallel opposed photon fields, latero–lateral for the brain and posterior–anterior for the spinal axis, using the moving junction technique. The total dose to the brain and spinal axis was 36Gy in 20 fractions . A boost to the posterior cranial fossa (18Gy in 10 fractions) was then delivered, resulting in a total dose to the tumor bed of 54Gy. Radiotherapy was well tolerated and the patient did not show any acute toxicity, with the exception of mild neutropenia and nausea.The patient underwent regular follow-up at the Radiation Oncology Department without evidence of recurrent disease. Eight years later, MRI showed a recurrence in the right cerebellum , causing a bulge in the IV ventricle. Caudally the lesion imprinted the right cerebellar tonsil, resulting in a reduction in the ipsilateral foramen of Luschka. The patient was in good general condition; neurological examination did not reveal any neurological deficit. Neurocognitive analysis obtained by submitting the patient to a standardized neurological examination using the Mini-Mental® State Examination (MMSE), Clock Drawing Test, Rey auditory verbal learning test and Trail Making Test (TMT) indicated a normal global cognitive function , with important disexecutive and attention deficits . No memory impairment was detected using the MMSE and Rey Auditory-Verbal Learning Tests., a salvage re-irradiation was considered.After multidisciplinary clinical discussion the patient underwent partial surgical resection of the recurrence. The post-surgical MRI evidenced a small nodule on the edge of the superior-medial cavity, showing post-contrast enhancement that was suggestive of residual disease. The patient then received salvage chemotherapy including carboplatin, procarbazine, etoposide, cisplatin, vincristine and cyclophosphamide according to the sequential trial of the French Society of Paediatric Oncology. After aThe patient signed an informed consent declaration, after being informed of the possible acute and late toxicities related to the re-irradiation. With the patient in the supine treatment position and immobilized with a thermoplastic mask, we obtained whole brain computed tomography (CT) with three-mm slices from the vault of the skull to C5. The volumes were defined on the planning CT and were co-registered and fused with the diagnostic MRI images Figure  a,b. TheThe treatment was well tolerated and the acute and sub-acute toxicities were mild; slight somnolence and asthenia disappeared within two months from the end of the treatment. The patient continued with follow-up at our institution. MRI, obtained at three months after re-irradiation, showed complete regression of the multiple nodules in the posterior fossa and no evidence of radionecrosis Figure  a,b. Unf2  of 200Gy, with at least one year between the two treatments; long-term complications related to the retreatment were seen in patients with a BED2>204Gy (α:β = 2 Gy)[The risk of toxicity can differ between brain regions with a highly collateralized blood supply and those perfused by only a few small branches. Importa = 2 Gy).In the largest published medulloblastoma re-irradiation series (13 patients), the dose prescribed for the second treatment was 30Gy (1.5Gy per fraction); the median cumulative dose reached 82.3Gy (range 73.8 to 98.4Gy). In thisThe fractionation regimen  was chosen to take advantage of the early-repair characteristics of medulloblastoma cells, as opposed to the late repair of healthy brain tissue. The theoretical cumulative BEDs for late and early effects on the healthy tissue of the posterior fossa  were of 74.3Gy and 78Gy, respectively  can be delivered, especially if given at low doses per fraction. The spinal cord can possibly be included in the treatment volume, even if it is not involved with the recurrence, to avoid early spinal progression.Because of concerns with regard to the risk of late central nervous system toxicity, especially radionecrosis that may occur several months to years after treatment, radiation oncologists have historically been cautious about retreating brain tumors. However, there is a lack of prospective data in this regard. Re-irradiation of brain tumors is attracting increasing interest. In fact, a greater understanding of brain tolerance to radiation, developments in tumor imaging and advances in radiotherapy planning and delivery techniques now make possible the achievement of better target definition and highly conformal treatments.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.BED: Biological efficacy dose; CT: Computed tomography; MMSE: Mini-Mental® State Examination; TMT: Trail Making Test.The authors declare that they have no competing interests.MB treated the patient with radiotherapy, analyzed and interpreted the data, coordinated all of the authors, and ideated and drafted the article. LT treated the patient with radiotherapy and drafted the article. SG treated the patient with chemotherapy and drafted the article. RL performed the magnetic resonance imaging and drafted the article. LB surgically treated the patient and drafted the article. SG performed neurologic evaluations and drafted the article. FB performed pathological evaluation. AT performed neurologic evaluations. LS carried out dosimetric evaluation and physical treatment planning, and drafted the article. SMM was responsible for patient treatment, analyzed and interpreted the data, revised the article and approved the final version. All authors read and approved the final manuscript.All of the authors are members of the Neuro-Oncology Multidisciplinary Group, Spedali Civili Hospital and Brescia University, Brescia, Italy.Appendix.Click here for file