Correction to: Light: Science & Applications10.1038/s41377-019-0188-0 published online 14 August 2019Nat Methods, 2014, 11(8): 861−867) and 3TAT : 421−435) have been reported to be able to deliver SNAP-surface 488 into live cells, our claim that “PV-1 is the first vehicle that can transfer cell-impermeable organic fluorescent probes into live cells with satisfactory efficiency for imaging, requiring only the coincubation of the probes with PV-1” is inaccurate. To address this discrepancy, we have made the following corrections to the article on the recommendations of the editor.10,11,14” should be “However, these methods present several limitations in terms of applicability and efficiency and are also time-consuming and technically demanding. Delivering organic fluorophores by non-covalently linked peptide vehicles is an alternative method that is more convenient and cost-efficient17,36. However, only a few organic fluorophores have successfully been made cell permeable following chemical modification or simple coincubation with peptide vehicles10,11,14,17,36.” to make the statement more accurate.On page 2, “However, these methods present several limitations in terms of applicability and efficiency and are also time-consuming and technically demanding. To date, only a few organic fluorophores have successfully been made cell permeable following chemical modificationOn page 2, “To date and to the best of our knowledge, PV-1 is the first vehicle that can transfer cell-impermeable organic fluorescent probes into live cells with satisfactory efficiency for imaging, requiring only the coincubation of the probes with PV-1.” should be “To date and to the best of our knowledge, PV-1 is the first vehicle that can simultaneously deliver up to three different probes into live cells with satisfactory efficiency for imaging, providing a new approach for labelling intracellular targets for multicolour live-cell imaging applications.” to make the statement more accurate.17 and 3TAT36.” to make the statement more accurate.On page 8, “Thus far, by simple coincubation, peptide vehicles can only efficiently deliver nucleic acids and proteins into live cells” should be “Thus far, by simple coincubation, most peptide vehicles can only efficiently deliver nucleic acids and proteins into live cells, except for dfTAT17,36.” to make the statement more accurate.On page 8, “To date, no effective cytosolic delivery of organic fluorescent probes by peptide vehicles via simple coincubation has been reported.” should be “The effective cytosolic delivery of a limited number of organic fluorescent probes by peptide vehicles via simple coincubation has been reportedOn page 8, “To the best of our knowledge, this is the first time that a peptide vehicle has been shown to transfer cell-impermeable organic fluorescent probes into live cells with satisfactory efficiency for imaging by simple coincubation.” should be “To the best of our knowledge, this is the first time that a peptide vehicle has been shown to simultaneously transfer up to three different probes into live cells with satisfactory efficiency for imaging by simple coincubation.” to make the statement more accurate.17. However, the live-cell imaging of subcellular structures requires a vehicle not only to deliver probes into live cells but also to deliver them with a sufficient delivery efficiency to obtain adequate signal-to-noise ratios that yield clear images. After coincubation of the cells with dfTAT and the probes , weak fluorescence signals from these probes were detected inside the cells, but they were insufficient for producing clear images of these subcellular structures  that achieved efficient cytosolic delivery of proteins or nucleic acids lost their excellent delivery capability for small-molecule organic fluorescent probes Fig. .” shouldBecause dfTAT .36. Brock, D. J. et al. Efficient cell delivery mediated by lipid-specific endosomal escape of supercharged branched peptides. We apologize for any inconvenience this may have caused.


For example, combination treatment with another substance could increase the effect on tumour tissue. Gemcitabine is a nucleoside analogue that has been shown to sensitise tumour cells to radiation. The aim of this study was to investigate potentially additive or synergistic effects of combining radiation with gemcitabine for treatment of MTC. Nude mice transplanted with patient-derived MTC tumours (GOT2) were divided into groups and treated with radiation and/or gemcitabine. Radiation treatment was given as 177Lu-octreotate or external beam radiotherapy (EBRT). The volume of treated and untreated tumours was followed. The absorbed dose and amount of gemcitabine were chosen to give moderate tumour volume reduction when given as monotherapy to enable detection of increased effects from combination treatment. After follow-up, the mice were killed and tumours were immunohistochemically (IHC) analysed. Overall, the animals that received a combination of EBRT and gemcitabine showed the largest reduction in tumour volume. Monotherapy with EBRT or gemcitabine also resulted in a clear detrimental effect on tumour volume, while the animals that received 177Lu-octreotate monotherapy showed similar response as the untreated animals. The GOT2 tumour was confirmed in the IHC analyses by markers for MTC. The IHC analyses also revealed that the proliferative activity of tumour cells was similar in all tumours, but indicated that fibrotic tissue was more common after EBRT and/or gemcitabine treatment. The results indicate that an additive, or even synergistic, effect may be achieved by combining radiation with gemcitabine for treatment of MTC. Future studies should be performed to evaluate the full potential of combining 177Lu-octreotate with gemcitabine in patients.Patients with medullary thyroid cancer (MTC) are often diagnosed with spread tumour disease and the development of better systemic treatment options for these patients is important. Treatment with the radiolabelled somatostatin analogue  RET proto-oncogene [Medullary thyroid cancer (MTC) accounts for about 1–2% of all thyroid cancers [oncogene . Many paoncogene , 6. Baseoncogene . Therefo131I) is a well-established treatment technique with high response rates [e.g. 111In-octreotide or 177Lu-octreotate, can be achieved in MTC [90Y-octreotide for patients with metastatic MTC was associated with long-term survival benefit [For distant metastases from the more common papillary and follicular thyroid cancer, systemic therapy with radioiodine  with gemcitabine for treatment of MTC. The study was performed in MTC-bearing mice.The aim of this study was to investigate the potentially additive or synergistic effects of combining ionising radiation . Radiolabelling was performed according to the manufacturer’s instructions. Instant thin layer chromatography (ITLC) was used to measure the amount of peptide bound 177Lu, which was determined to be over 99%. All syringes containing 177Lu-octreotate were measured in a well-type ionisation chamber  before and after injection to determine the amount of injected radiopharmaceutical into each animal.RET-driven MTC in Gothenburg. By serially transplanting GOT2 tumours to new generations of mice, we have used this patient-derived xenograft model for studies of MTC for over 15 years. The collection of tumour tissue from the patient was performed during surgery in 2001. At that time, formal ethical approval by an ethics committee was optional according to Swedish law, but was in this case not regarded necessary. The patient provided oral informed consent for the collection according to standard procedure at the time. The consent was verified by two surgeons, one pathologist and one medical physicist. The principles expressed in the Declaration of Helsinki was followed.GOT2 are MTC cells that have been successfully transplanted to nude mice . Originaca. 1x1x1 mm3) were transplanted subcutaneously in the neck of 4–5 weeks old female BALB/c nude mice  under anaesthesia by intraperitoneal (i.p.) injection of Ketaminol® vet.  and Domitor® vet. . An i.p. injection of Antisedan® vet.  was used as antidote to anaesthesia. After about 2 months, tumours appeared close to the transplanted location. Digital callipers were used to measure the tumour length, width, and height. Then, the tumour volume was calculated by assuming an ellipsoidal shape. The experiments were initiated when the tumours reached a volume of about 200–2000 mm3 . All mice were given water and autoclaved food ad libitum. The experiments were approved by the Ethical Committee on Animal Experiments in Gothenburg, Sweden (permit no. 107–15).In this study, small GOT2 tumour tissue samples  were divided into groups of 5–12 mice per group. The tumour size distribution within each group was kept as similar as possible. Three groups were used for combination studies of  animals . The 177177Lu-octreotate  was administered by intravenous injection in the tail vein as a single treatment on day 0. Gemcitabine  was obtained as a powder, which was dissolved in saline solution, and 60 or 125 mg/kg was administered by i.p. injection twice a week for a total of 2.5 weeks starting on day 0. Prior to each injection of gemcitabine, the mice were weighed and the injected volume (0.15–0.20 ml) was adjusted to administer the correct dose to each mouse. No symptoms of toxic side effects were seen for the animals receiving 177Lu-octreotate. However, the initial twice weekly gemcitabine dose of 125 mg/kg for the groups used for combination treatment studies of 177Lu-octreotate and gemcitabine resulted in weight loss for many animals. Therefore, the gemcitabine dose was lowered to 60 mg/kg from day 10 for these animals. Unfortunately, this change did not improve the condition of the animals and this treatment group was only followed for 16 days ( 16 days . For thei.p. injection of Ketaminol® vet. and Domitor® vet.), placed on their side on a tissue-equivalent polystyrene bed and individually irradiated using a Varian linear accelerator with 6 MV photon beam  at a gantry angle of 0 degrees . To obtain a relatively uniform dose distribution in the tumour and to minimise air gaps, tissue-equivalent material was fitted around the mouse and the tumour. The tumour was covered with 15 mm tissue-equivalent material and the centre of the tumour was positioned at isocenter at a depth of about 20 mm (depending on the size of the tumour). A 30x30 mm2 irradiation field was then used to deliver 5 Gy to the tumour. Using this setup, adjacent normal tissues were irradiated to some extent . However, the animals showed no apparent signs of side effects from the EBRT during the follow-up time of the animals.For EBRT, the animals were anaesthetised  formalism [Ã is the time-integrated activity, the product EiYi is the energy emitted per decay for the ith nuclear transition, ϕi is the absorbed fraction and M is the mass of the tissue of interest. Only the contribution from beta particles was considered. Therefore, ΣiEiYi was set to 147.9 keV and the absorbed fraction was set to 1 [177Lu-octreotate in BALB/c nude mice carrying GOT2 tumours, by fitting a mono-exponential curve to the time-activity concentration data [For ormalism :D=Ã∑iEiset to 1 , 31. Theion data .ca. 3–4). In addition, a mouse that met one of the following criteria was killed: 1) the tumour reached a volume corresponding to more than 10% of the total body weight, 2) the body weight was reduced by more than 10%, or 3) the mice showed any signs of lower health status. The mice were killed by cardiac puncture under anaesthesia  and tumours were fixed in formalin for histological studies. The follow-up time for each group can be seen in After start of treatment, the animals were monitored and tumour volume was measured twice a week. For each mouse, the relative tumour volume was defined as the tumour volume at a given point-in-time divided by the tumour volume at day 0 (start of treatment). Each group was followed until most of the tumours in a group had regrown  and Masson’s trichrome (MT) for examination of morphology and fibrosis, respectively.™ FLEX Target Retrieval Solution (high pH) using a PT-Link . The IHC staining was performed using an Autostainer Link using EnVision™ FLEX according to the manufacturer’s instructions (Dako). In each run, positive and negative controls were included. To analyse cell proliferation, a primary antibody for Ki67  was used. To verify MTC origin of the tumours, tumour tissue sections from the untreated control group were stained using antibodies for MTC markers chromogranin A , synaptophysin , and calcitonin . Imaging for figure presentation of MTC markers was performed using a microscope  equipped with a camera .For immunohistochemical (IHC) analysis, tumour sections were placed on glass slides and treated with EnVision2) using an in-house developed MATLAB tool . In brief, tumour tissue was delineated by drawing a region of interest (ROI) around the tumour, excluding the tumour capsule. Thereafter, colour thresholds were applied to segment and remove regions of cracks (background) or folds caused by histological processing, and to segment positively stained tumour tissue. Necrotic tumour regions were easily discernible in the MT images (and verified on H&E sections), which facilitated automatic segmentation of tumour tissue into necrotic or non-necrotic tumour (denoted as viable). By aligning the digital Ki67 and MT sections by semi-automatic image registration (MATLAB control point selection tool), the segmentation mask for viable/necrotic tumour tissue defined from MT staining was applied also to the Ki67 sections. The colour thresholds and corresponding segmentation of tumour tissues were approved by a board-certified pathologist (O.N.). Ki67 was quantified only in viable tumour, whereas MT (blue regions representing collagen) was quantified for the entire tumour section (excluding background and artefacts).Tumour sections stained for Ki67 and MT were digitalised using a digital slide scanner at 40x magnification . Then, quantitative analysis of histological features were performed on these digitalised sections  and with gemcitabine, respectively [mono and RTVcontrol is the mean relative volume in a monotherapy group and in the untreated control group, respectively. Lastly, Fadd was compared with the measured effect in the combination therapy group. The measured effect was assumed to be synergistic, additive or antagonistic if it was larger than, equal to or smaller than Fadd, respectively.The interaction effect between radiation and gemcitabine was analysed using the Bliss independence model . Based oectively . The fra177Lu-octreotate in this study resulted in a mean absorbed dose to tumour of 0.13 Gy. Tumour volume vs. time curves for nude mice carrying the patient-derived MTC model GOT2 treated with 10 MBq 177Lu-octreotate and/or 125 mg/kg gemcitabine  are shown in 3) are provided in The administered activity of 10 MBq 177Lu-octreotate showed similar response as the untreated control animals (p = 0.249 at day 16). Consequently, the animals treated with a combination of both 177Lu-octretoate and gemcitabine showed no statistically significant difference in tumour volume compared with the animals treated with gemcitabine only (p = 0.430 at day 16).Given as monotherapy, gemcitabine resulted in a detrimental effect on tumour growth. At day 13, the relative tumour volume for the animals treated with gemcitabine was 51% of the relative volume for the untreated control animals  are provided in Tumour volume Given as monotherapy, both gemcitabine and EBRT resulted in a clear detrimental effect on tumour growth. Compared with the tumour volume in the untreated control group, the largest effect of the gemcitabine treatment was seen after 20 days when the relative volume was 48% of the relative volume in the untreated group (p<0.001). For the animals treated with EBRT, a clear initial tumour volume reduction was seen. Seven days after treatment, the mean relative tumour volume was 0.97 and reached a minimum after 13 days when the relative volume was 0.57, corresponding to 29% of the relative volume in the untreated group (p<0.001). Thereafter, the tumours started to regrow with a growth rate similar to what was seen in the gemcitabine group and in the untreated group .Overall, the animals that received a combination of EBRT and gemcitabine showed the largest reduction in tumour volume over time of all groups –Fig 2. OThe PFS curves showed a substantially prolonged TTP for the animals treated with EBRT monotherapy, with a median TTP of 27 days . HoweverThe stained tumours in the untreated control group showed a high and specific expression of MTC markers chromogranin A, synaptophysin, and calcitonin, and were morphologically consistent with MTC .The percentage of Ki67-positive tissue (in viable tumour areas) was similar in all groups with an overall median of 64% . The pere.g. repeated or differently scheduled treatment, would probably have resulted in a much higher effect on tumour volume in each of the monotherapy groups.In this study, we have investigated the therapeutic effect of combining ionising radiation with gemcitabine for treatment of medullary thyroid cancer (MTC). Using a patient-derived MTC animal model (GOT2), we have provided results that indicate that an additive, or possibly even synergistic, combinatorial effect could be achieved. Furthermore, given as monotherapy, both gemcitabine and irradiation appeared to have a clear detrimental effect on tumour volume. It should be noted that suboptimal monotherapy doses and treatment protocols were used in order to enable detection of an increased effect from the combination treatment. Higher doses or a more optimal treatment protocol, e.g. 177Lu-octreotate) instead of EBRT. Even though the 177Lu-octreotate monotherapy group showed similar response as the untreated control group in this study, previous data indicate that 177Lu-octreotate treatment is an option for patients with metastatic MTC with high SSTR expression [177Lu-octreotate in GOT2 nude mice has previously been studied [177Lu-octreotate treatment. Furthermore, it is possible that the administered amount of 10 MBq 177Lu-octreotate used in this study was too low (absorbed dose to the GOT2 tumours was only 0.13 Gy). In a similar study, where 177Lu-octreotate was evaluated for treatment of small-intestine NETs in a xenograft animal model (GOT1), a clear effect on tumour volume was seen after administration of 15 MBq 177Lu-octreotate (resulting in 2.7 Gy to the tumour) [177Lu-octreotate monotherapy could have been significantly improved if a higher absorbed dose would have been achieved. Because of the relatively low SSTR-expression in the GOT2 tumours, it would have been difficult to increase the absorbed dose by simply increasing the administered activity, due to tumour SSTR saturation. Unfortunately, there is, to our knowledge, no other MTC animal model available with higher SSTR-expression to better reflect the high uptake seen in some MTC patients; GOT2 is, as far as we know, the model with the highest uptake [For the combination of irradiation and gemcitabine to be of value for patients with metastatic disease, systemic radionuclide therapy is needed  , 37. Sint uptake . Therefoe.g. a primary tumour and its metastases, and in both situations, using more than one drug may be crucial to be able to target different populations of tumour cells and kill all cancer cells in the tumours if some cancer cells are not affected by only one drug. Furthermore, given a patient-to-patient variability, the use of drug combinations give each individual patient a better chance that at least one drug will be effective [Combination therapy offers many potential benefits for patients and can still be warranted even if there is no synergistic, but instead an additive or even antagonistic, effect between the drugs combined . Firstlyffective .177Lu-octreotate and would be interesting for future investigations.The results in this study show that an additive, or possibly even synergistic, effect could be achieved when combining irradiation and gemcitabine for treatment of MTC. A synergistic interaction effect could be explained by the mechanism of action of gemcitabine. By acting as a nucleoside analogue of deoxycytidine, gemcitabine can inhibit DNA synthesis and cause cell death . ThroughThe IHC analyses showed high expression of all MTC markers and a morphology consistent with that of MTC, verifying the MTC origin of the GOT2 tumours. The percentage of viable tumour area positively stained for Ki67 (indicating proliferative activity) was similar in all groups. This can be explained by the fact that the tumours were harvested for histological analysis at the end of follow-up (day 30–58), when all tumours had started to regrow. The amount of MT staining was also similar on a group level. However, some tumours in the treatment groups had a higher percentage of area positively stained for MT compared with untreated tumours. High amounts of positive MT staining indicates high levels of fibrosis, which in turn could be a sign of scarring due to treatment-related effects.2) was used to treat two patients with MTC, no apparent effect was seen [2 on days 1 and 8 of each 21 day cycle [2 can be used, minimizing the side effects while still increasing the therapeutic effect on tumour tissue. In a study on locally advanced head and neck cancer, gemcitabine appeared to be a strong radiosensitiser at doses much lower than those required for cytotoxic effects [2 for humans [2. Therefore, we believe that patients with MTC could benefit from the suggested combination therapy, while still being able to limit the dose of gemcitabine to keep side effects at an acceptable level.The experience of using gemcitabine as a treatment for patients with MTC is limited. In a study where gemcitabine  for MTC is relatively limited but larger than that of gemcitabine for MTC [177Lu-octreotate has become more commonly used than 90Y-octreotide and in 2017 and 2018, respectively, the European Medicines Agency (EMA) and the FDA, approved the use of 177Lu-octreotate for the treatment of gastroenteropancreatic NETs.The clinical experience of PRRT  and/or 125 mg/kg gemcitabine twice weekly (Gem high), and (B) 5 Gy external beam radiotherapy (EBRT) and/or 60 mg/kg gemcitabine twice weekly (Gem low). Error bars show SEM.GOT2-carrying nude mice were treated with (A) 10 MBq (TIF)Click here for additional data file.


Mini-grants have been used to stimulate multisector collaboration in support of public health initiatives by funding non-traditional partners, such as economic development organizations. Such mini-grants have the potential to increase access to healthy foods and places for physical activity through built environment change, especially in small and rural towns in the United States. Although a promising practice, few mini-grant evaluations have been done. Therefore, our purpose was to conduct an Evaluability Assessment (EA), which is a process that can help promising programs that lack evidence advance toward full-scale evaluation. Specifically, we conducted an Evaluability Assessment of a statewide mini-grant program, called “Growing Healthy Communities” (GHC), to determine if this program was ready for evaluation and identify any changes needed for future implementation and evaluation that could also inform similar programs.Telephone interviews with directors of six past mini-grant recipient organizations were conducted to assess implementation and evaluability. The six interviews were split equally among agencies receiving funding for food-oriented projects and physical activity-oriented projects. Within- and cross-case thematic analyses of interview transcripts were conducted.Organizational capacity was a universal theme, reflecting other key themes (described in detail in the manuscript) that affected program implementation and evaluation, including collaboration, limited time and measurement integration. Conclusions. The EA process provided pilot data that suggest that other state, regional, and national funders should provide centralized assistance for data collection and evaluation from the outset of a mini-grant award program.The online version of this article (10.1186/s12889-019-7156-8) contains supplementary material, which is available to authorized users. Healthy People 2010. The purpose of mini-grants  is to support government entities, non-profit organizations, small businesses, or other interest groups to stimulate quick, positive changes in communities. These are possible to implement when awarded to trusted, community-based organizations that can facilitate multi-disciplinary solutions to problems because of strong existing collaborations, communication, and engagement among collaborators  focus towards healthy communities, healthy life style development, is a really, really good idea. And a good way to go for West Virginia in particular, but in terms of economic development, it is what people want. limited time and measurement integration together were identified as barriers. Regarding time, all of the local MSWV directors expressed concern with GHC mini-grant funding timelines, project planning, and/or the mistiming of the grant cycle with food growing or construction seasons. To overcome this barrier, most sites relied on other non-profits or the local municipality for funding or personnel to extend the GHC funding cycle. This is represented in the following quote:I think the [GHC funding] time frame should be a little longer, because in [site county name] we really haven’t started farmers market until June. That is because of the weather. So, I have to, you know, kind of figure out how to do that, but it will be carried out throughout the whole summer. Even though it will end … we will be doing it throughout the summer. (Food Site 3)Parallel to this strength, measurement integration was an area in need of improvement. Although important, health outcome evaluation was not integrated into the grant timeline or trainings for the local organizations that were not traditionally focused on measuring health outcomes. The combined effect of funding time constraints and the organizational focus of Main Streets on economic development was that capturing outcome indicators, especially health-related ones, became secondary or was neglected because action steps for project implementation took center stage. This was not due to a lack of organizational willingness. Rather, as one interviewee noted:“I don’t know whether we see that part we haven’t really got into it because I am not an expert on the health issue of it, we are looking at it as an economic driver…” (Food Site 1)Of great importance to evaluating these mini-grants, all of the local MSWV directors expressed that organizational capacity. The sites’ perseverance to handle tight funding deadlines with limited organizational capacity is a testament to their passion for their communities. However, this passion can lead to personnel “burnout,” which was interlaced throughout the interviews. This suggests that MSWV programs in rural communities, especially those with only part-time staff, are tenuous and that components outside of sites’ primary focus, such as health outcome evaluation, tend to be neglected. Thus, there is a need for centralized resources and technical assistance to enhance organizational capacity as illustrated in the following quote:These cross-case themes highlight a unifying concept of MSWV sites’ We think that this [GHC] is amazing. To have these plans, to have these projects, and move it down the road separately. We were funded for this resource to kind a head the charge on, but this is not here yet [planned activities] and the grant is on a, you know, very short span or time-frame. So, we’re scrambling to have capacity and meet the deadlines while competing with our day jobs. It’s another project and, uh, so having all of that in the line. It would be great if we had this resource on board already … (Food Site 3)after the grant funding period of 4–6 months which was almost exclusively focused on environment changes .The EA of the GHC mini-grant funding program accomplished the common elements of frequently used EA models , 20, 21,The second purpose of this study was to inform the implementation and evaluation of this and other programs. Due to the factors noted in the previous paragraph, any evaluation of GHC or similar mini-grants should focus on process evaluation to assess activities accomplished during the grant period, such as linear feet of trail constructed or square feet of community garden plots built. There will be a delayed effect on behavioral outcomes, such as physical activity and fruit and vegetable consumption, which could be measured in subsequent years. Critical, however, is capturing baseline outcome measures at the inception of the grant against which subsequent measures could be assessed.Other community development entities such as MSA or the Federal Reserve Bank , when coDespite important results, the findings should be viewed in light of limitations in the data collection and analysis methods. First, the interview responses may not capture the full extent of the requested information. Therefore, the discussion may be limited with respect to forming a fully comprehensive set of codes and themes for each MSWV site. Though applicable to MSWV, our findings may not be applicable in communities dissimilar in size or sociodemographic characteristics or to MS organizations of different staff size or structure. Findings should be interpreted with caution, as responses may be limited in scope and/or applicability based on the subjective methods used to identify the MSWV sites interviewed. However, based on the consistency found in themes and codes across different interviews, the results were deemed reliable and usable.As Schmidt  noted, mOther mini-grant funding organizations could overcome this critical barrier by dedicating resources from the moment a Call for Proposals is released to facilitate collaboration with evaluation experts to help recipients plan for, implement, and conduct outcome evaluation data collection from the outset of a grant award. Though limited, the recent public health mini-grant literature highlights the use of a central entity to evaluate the effectiveness and cost-effectiveness of multi-site, community-based mini-grant programs –6, 32. IAdditional file 1:Growing Healthy Communities Mini-grant Recipient Interview Script. (DOCX 18 kb)


The following funding information and acknowledgment should have appeared in the article.Volume 9, issue 2, e00626-17, 2018, Artur Matysik was supported by the National Medical Research Council under its Clinical Basic Research Grant (NMRC/CBRG/0086/2015).


Ex-situ normothermic machine perfusion (NMP) of transplant kidneys allows assessment of kidney quality and targeted intervention to initiate repair processes prior to transplantation. Mesenchymal stromal cells (MSC) have been shown to possess the capacity to stimulate kidney repair. Therefore, the combination of NMP and MSC therapy offers potential to repair transplant kidneys. It is however unknown how NMP conditions affect MSC. In this study the effect of NMP perfusion fluid on survival, metabolism and function of thawed cryopreserved human (h)MSC and porcine (p)MSC in suspension conditions was studied. Suspension conditions reduced the viability of pMSC by 40% in both perfusion fluid and culture medium. Viability of hMSC was reduced by suspension conditions by 15% in perfusion fluid, whilst no differences were found in survival in culture medium. Under adherent conditions, survival of the cells was not affected by perfusion fluid. The perfusion fluid did not affect survival of fresh MSC in suspension compared to the control culture medium. The freeze-thawing process impaired the survival of hMSC; 95% survival of fresh hMSC compared to 70% survival of thawed hMSC. Moreover, thawed MSC showed increased levels of reactive oxygen species, which indicates elevated levels of oxidative stress, and reduced mitochondrial activity, which implies reduced metabolism. The adherence of pMSC and hMSC to endothelial cells was reduced after the thawing process, effect which was particularly profound in in the perfusion fluid. To summarize, we observed that conditions required for machine perfusion are influencing the behavior of MSC. The freeze-thawing process reduces survival and metabolism and increases oxidative stress, and diminishes their ability to adhere to endothelial cells. In addition, we found that hMSC and pMSC behaved differently, which has to be taken into consideration when translating results from animal experiments to clinical studies. As the outcome of kidney transplantation has improved, the demand for kidney transplantation has increased and the donor organ pool to date is too small to supply the current need for transplant organs. The shortage in available donor kidneys  has led Currently, several techniques are being employed to improve the quality of expanded criteria kidneys and discarded kidneys to make them suitable for transplantation, including machine perfusion. Hypothermic machine perfusion of donor kidneys implies connection of the organ to a pump that perfuses the organ with a solution that provides the required components needed to maintain viability while also removing waste products released as a result of the metabolism and perfusion injury of the organ.A more physiological way to assess viability of donor organs is continuous perfusion at normothermic temperature at 37°C with proper oxygenation and in the presence of necessary nutrients. A few years ago, the clinical feasibility and safety of 1 h normothermic machine perfusion (NMP) was demonstrated . Other gin vitro attachment to plastic, expression of several cell surface markers including CD29, CD44, CD90, the absence of endothelial and hematopoietic markers and the capacity to differentiate into cell types of mesodermal origin  are multipotent cells which are found in adult tissues where they support function and repair . The Intl origin . MSC prol origin , 12. Moriologies .ex vivo stand-alone circuit may overcome this dilemma. The idea to combine NMP and MSC therapy has generated an interest in the area of kidney transplantation  administered, which inevitably leads to accumulation in the lungs and poor delivery to target organs , 15. Obvantation –18. It iin vitro and administered directly from the culture flask to laboratory animals when the cells are ready for infusion. In the human setting, large numbers of MSC are needed to treat a patient and cells are often produced at locations distant from the place of administration. This requires storage of cells in a frozen state and infusion following a delicate thawing process  and from human (hMSC) origin.n = 5) that became available during kidney donation procedures after obtaining written informed consent as approved by the Medical Ethical Committee of the Erasmus University Medical Center Rotterdam (MEC-2006-190). pMSC were isolated from subcutaneous adipose tissue (n = 5) collected from male pigs, which were subjected to surgery for teaching purposes, as a waste product. hMSC and pMSC were isolated as described previously   supplemented with penicillin (100 IU/ml), streptomycin (100 mg/ml) , 2 mM L-glutamine (Lonza) and 15% fetal bovine serum . HUVEC were cultured in endothelial growth medium 2 . PAOEC were cultured in porcine endothelial cell media . MSC were used at passage 3-6, HUVEC were used at passage 4-8 and PAOEC at passage 3-6.The perfusion fluid was a RBC-based solution with albumin as colloid adapted from NMP experiments used by several groups and allowing stable NMP of kidneys , 26, 27.MSC were trypsinized from the culture flasks at 90% confluency or thawed after cryopreservation and re-suspended either in complete culture medium or perfusion fluid at a concentration of 500,000 MSC/ml. MSC were incubated in perfusion fluid in polypropylene tubes to avoid attachment of MSC to plastic. After 30 min or 2 h in suspension, MSC were submitted to a RBC lysis process to remove the large amount of RBC present in perfusion fluid. MSC incubated in suspension with culture medium were also subjected to RBC lysis to treat both groups in the same way. Briefly, 3 ml of red blood cell lysis buffer  was added to MSC and incubated for 20 min at room temperature (RT). MSC were then washed with PBS and stained with Annexin-V (PE) and ViaProbe (PercP) to assess the number of early and late apoptotic cells. Perfusion fluid was also added to attached MSC and incubated for the same time and then trypsinized and stained as mentioned. Cells were analyzed by flow cytometry  and data were analyzed using Kaluza Analysis 1.5a .MSC metabolic activity was measured by a colorimetric assay based on the reduction of XTT  by nicotinamide adenine dinucleotide (NADH) . The reagent is reduced by NADH produced during mitochondrial metabolism which results in a color change of the XTT reagent detectable by a spectrophotometer. The concentration of the reagent is measured by absorbance measured at a wavelength of 450–500 nm. This assay was performed on MSC that were in suspension for 30 and 120 min in perfusion fluid or culture medium and on attached MSC. In addition, oxidative stress of MSC was measured using CellRox reagent (ThermoFisher) according to the manufacturer's manual. CellRox is oxidized by reactive oxygen species (ROS) and emits a fluorescent signal that is measured by flow cytometry.In order to assess the effects of perfusion fluid on cell proliferation, MSC were fluorescently labeled with carboxyfluorescein succinimidyl ester (CFSE) (ThermoFisher) which was added to the cells at a concentration of 5 mM. The cells were then incubated at 37°C for 15 min in the dark. Staining was stopped by the addition of twice the volume of FBS-containing culture medium and incubated for 5 min at RT. Cells were washed and exposed to perfusion fluid for 30 and 120 min at 37°C. After incubation with perfusion fluid, MSC were seeded in a 6-well-plate with regular culture medium and proliferation was measured at 24, 48, and 72 h by flow cytometry.A custom-made Luminex Multiplex Assay  was designed to measure the release of the following cytokines and growth factors by hMSC after incubation with perfusion fluid:Angiopoietin-1 (ANG-1), Angiopoietin-2 (ANG-2), epidermal growth factor (EGF), hepatocyte growth factor (HGF), interferon-gamma (IFN-γ), interleukin 10 (IL-10), interleukin 6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), programmed death ligand 1 (PD-L1), platelet derived growth factor AA (PDGFAA), Thrombospondin-2, tissue inhibitor of metalloproteases 1 (TIMP-1), tumor necrosis factor alpha (TNF-α), soluble tumor necrosis factor receptor 1 (sTNF-RI) and vascular endothelial growth factor (VEGF).Subconfluent (90%) cultures of hMSC were washed and cells were incubated in perfusion fluid for 30 and 120 min. Wells were washed again and culture medium was added. After 24 h supernatants were retrieved and the array was performed according to the manufacturer's protocol. Fluorescence was measured on a Luminex 100/200 system  using Xponent software. Due to the lack of pig-specific reagents this assay was only performed in with hMSC.Confluent monolayers of endothelial cells were cultured in 24-well-plates. Culture medium was replaced by either a 1:1 mix of MSC and endothelial cell (EC) medium or perfusion fluid. 200,000 MSC were fluorescently-labeled with PKH26 (Sigma) and added to each well. After 10, 30, 60, 120, and 240 min supernatant was removed and wells were washed to eliminate all non-adherent MSC. Attached cells were trypsinized and analyzed by flow cytometry. Fluorescent signal detected by flow cytometry allowed the determination of the percentage of MSC attached.To examine the survival of MSC, fresh and cryopreserved MSC were incubated in suspension in perfusion fluid. Survival rates of freshly cultured and thawed pMSC were <40% after 30 min and decreased to 30% after 2 h in culture medium. Perfusion fluid had no negative impact on pMSC survival compared to culture medium except on thawed MSC after 30 min in perfusion . hMSC weTo assess the function of surviving MSC in perfusion fluid, the capacity of MSC to adhere to EC was tested. In culture medium, fresh and thawed pMSC started to attach to PAOEC already 10 min after seeding. After 4 h, almost 100% of freshly cultured pMSC were attached to PAOEC while only 80% of thawed pMSC adhered . PerfusiThe reduced adhesion of thawed MSC to EC could be explained by higher oxidative stress of MSC after the thawing process. The accumulation of ROS derived from the thawing process might induce damage in thawed MSC. An increased production of ROS by thawed pMSC and hMSC was observed at 30 min and 2 h after thawing both in medium and perfusion fluid . Thawed ROS production leads to mitochondrial damage which results in reduced metabolic activity of cells. It is possible that MSC survive in suspension but are less metabolically active, which may explain the different capacity of MSC to adhere to EC after thawing or in perfusion fluid. To determine the effect of perfusion fluid and the freeze-thawing procedure on MSC metabolic activity, the conversion of XTT to its reduced state by mitochondria was measured in MSC. pMSC mitochondrial activity showed to be very stable and was not affected by cryopreservation . HoweverProliferation of MSC was measured after initial incubation in suspension in perfusion fluid or medium for 30 min and 120 min, followed by 72 h culture in culture medium. Incubation in perfusion fluid did not affect pMSC proliferation . FreshlyMSC are adherent tissue cells and the suspension conditions in the previous experiments may affect their phenotype and function. To examine how adherent MSC respond to perfusion fluid, morphology, survival, metabolic activity and proliferation of attached MSC were studied. The morphology of MSC in culture was not affected after 30 min or 2 h culture in perfusion fluid . AdherenThe secretion of growth factors and cytokines is an important mechanism of action of MSC. To examine whether perfusion fluid would preserve the secretory profile of adherent hMSC and furthermore whether perfusion fluid induced an inflammatory response in hMSC, hMSC were incubated in perfusion fluid for 30 min or 2 h and growth factor and cytokine secretion was analyzed. We observed that the secretion of the angiogenic factors VEGF, PDGF, ANG-1, HGF and Thr2 was unaffected in perfusion fluid . Inflammex vivo NMP of kidneys on MSC of both pig and human origin. Our work involved the use of cryopreserved MSC for logistic purposes, suspension conditions to deliver MSC via the renal artery using NMP and the use of an RBC-based perfusion fluid which may affect the survival and function of MSC. In order to mimic the conditions of a potential novel MSC therapy to stimulate the repair of injured kidneys while these are connected to NMP, MSC were thawed after cryopreservation and incubated in perfusion fluid in suspension. Actual infusion of MSC using NMP was not carried out as the purpose of the study was to assess the individual effect of each of the aforementioned conditions separately. In case that NMP conditions did not support MSC survival and function, future planned experimentation in the NMP setup could have been stopped, reducing economic and time costs. Nevertheless, the results of this study allow to take the next step and study MSC infusion through an NMP system, which is already planned to be carried out.In the present work we have assessed the effect of a period of incubation in a perfusion fluid required for robust longer term In the field of clinical MSC therapy, temporary cryopreservation and thawing along with vehicle solutions to deliver the therapy are important factors that can determine the success of MSC therapy , 28–30. in vitro under flow conditions as previously shown, especially when endothelial cells have been treated to recreate an inflammatory environment. Attachment of MSC to endothelial cells was shown to be reduced under flow compared to static conditions (ex vivo NMP will depend, among other factors, upon the composition of the perfusion fluid. A recent study perfused kidneys for 24 h using NMP. They infused MSC at different concentrations however, after 24 h they found 95% of infused MSC back in their perfusion solution (The bespoke NMP perfusion fluid used in this study supports the survival of pig and human MSC. However, the function and metabolism of these cells are affected by the suspension conditions MSC were kept in. Specifically, perfusion fluid inhibited the adhesion of pMSC in suspension to PAOEC and hMSC to HUVEC. MSC have the capacity to adhere to endothelial cells nditions . Being inditions  and the nditions , 35. Thesolution , which iHuman and porcine MSC showed a negative response at several levels to cryopreservation, thawing and re-suspension in perfusion fluid. The effect of cryopreservation and thawing of hMSC has been a concern for the community as it can decrease the presumed efficacy of MSC therapy , 30, 37.In vivo, MSC are tissue resident cells and are found in perivascular niches in a wide variety of tissues (In vitro, MSC are strictly grown as adherent cells, and for this reason MSC are cultured allowing plastic adherence to expand them ( tissues –43. In vand them . We demoand them . These rand them –46. We hCryopreservation, thawing and suspension of MSC are inevitable conditions to infuse GMP-grade MSC to kidney transplants via NMP. These conditions are necessary to bridge differences in time and location between MSC preparation and NMP. The disadvantageous effect of these conditions on MSC has to be taken into account for the evaluation of the suitability of MSC therapy for NMP. In order to improve the viability of MSC in NMP conditions, the composition of the perfusion fluid may be adapted to provide better support for MSC survival. Another possibility is to recover MSC after thawing under favorable conditions to improve the resistance of MSC to NMP. An alternative would be to take the loss of MSC under NMP conditions into account and use higher numbers of cells, although the effect of administering large numbers of non-viable MSC to the kidney is uncertain.in vitro studies with porcine cells toward the behavior of human cells. A safe translation from swine pre-clinical models to clinical studies is challenging when results are not reproducible between species.Pre-clinical work in the field of transplantation is often performed using porcine models to better understand the possible behaviors of new therapies in patients. Therefore, we investigated MSC of porcine and human origin in order to determine if results can be translated. The response to the thawing process as well as to suspension conditions and perfusion fluid was quite different between MSC from both origins for the parameters studied. We are aware that differences could be related not only to species but also to age and gender, as well as site of adipose tissue harvesting from the donor. However, our results indicate that caution should be taken when interpreting Summarizing, NMP conditions will affect MSC but show sufficient support of their function and survival to consider MSC administration through NMP as a viable option in pursuit of a potentially beneficial cell therapy for the regeneration of injured organs. After these essential preliminary experiments, further study is now underway to determine the best way of joining these two exciting techniques in an optimal manner.BJ, CB, ME, RP, and MH: conception of the study; JS, AM, and MH: designed the experiments; JS: performed the experiments and the analysis of the data; JS, AM, and MH: interpreted the data. All authors contributed to writing and reviewing the manuscript.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.


Twitter has become an important platform for geo-spatial analyses, providing high-volume spatial data on a wide variety of social processes. Understanding the relationship between population density and Twitter activity is therefore of key importance. This study reports a systematic relationship between population density and Twitter use. Number of tweets, number of users and population per unit area are related by power law functions with exponents greater than one. These relations are consistent with each other and hold across a range of spatial scales. This implies that population density can accurately predict Twitter activity, but importantly, it also implies that correct predictions are not given by a naive linear scaling analysis. The observed super-linearity has implications for any spatial analyses performed with Twitter data and is important for understanding the relationship between Twitter use and demographics. For example, the robustness of this relationship means that we can identify ‘anomalous’ geographic areas that deviate from the observed trend, identifying several towns with high/low usage relative to expectation; using the scaling relationship we are able to show that these anomalies are not caused by age structure, as has been previously proposed. Proper consideration of this scaling relationship will improve robustness in future geo-spatial studies using Twitter. Twitter is an important source of data for a variety of academic fields, including many that explore relationships between social behaviours (manifested on Twitter) and spatial/geographic features (identified by geo-location of Twitter users and content). Yet there remains a lack of understanding around the key underpinning relationship between Twitter usage and population density, which is a primary determinant of many social phenomena.In this work we show there is a power law relationship between population density and social media activity which is consistent across a range of spatial scales. This has important implications for any large-scale spatial analyses of social media data, e.g. normalising background activity for event detection, or accounting for common co-variates in demographic studies. We show that controlling for this non-linear relationship destroys the apparent correlation, for example, between proportion of people under 35 and social media activity. Finally, in the course of our analysis we find that GPS-tagged tweets originate from a highly atypical set of sources when compared to other tweets. Given the frequency with which geo-tagged tweets are used as a basis for spatial analysis in the literature, this is a significant finding by itself.Background and then outline our approach to collecting and aggregating tweets in Section Materials and methods. Our main results related to scaling laws are shown in Figs Checking the consistency relation verifies the consistency of the scaling relationships across a range of measurement scales and their self consistency according to Finding Anomalies explores when and why these scaling relationships fail to hold. Our second main result is in this section, which explores the local anomalies that remain in observed Twitter activity after accounting for the effects of population density. We then show that the density of young people, argued elsewhere to be a factor in the amount of Twitter activity, has no additional effect once the common correlation of both variables with population density is removed. We conclude in the Discussion section where we explore the significance of our findings and the potential for future work.The outline of this paper is as follows. We begin with a short survey of related work in Twitter is a social media platform whose open access API makes it very popular with researchers. Twitter data has been used extensively in the disaster response literature to study earthquakes , wildfirdensity, by taking county area into consideration, as we do in this work.Studies of demographic bias find users in cities and urban areas over-represented in collections of Twitter data. Mislove et. al.  found “…It is the aim of this paper to determine how Twitter usage is related to population density. In particular, we are motivated by work of Takhteyev et. al.  and StepThe search for systematic effects with population density and urban bias of Twitter usage relates closely to work on scaling laws in cities  21] [22 [2221]  and pattcity is crucial and simple changes can cause apparent scaling relationships to vanish. By using population density we avoid the issue of defining a city and can use arbitrary sampling areas. This allows us to vary the spatial sampling distribution, so that our results do not depend on particular definitions of city or town, as well as to vary the size of the sampling areas, showing us the range of spatial scales where our results hold. This makes our results more robust, given they don’t depend on the specifics of the sampling procedure, and reliable, given we can see where they apply and where they break down.Social media activity, here Twitter accounts and tweets, is the output of a social network. Thus, like patents, wages or other social outputs, according to the scaling law literature we would expect their number to grow super-linearly with population. The work of  and 28]28] who sThe literature on the science of cities also provides important guidance on how to deal with non-linearity. The work of Bettencourt  and otheWe collected tweets from the South-West UK by passing a geographical bounding box with longitude from -5.8 to -1.2 and latitude from 49.9 to 52.2 to the Twitter Streaming API. All data was gathered in compliance with all applicable Twitter API policies and terms of use  and in aThe collection ran from 11/4/2016 to 10/4/2018 with some gaps in the collection (due to machine downtime) covering all of December 2016 and all of March 2017. The Twitter Streaming API is free of cost and easy to access compared to the full Twitter datastream. This makes it the usual choice for research purposes. The Streaming API is rate limited, only allowing us to collect a restricted number of tweets, at most 1% of all the tweets on Twitter . Since wWe use two metadata properties of each tweet to locate it: geo-tags and place-tags. Geo-tags are GPS co-ordinates added by the user’s mobile device that give a precise location for a particular tweet. According to the Twitter API documentation : “PlacesWe will show geo-tagged tweets are a minority (as other studies have found e.g. ) and havX × X grid. Since our original bounding box is not square, these grid cells are rectangular. As some grid boxes are over coastal areas, not all grid boxes cover the same land area. Let A be the land area of a grid box. To calculate A the shape-files for the UK were obtained from the GADM database of Global Administrative Areas  [28 [2827]  [26 [26α, β We do not see these effects when using geo-tagged tweets and have shown that geo-tagged tweets are very different in nature to place-tagged tweets. Geo-tagged tweets often originate from automated accounts or via shares from other social media platforms. Due to the large number of studies utilizing geo-tagged tweets this is an important consideration for future research.Our study builds on previous studies which ask about sampling biases in Twitter data. Those studies consistently find a bias towards urban areas, i.e. areas with large population density. We have demonstrated that this systematic bias can be modelled with a simple power law function. The number of tweets grows super-linearly with number of users, in line with work on other scaling laws in cities. Since Twitter is such a rich and useful data source we believe this study will enable better, more representative, sampling of tweets and a better interpretation of research based on Twitter.T = Aρα might be interpreted in terms of the effect of population density on interaction frequency, which is harder to infer from a similar relation with absolute population size. Contrast 10000 people spread across a large rural area with 10000 people in a small town. The population size is identical, but the likelihood of interaction is much greater in the town, which by the argument above might lead to more social media activity.We also believe that social media data can be a powerful test of models developed to explain super-linear scaling e.g.  since thT) and population (ρ) is key. If a linear relationship was assumed we would under-correct for the dependence of T on ρ and discover a spurious relationship between the variables.Finally, in section we have shown that while the ‘youth’ of an area (number of 18 to 35 year-olds) is correlated with high Twitter usage, when we correct for the common association of both variables with population density and correlate the residuals, the association vanishes. This is an important lesson for other demographic studies and here the non-linear relationship between tweets Click here for additional data file.S2 Text(PDF)Click here for additional data file.


This previously unidentified fluid focusing mechanism bridges the gap between observations and established conceptual models for overcoming and destroying assumed impermeable barriers. This mechanism therefore has a profound impact on assessing the evolution of leakage pathways in natural gas emissions, for reliable risk assessment for long-term subsurface waste storage, or CO2 sequestration.Ubiquitous observations of channelised fluid flow in the form of pipes or chimney-like features in sedimentary sequences provide strong evidence for significant transient permeability-generation in the subsurface. Understanding the mechanisms and dynamics for spontaneous flow localisation into fluid conductive chimneys is vital for natural fluid migration and anthropogenic fluid and gas operations, and in waste sequestration. Yet no model exists that can predict how, when, or where these conduits form. Here we propose a physical mechanism and show that pipes and chimneys can form spontaneously through hydro-mechanical coupling between fluid flow and solid deformation. By resolving both fluid flow and shear deformation of the matrix in three dimensions, we predict fluid flux and matrix stress distribution over time. The pipes constitute efficient fluid pathways with permeability enhancement exceeding three orders of magnitude. We find that in essentially impermeable shale (10 In the shallow subsurface, distinct features are observed16 of vertical chimneys associated with localised continuous or episodic buoyancy-driven fluid flow. These vertical fluid migration pathways are located in most of the studied sedimentary basins. As these regions are of economic interest, they are widely screened with geophysical methods, which provide excellent data that clearly exhibit fluid migration pathways in three dimensions17. These features are particularly recognisable by their specific signature on seismic cross-sections  Vertical chimneys occur in various sedimentary basin lithologies21 and appear unaffected by rock composition, refuting chemical reactions as the main formation mechanism. (2) The vertical chimneys develop through existing formations and are not influenced by sedimentary layering or structural features such as faults22. These observations also suggest that they mainly do not form by reactivation of pre-existing structures, but are the outcome of a self-sustaining dynamic process independent of the inherited geological setting. However, pipes or chimneys may depart from fault planes or root at a similar stratigraphic level. In addition, we do not exclude chemical reactions and hydro-chemo-mechanical coupling to contribute to the generation and propagation of pore fluids25. Since chimney formation cannot be inferred from static models nor geological history, there is a need to develop predictive models to resolve their spontaneous formation and to better constrain the parameters that govern their propagation.Fluid and melt expulsion from porous rocks occurs at many scales on Earth, from the mantle to the shallow subsurfaceons Fig. , while pons Fig.  are theions Fig. . These f26. We use a continuum mechanics model to compute the porous matrix deformation using a non-linear bulk and shear rheology. Resolving the mechanics is required to calculate the total pressure, strain rates and corresponding stresses, which implies evaluating the entire deviatoric stress tensor. We couple the mechanical solver with a Darcy flow solver and calculate the fluid fluxes using the non-linear Carman-Kozeny relation27 for dynamic permeability. We streamline a supercomputing approach to resolve the coupled fluid-rock interactions in high resolution28. This hydro-mechanical model permits us to predict high-permeability chimney formation and propagation, and the stress distribution in the deforming poro-visco-elastic matrix in three dimensions  chimneys from a fluid-enriched source region in three dimensions Fig. , which crix Fig. . Variatientclass1pt{minima−19 m2, representative of typical clay-rich sealing sequences37, we predict that the formation of high-permeability chimneys leads to maximal flow rates up to 0.1 m/yr (mean flow rates of about 0.01 m/yr) through a 1 m2 horizontal cross-section of clay-rich shale . We utilised a high-resolution numerical grid of 500 × 500 × 1,000 grid points in x, y and z direction, respectively, required to accurately resolve chimney formation and propagation.26 to model the formation and the evolution of high-porosity chimneys over time as a natural outcome of the coupling of fluid flow to the deformation of a viscous porous matrix. The mass balances for fluid and solid phases, assuming constant fluid and solid densities, were:27 defines the porosity 1 that quantifies the ratio of compaction We employed a system of two-phase equationsThe hydro-mechanical simulation initial configuration consists of a rectangular box with a dimensionless extent of 30 × 30 × 60 in the x, y  and z (depth) directions, respectively. The initial porosity follows an anisotropic Gaussian random-field distribution, with a standard deviation equal to 1, and correlation lengths of 5, 5, 1 in the x, y and z directions, respectively. Further, a high-porosity cylindrical ellipse is located at the first ¼ of the domain height and represents a fluid-rich source region or reservoir. Permeability values within the source region were nine times higher compared to normalised background values of 1  libraries to parallelise the application on distributed memory machines and supercomputers. We calculated the presented high-resolution numerical results in a five-day run on 128 GPUs (Nvidia GTX Titan X Maxwell) in parallel on the octopus supercomputer, in-house designed for such high-performance computations, hosted by the Swiss Geocomputing Centre, Institute of Earth Sciences, University of Lausanne, Switzerland.We reached an implicit solution of the coupled set of equations using an iterative pseudo-transient relaxation approach. Acceleration of the residual convergence rates was achieved via a problem-specific damping strategy51. We used three independent scales:entclass1pt{minimaSupplementary Information guideMovie 3Movie 4Movie 5


Traumatic injuries pose a global health problem and account for about 10% global burden of disease. Among injured patients, the major cause of potentially preventable death is uncontrolled post-traumatic hemorrhage.This review discusses the role of prehospital trauma care in low-resource/remote settings, goals, principles and evolving strategies of fluid resuscitation, ideal resuscitation fluid, and post-resuscitation fluid management. Management of fluid resuscitation in few special groups is also discussed.Prehospital trauma care systems reduce mortality in low-resource/remote settings. Delayed resuscitation seems a better option when transport time to definitive care is shorter whereas goal-directed resuscitation with low-volume crystalloid seems a better option if transport time is longer. Few general recommendations regarding the choice of fluid are provided. Adhering to evidence-based clinical practice guidelines and local modifications based on patient population, available resources, and expertise will improve patient outcomes. Traumatic injuries account for nearly 10% of the global burden of disease . In trauThis review discusses the role of prehospital trauma care (PTC) in low-resource/remote settings; goals, principles and evolving strategies of fluid resuscitation, particularly those before blood products are available; and post-resuscitation fluid management. Management of fluid resuscitation in some special groups is also discussed.PTC aims at the stoppage of bleeding to minimize further blood loss, initial resuscitation to maintain mental status and peripheral pulses, and fast transportation to hospital for definitive treatment . PrehospThe goals of fluid resuscitation include controlling bleeding, restoring lost blood volume, and regaining tissue perfusion and organ function. Different target systolic blood pressure (SBP) values may be considered for different traumas: 60–70 mmHg for penetrating trauma; 80–90 mmHg for blunt trauma without traumatic brain injury (TBI); 100–110 mmHg for blunt trauma with TBI .Fluid administration is beneficial only if it increases the stroke volume (SV) and thereby, the cardiac output. Patients are considered fluid responsive if SV increases by at least 10% after a fluid challenge of 500 mL of crystalloid . Pulse pResuscitation strategies are based on volume, rate, and time of fluid administration. Earlier, immediate aggressive fluid resuscitation in trauma patients was the standard approach to restore circulating volume and maintain organ perfusion. However, it may dislodge soft clots and cause dilutional coagulopathy thereby increasing hemorrhage and mortality . TreatinTwo strategies were proposed to avoid clot disruption and dilutional coagulopathy: delayed resuscitation strategy where fluid is given after bleeding is controlled and permissive hypotension strategy, where fluid is given to increase SBP without reaching normotension. In penetrating trauma patients with hypotension , delayed resuscitation shows better survival rates compared to immediate resuscitation . IncreasThe low volume fluid resuscitation during permissive hypotension maintains low tissue perfusion but is adequate for short periods. Permissive hypotension is achieved by goal-directed resuscitation [SBP/mean arterial pressure (MAP) is targeted based on patient physiology] or controlled resuscitation (predetermined fixed rates are infused such that normotension is not achieved) , 14. In Permissive hypotension is associated with decreased blood loss, intra-abdominal bleeding, risk of intra-abdominal hypertension, acidemia, hemodilution, thrombocytopenia, coagulopathy, apoptotic cell death, tissue injury, sepsis, volumes of crystalloid administration needed, and blood product utilization, and improved organ perfusion and survival , 15. HowInternational guidelines recommend restrictive volume replacement approach to achieve target blood pressure (BP) until bleeding is controlled . PenetraMortality is increased in patients with head injury and low cerebral perfusion pressures . Small aWhen PTT to definitive care is shorter < 10–15 min) and patients are well-selected, delayed resuscitation seems a good option , 14. Adv min and In severely injured patients, the lethal triad of hypothermia, acidosis, and coagulopathy exacerbates hemorrhage. Damage control resuscitation combats this and comprises of permissive hypotension, hemostatic resuscitation, and damage control surgery (DCS).Permissive hypotension, as discussed, prevents further bleeding from recently clotted vessels. Hemostatic resuscitation involves early use of blood and blood products to minimize coagulopathy, prevent dilutional coagulopathy, and improve survival , 21. It DCS restores physiology instead of providing definitive anatomical repair. It consists of bleeding control, decontamination, quick body cavity closure to rewarm the patient, and planned re-operation for definitive repair .Though crystalloids and colloids are widely used for fluid resuscitation, the ideal choice of fluid is debated. Hypotonic fluids do not stay intravascular. Therefore, isotonic and hypertonic crystalloids are used for fluid resuscitation. Lactated Ringer’s (LR) or normal saline (NS) is the primary resuscitation fluids . AlbuminThe Saline versus Albumin Fluid Evaluation (SAFE) study compared 4% albumin and NS. Both showed clinically equivalent efficacy. The volume of fluid administered was less with albumin than with NS (1:1.4) . Howevern = 1414; gelatins, dextrans, hydroxyethyl starches, or 4% or 20% of albumin) or crystalloids  for fluid resuscitation  used for fluid resuscitation in burn patients does not compensate for depth . Deeper Enteral resuscitation is started during the initial 6 h . Oral reIn pregnant patients with burns, both Parkland formula and clinical signs such as vital signs, urine output, and fetal heart rate are considered to prevent under-resuscitation because the intravascular volume is increased during pregnancy .Pregnant patients tolerate blood loss better due to increased circulating blood volume and cardiac output , 51. SupBoth fluid overload and fluid composition affect the kidneys. Compared to buffered crystalloids, isotonic saline reduces renal perfusion and increases the risk of AKI . BalanceExcessive fluid administration in patients with decreased LV compliance worsens lung congestion and non-cardiogenic pulmonary edema resulting in pulmonary hypertension, right ventricle dysfunction, and further decrease in LV volumes . EchocarCirrhotic patients have elevated cardiac output, decreased systemic vascular resistance, and low BP . This isIn trauma patients with cirrhosis, fluid loading may be needed. However, the fluid load may worsen organ function and contribute to ascites. Therapeutic paracentesis is recommended in patients with tense ascites and MAP ≥ 60 mmHg is appropriate in cirrhotic patients . PulmonaIn patients with massive blood loss, permissive hypotension prevents progression to dilutional coagulopathy of trauma . In seveNew generation gelatins like polygeline may maintain circulation until blood is available. Improvements in BP, MAP, pulse rate, respiratory rate, and blood pH are noted within 1 h of administration in hypovolemic trauma patients with sustained benefits even after 24 h , 43, 59.Fluid resuscitation strategies have evolved with time. Different traumas need different fluids and different resuscitation strategies. Prehospital trauma care reduces mortality in rural/remote settings. Delayed fluid resuscitation is preferred when transport time to definitive care is shorter whereas goal-directed resuscitation with low-volume crystalloid is preferred if transport time is longer. Adhering to evidence-based clinical practice guidelines and local modifications based on patient population, available resources, and expertise may improve patient outcomes.


Spontaneous regression of a primary testicular germ-cell tumour (GCT), over time known as ‘Burned out’, ‘Shrinking Seminoma’, ‘pT0’, ‘Burnout’ or ‘Spontaneous Regression’, is an uncommon, generally metastatic phenomenon, which may present elevated tumour markers and a suspicious testicular ultrasound image. The histological study of the testicle demonstrated morphological changes of complete or partial tumour regression and found fibrous scarring and other characteristic changes of this phenomenon, which in some cases include vestiges of GCT.There are few publications on testicular GCT tumour regression and those that exist present limited data on the biology of the disease and its etiopathogenesis. This entity was recently recognised in the latest edition of the World Health Organization’s (WHO) Classification of Tumours.We present our clinical, imaging, laboratory, cytohistological and management experience, as well as a historical review of the literature. Spontaneous tumour regression has been reported in various neoplasias , 2. In tWe present our experience and carry out a historical review of the literature.Clinical records of patients from the Urology Service of the Regional Institute of Neoplastic Diseases North, in Trujillo, Peru, were reviewed, from January 2010 to June 2018. We identified the cases with a diagnosis of testicular GCT regression and proceeded to collect the data in a digital card developed for this purpose.We obtained data such as age, pathological history, time of illness, signs, symptoms and information from the physical examinations.Imaging information obtained was confirmed by evaluating existing material in the Radiology services’ records . Tumour marker data such as alpha fetoprotein (AFP), human chorionic gonadotrophin (HCG) and lactic dehydrogenase (LDH) were collected and correlated with the histological findings.We collected data regarding the type of treatment , as far as primary, metastasis and/or recurrence.Of the testicle.Of the metastasis.We reviewed the cytological and histological material, classifying it according to the World Health Organization’s (WHO’s) 2016 classification of testicular tumours and paratesticular tissue. This edition recognises GCT regression as an entity .We used the 8th Edition American Joint Committee on Cancer (AJCC) staging system based on the study of tumour (T), lymph node involvement (N), the presence of metastasis (M) and serum tumour marker (S) .Upon reviewing the medical history, we obtained the dates of the last check-up and the state of the disease. In the cases with no recent data, we located patients.Bibliographic searches were conducted on Scopus, Medline, EBSCO and BVS from 2000 to the present. The data obtained were analysed, compared and discussed.In the review of the medical records, five  cases welymphoma. A core percutaneous biopsy was performed and had inconclusive pathological results. As part of imaging studies, a scrotal ultrasound was requested and a 23 mm × 26 mm hypoechogenic nodule was found in the right testicle. The AFP and HCG tumour markers were found in normal parameters and the LDH in 2480UI. Radical orchiectomy was performed and fibrous scarring associated with histological changes of regression was found. He underwent exploratory laparotomy and subtotal resection of the retroperitoneal tumour with a cytological, histological and immunohistochemical study consistent with a seminoma-type GCT. He received chemotherapy (chemo) with complete tumour remission and tumour markers within normal values; during treatment, he developed deep vein thrombosis (DVT) in both lower extremities (LEs), which was managed medically. At the 60-month follow-up, he shows no evidence of disease.A 54-year-old patient without any significant history, with a 6-month disease time, characterised by weight loss, abdominal tumour and lumbar and abdominal pain. Upon physical examination, no peripheral adenopathies were found, a hard fixed mass was felt in the abdomen located in the mesogastrium and testicles with no notable particularities. On the CT scan of the chest, abdomen and pelvis (CT-CAP), a retroperitoneal 17 cm × 10 cm × 8 cm tumour was observed. It encompassed the abdominal aorta, collapsed the vena cava and projected to the iliac arteries; it was initially classified as lymphoma. A fine-needle aspiration biopsy (FNAB) of the supraclavicular adenopathy was performed, revealing cytological features of a malignant round-cell neoplasm, likely seminoma. The scrotal ultrasound showed the right testicle with microcalcifications and an 8 mm × 7 mm hypoechogenic nodule. Tumour markers were AFP 4.5UI/l, HCG 8.02UI/L and LDH 3637UI/L. A radical orchiectomy was performed, with a histological report of the fibrous nodule and regression changes, among them germinal neoplasia in situ. A supraclavicular tumour biopsy was performed with histological and immunohistochemical study consistent with seminoma-type GCT. The patient received chemo with complete tumour remission at the supraclavicular and mediastinal level; the remission was partial in the retroperitoneum, therefore, a positron emission tomography was performed, reporting para-aortic tissue of 35 mm, inactive residual appearance, which diminished in subsequent tests. At the 42-month follow-up, there is no evidence of active disease.A 58-year-old patient with a 2-month disease time, characterised by bilateral lumbar pain, sensation of thermal rise, volume increase in LEs and weight loss. Physical examination revealed a left supraclavicular lymph node conglomerate of hard consistency, a mass in mesogastrium, hard edema in both legs and no other peripheral adenopathies. The abdomen and testicles were without any particular features. Supraclavicular, subclavian, mediastinal and retroperitoneal adenopathies were observed via CT-CAP, the latter being in a 15 cm × 13 cm × 9 cm conglomerate, which includes the great vessels, as well as thrombus in vena cava and iliac vessel. It was also initially classified as A 23-year-old patient with a history of thoracic trauma and haemoptysis underwent a thoracic tomography, in which multiple nodules were observed in both pulmonary fields. Subsequently, he developed hemiplegia on the left; in the supplemental CT, a 48-mm hypodense image in the brain was found in the right fronto-parietal region; also in the retroperitoneum, a 11cm × 7 cm × 4cm ganglion conglomerate. Physical examination revealed no peripheral adenopathies, resistance to palpation in the abdomen at the level of mesogastrium and testicles without particular features. The scrotal ultrasound revealed a left testicle with multiple microcalcifications and a 9 mm × 8 mm heterogeneous, hypoechogenic nodule. The tumour markers were AFP 0.9UI/L, HCG 19609UI/L and LDH 561UI/L. Radical orchiectomy was performed with pathology that showed the testicle with fibrous scar associated with histological regression changes. The patient underwent a pulmonary nodule FNA with cytological study compatible with choriocarcinoma-type GCT. Due to brain metastasis, he received radiotherapy (RT) and started chemo. During the treatment, he presented seizures, anaemia and febrile neutropenia; the poor clinical response was observed in imaging studies  and in the tumour markers . Chemo was not completed due to complications and the patient died 7 months after the initial diagnosis with the evidence of disease progression.A 36-year-old patient with no relevant history, with a 7-month disease time characterised by left lumbar pain, abdominal mass, weight loss and increase in volume of the left leg. Upon physical examination, no peripheral adenopathies were found. In the abdomen, a hard and fixed mass was felt in the mesogastrium, testicles had no particularities and an increase in volume in the left leg. On the CT-TAP, a retroperitoneal tumour of 16 cm × 9 cm × 9 cm was observed, which includes the aorta and the cava. Doppler ultrasonography reported DVT in the iliac, femoral and popliteal veins of the left leg. On the scrotal ultrasound, a 30-mm hypoechoic nodule was found in the left testicle, associated with multiple microcalcifications. The tumour markers were AFP 0.69UI, HCG 0.44UI and LDH 1240UI. Radical orchiectomy was performed with pathology that reported parenchyma with a fibrous scar and tubular hyalinization. Percutaneous biopsy of the retroperitoneal tumour with histological and immunohistochemical exam was compatible with the seminoma-type GCT. Full chemo with partial remission of the disease and tumour markers were within normal parameters. The residual tissue has progressively decreased in volume. Currently, at 20 months of follow-up, there is no evidence of active disease.A 20-year-old patient without any significant history, with a 6-month disease time, characterised by weight loss, abdominal tumour and pain. Upon physical examination, no peripheral adenopathies were found, a hard fixed mass was felt in the abdomen located in the mesogastrium, testicles had no notable particularities. On the chest, abdomen and pelvis CT, a retroperitoneal tumour was observed predominantly in the left iliac region measuring 16 cm × 15 cm × 9 cm, which includes iliac vessels and left renal agenesis. Suspecting a metastatic GCT, a scrotal ultrasound was performed and found a 5 mm × 10 mm isoechogenic nodule in the left testicle. Tumour markers were 1745UI AFP, HCG 3705UI and LDH 2948UI. Radical orchiectomy was performed and fibrous scarring associated with histological changes of regression was found. A percutaneous biopsy of the retroperitoneal tumour was performed, with a cytological and histological report of mixed GCT . During the evolution of the disease, one of the complications presented was a bowel obstruction, resolved with sigmoidectomy and block retroperitoneal lymphadenectomy. Patient was currently in chemo with partial remission.The cases presented between 20 and 58 years with an average age of 38 years. No significant data were found with regard to history. The average disease duration was 3.8 months (range 1–7 months), mainly characterised by abdominal and/or lumbar pain, weight loss and abdominal tumour in three cases and one supraclavicular tumour. On examination of testes, tumours were not felt on palpation in any patient. In two cases, the initial diagnosis was lymphoma The results of the imaging studies conducted are summarised in The initial clinical suspicion in the first three cases was different from primary testicular metastatic GCT, therefore, the diagnostic work-up included aspiration and/or surgical biopsies of retroperitoneal, supraclavicular and lung masses, respectively. Laboratory studies and images were supplemented with histological findings and radical orchiectomy was performed. In the last two cases, where GCT was suggested from the beginning, the pathology of the orchiectomy was consistent with tumour regression, having found evidence of germinal neoplasia in the study of the metastasis.With the anatomopathological diagnosis of testicular tumour regression and metastatic staging, all patients received chemo and had favourable responses corroborated through imaging studies and MT, except for the third case, who also received RT due to brain metastasis and progressed to death .The existing material was reviewed and classified according to WHO classification of testicular tumours and paratesticular tissue (4).testicle product of radical orchiectomy, macroscopically, all cases presented a whitish fibrous scar, located close to the to rete testis. The surrounding testicular parenchyma did not present significant alterations. For histological interpretation, we divide the testicles into two regions: the scar and the area adjacent to the scar , whose characteristics are described in In the metastasis, four of these were evaluated initially with cytology, two with FNAB and two with intraoperative cytology. The results of these showed germ-tumour cytology, allowing identification of the types seminoma, choriocarcinoma and embryonal carcinoma. Subsequently, all cases were subjected to conventional histological and immunohistochemical study, corroborating diagnoses of germinal neoplasia; the latter presented teratoma and yolk sac tumour in addition to embryonal carcinoma. With regard to The five cases were classified as metastatic stage pT0, with findings of testicular tumour regression.Performing a search from January 2000 to June 2018, 159 cases were found in 57 articles. The cases arose between 17 and 67 years old with an average age of 35.96 years; only in nine cases (15.8%), cryptorchidism was reported as a precedent. 96.8% of the patients had metastatic disease and 71.7% had complete regression.Likewise, the time frame of the review found that there is a discrete increased frequency of regression in the right testicle (47%) and the more common histological type is seminoma (50.8%). The publications by author’s locations were as follows: Europe 22 38.6%), Asia 18 (31.6%) and America 17 (29.8%). The summary of the data is presented in %, Asia 1In the review conducted, there are some publications of cases reported as ‘burned out’, the same as the clinic and/or imaging studies and/or laboratory are compatible with GCT; however, they were not considered because they do not have complete data, especially histological findings of the testicle.Over time, the ‘phenomenon’ of tumour regression has been described in different pathologies such as melanoma, breast cancer, lymphoma, renal carcinoma, among others , 2. It iet al [There are not many publications on the regression of testicular GCTs; this entity has only recently been recognised in the last edition of the WHO’s book on Tumours of the Urinary System and Male Genital Organs (2016), in the chapter on testicular tumours and paratesticular tissues . It is cet al  reportedet al [burned out primary…’. This is likely the first publication to use this terminology to define tumour regression in germ cells.In 1955, Slater et al  reportedet al [ pattern of regression in a testicular seminoma with viable metastasis. The author provides a comprehensive description of typical histological findings, as well as a comparison with choriocarcinoma [In 1961, Azzopardi et al  publishearcinoma .et al [In 1970, Veragut et al  reportedIn 1990 and 2000, two works were published, titled ‘Shrinking Seminoma’ and ‘Shrinking Seminoma—Fact or Fiction?’, describing volume reduction in the testicle with seminoma, where the mechanism is fundamentally ischemia—necrosis secondary to intermittent testicular torsion. Other possible causes also described were chronic inflammation and hormonal disorder. In the aforementioned phenomenon, depending on the stage at which it is diagnosed, a testicle ‘shrunken’ in size, with or without a residual tumour may be found, which is why in a patient with a retroperitoneal, mediastinal or other mass that also presents testicular shrinking, a GCT should be suspected , 36.Near the end of the 20th century, various publications report on probable EGCT, in which lesions were found at the testicular level, consistent with spontaneous regression, correlating to the primary –9. Accorimmunological response mediated by cytotoxic T lymphocytes that recognise tumour antigens and destroy malignant neoplastic cells, with subsequent fibrosis replacement; and those related to an ischemic response in the neoplasia, secondary to the blood supply deficit due to high metabolic rates and/or intermittent testicular torsion (Shrinking Seminoma). Another hypothesis indicates that when seminomas become metastatic, the organism produces antibodies that attack the metastasis, as well as the primary testicular tumour, which shrinks and may even be destroyed, leaving only traces. This foundation refers to the immunological theory of regression [Although the mechanism behind primary tumour regression has not been determined, there are several hypotheses. The two main hypotheses are: those related to an gression , 39–43.Clinical manifestations generally depend on the metastatic disease –52; onlyExamination of the scrotal sacs through palpation is insufficient to exclude testicular tumours. The findings depend on the size of the tumour, its relation to the size of the testicle, the placement, consistency and/or associated pathologies, such as hydrocele, cysts or others , 56, 58.The sensitivity of the testicular ultrasound for diagnosing GCT is close to 100%, so all young patients with a retroperitoneal or mediastinal mass should undergo this test. The characteristics of testicular tumour regression are not specific, as there have been findings of hyperechogenic, hypoechogenic and mixed lesions, nodular and linear areas, signs of testicular atrophy and/or acoustic shadowing reflecting calcifications or fibrosis , 59, 60.Tumour markers are fundamental to the diagnostic approach, staging, treatment and follow-up; they show up in varying forms depending on the histological lineage and the response to treatment. In our report, during the initial diagnostic, LDH was increased in all of the cases in relation to the metastasis and tumour burden, in two patients with HCG and in one with AFP , 38, 60.The five reported cases were metastatic and diagnosed using the cytohistological results of the lesions, which, in addition to the clinical findings, laboratory and imaging studies, allowed us to formulate the primary testicular diagnosis and to indicate the corresponding radical orchiectomy. In the various reports, the macroscopic description of the testicles showing the partial or total tumour regression phenomenon, the presence of lesions that are hardened, whitened, fibrose, of scar aspect, in the form of nodules (singular or multiple), banded, linear or starred, is reported , 63–65. in situ (approximately 50%), hyperplasia in Leydig cells (45%), intratubular microlitres (30%). The literature mentions intertubular calcifications and germinal neoplasia in situ as pathognomonic signs [Microscopically, our findings coincide with the WHO description defining the diagnostic criteria for testicular tumour regression, including inflammatory lymphoplasmacytic infiltrate (present in around 90% of cases), tubular hyalinization (around 70%), increase in vascularity (50%), hemosiderin 44%) and thick intratubular calcifications. The peripheral area was observed to have atrophy and sclerosis of the seminiferous tubules (100%), germinal cell malignancy rolitres 0%. The l4% and thic signs .It is known that all germinal tumours have the potential for regression; however, there is evidence in the literature that disagrees on the frequency of the histological subtypes that demonstrate this phenomenon. Historically, choriocarcinoma has been considered the most prone to regression, but recent reports, as well as ours, confirm that seminoma has the most common histology, with exception of the spermatocytic type, which is now a separate entity. The teratomas are classified as the histology group with the lowest probability of regression , 66, 67.Various publications agree that chemotherapy is not completely effective for the testicles due to the haemato-testicular barrier and thus the necessity of surgical resection (orchiectomy) on the ‘regressed’ primary is essential and represents the cornerstone of the burned out definition, in addition to being the foundation for proper treatment. The management is, in general, similar to that for primary GCT testicles , 68–71.For a large majority of patients, the diagnostic work was performed based on the symptomatology of the metastasis, not infrequently with an approach different to that of burned out GCT that shows the delay in proper diagnosis, the risk of complications due to time and progression of the disease, as well as procedures that some may have undergone. The incorrect extragonadal GCT diagnosis implies not treating the primary testicle, which may present partial regression, and, thus, in not responding to the systemic treatment and being maintained in a safe haven by the haemato-testicular barrier, it may become one of the most important factors determining recurrence and prognosis.In our review of the literature, we found no conclusive publications on survival, the persistence of the disease or recurrence when compared to burned out GCT versus Gonadal and/or extragonadal GCT. In our case, the reported patients began a long-term follow-up protocol in order to carry out collaborative projects with other institutions in an attempt to answer the questions raised.Throughout time, the evidence regarding the ‘phenomenon’ of testicular tumour regression has been described in various publications, which has allowed for its current definition as an entity with its own diagnostic criteria.Etiopathogenesis is still not well defined, nor if the tumoural regression tumour itself has some value in the prognosis. What is well defined is the indication for treatment in accordance with GCT protocols.‘Burned out’ GCTs are classified as metastatic or non-metastatic with complete or partial regression. The most frequent ones were metastatic with complete regression and the most common histological type was seminoma.In all tumours with GCT clinical and/or histology, the primary testicular tumour should be ruled out before classifying it as an extragonadal GCT.The authors declared no conflicts of interest.The main idea and literature review were by JCA and MAA; the collection of data was done by FMA, BAL and JLD. Manuscript revision and approval of the final copy was done by all.