The data presented here are associated with the article “The blister fluid proteome of paediatric burns”  [1]. Bur Specifications Table•First and most comprehensive proteome of burn blister fluid that can be used to compare against other disease states/patient populations.•These data provide a reference list of known, observed proteins within paediatric burn blister fluid, which will be of interest for the burn wound research community and clinicians.•Qualitative evaluation of the biochemical differences between burns of different depths will enable future targeted quantitative analyses of protein abundance based on burn depth.•The dataset allows for extensive Gene Ontology (GO) term analysis of the burn blister fluid proteome and the interaction/interrogation of this proteome through the Cytoscape data file provided herein.wound healing’ and ‘response to stress’ are shown in Presented in this publication is an inventory of proteins identified in paediatric burn blister fluid (1% FDR corrected), and the depths at which they were detected , molecular functions (MF), and cellular components (CC) were determined through Gene Ontology (GO) enrichment analysis of the whole blister fluid proteome using the BiNGO app within Cytoscape  (


A large body size is associated with larger breast cancer tumours at diagnosis. Standard regression models for tumour size at diagnosis are not sufficient for unravelling the mechanisms behind the association.Using Swedish case-control data, we identified 1352 postmenopausal women with incident invasive breast cancer diagnosed between 1993 and 1995. We used a novel continuous tumour growth model, which models tumour sizes at diagnosis through three submodels: for tumour growth, time to symptomatic detection, and screening sensitivity. Tumour size at other time points is thought of as a latent variable.P value = 5.0 × 10−5 and individual P values = 0.089 and 0.022). We also quantified the role of mammographic density in screening sensitivity.We quantified the relationship between body size with tumour growth and time to symptomatic detection. High body mass index and large breast size are, respectively, significantly associated with fast tumour growth rate and delayed time to symptomatic detection (combined The times at which tumours will be symptomatically detected may vary substantially between women with different breast sizes. The proposed tumour growth model represents a novel and useful approach for quantifying the effects of breast cancer risk factors on tumour growth and detection. Among postmenopausal women, a high body mass index (BMI) is known to be associated with an increased risk for breast cancer . High BMTo evaluate the full impact of body size on tumour growth/symptomatic detection, it is important to incorporate mammographic density at the analysis stage. Mammographic density refers to the tissue composition of a woman’s breast as seen on a mammogram. Fibro-glandular tissue is radiodense and appears white on a mammogram, whereas fat is radiolucent and appears dark on a mammogram. Body size, as measured by BMI, is negatively correlated to percentage mammographic density (PMD) (the percentage of the breast area appearing ‘dense’ on a mammogram), which in turn is associated with larger tumour size because of lower screening sensitivity –7.The hypothesis that BMI is associated with tumour growth is supported by studies with molecular markers. In a retrospective cohort study of women enrolled in a screening programme in western Washington state, it was reported that obese women had significantly faster-growing tumours, as measured by Ki-67 . High BMAlthough standard regression techniques can be used to evaluate  associations between tumour size and body size covariates, more sophisticated approaches are needed to shed light on the mechanisms underlying these associations. In this article, we quantify the relationships between body size covariates (BMI and breast size) and tumour growth/symptomatic detection as well as the role of mammographic density in screening sensitivity Fig. . We do tThe extension of the model that we present in this work makes it possible to include covariates in all submodels, enabling us to jointly evaluate how particular factors, such as body size, affect tumour growth, symptomatic detection, and screening sensitivity. To the best of our knowledge, ours is the first approach to enable this.The study population consists of postmenopausal women born in Sweden with a primary invasive breast cancer. It originates from the cases in a case-control study of women who were between 50 and 74 years old and whose cancer was diagnosed between 1 October 1993 to 31 March 1995 . ThroughInformation about tumour size and other tumour characteristics, as well as neoadjuvant treatment and reason for diagnostic mammography, was retrieved from surgical and oncological patient records throughout Sweden. Through self-reported questionnaires, we collected information about breast cancer risk factors, such as height and weight.Of the 3979 women identified as cases, 84 % (3345) chose to participate. However, 320 participants were excluded because of a noninvasive breast cancer diagnosis, a previous or other cancer diagnosis, or the lack of written consent or because their diagnosis was made before or after the study period. Furthermore, 177 premenopausal women were excluded, as were 168 women with unknown age at menopause. Screening history was missing on 626 cases, so these women were also excluded, as were 50 cases with unknown detection mode . Also, 24 women had no information on tumour size, and 611 women were lacking available mammograms and therefore were also excluded here. Six cases had no information on BMI, calculated from height and weight, and 11 women received neoadjuvant treatment before the pathologic measurement of the tumour size and therefore were excluded. From the original case-control study, 1352 women are included in the analyses here; 937 were screening cases and 415 were symptomatic cases. Of the symptomatic cases, 292 women (70 %) had a tumour diagnosed within 2 years of a negative screening. Prior to the exclusion of women without screening history or available mammograms, data for 2680 women were available for analysis. The age and tumour size (diameter) distributions—25th, 50th, 75th percentiles of  for age (years) and  for tumour size (millimetres)—in this group are similar to those in the group of women included in the analysis presented in this article:  for age (years) and  for tumour size (millimetres). Ethical approval for the study was obtained from the Regional Ethics Review Board in Uppsala at Uppsala University (reference number 155/93), and ethical approvals of extensions of the study were obtained from the Regional Ethics Review Board in Stockholm at Karolinska Institutet (reference numbers 98-226 and 99-338). All participants provided written informed consent.We first carried out an explanatory data analysis, tabulating relationships between the key variables tumour size, BMI, and TBA. Subsequent analyses were based on our tumour growth model, which we describe below.Our main approach is based on modelling the size of a woman’s tumour (at detection), conditioning, explicitly, on her screening history (dates of previous mammography screens), and how her tumour was detected (screening/symptomatic detection). We also allow tumour size (at detection) to be dependent on body size and PMD. This is done by specifying and using three submodels (described below) for tumour growth, time to symptomatic detection, and screening sensitivity (as functions of body size covariates and PMD). Although calculations are complex, only eight parameters are estimated. The unknown parameters are estimated by optimising a likelihood function. The model described in this article is based on a non-trivial extension of the approach of Abrahamsson and Humphreys  , which will diminish their individual associations with tumour size. This has been shown for standard regression models in a study of clinical stage with BMI and breast size as covariates . In the α2. There are two other parameters, α1 and σ2, which are used to specify the general form of the tumour growth function (see the Appendix). The tumour growth model, which has been chosen partly because of mathematical tractability, has also been used by Bartoszyński et al.  and the percentile method for 100 bootstrap replications was used to plot 95 % confidence regions for the screening sensitivity estimates.Key characteristics of the data are presented in Table P value associated with adding both BMI and TBA to the model was small (5.0 × 10−5). The P value from testing the  association between BMI and tumour growth was 0.089 (likelihood ratio test), whereas the P value for TBA and time to symptomatic detection was 0.022. The main purpose of our tumour growth model analysis is to learn about the direct role of BMI/TBA in tumour growth/time to symptomatic detection. In Fig. Parameter estimates  for the tumour growth model described in the Methods section are displayed in Table Figure To assess model fit, we graphically compared observed and fitted tumour size group proportions, the latter being calculated by summing fitted, individual probabilities. We did this within groups of women defined according to body size and detection mode so that we could also visualise differences between fitted tumour size distributions according to these factors Fig. . The “laIn this study, we have quantified the relationship between body size covariates  with tumour growth and time to symptomatic detection, as well as the role of PMD in screening sensitivity, using a novel continuous tumour growth model. Owing to the extension of the model that we present, this is the first time that breast cancer risk factors have been included in all parts of a statistical growth model which jointly studies tumour growth, screening sensitivity, and time to symptomatic detection. Only a few earlier studies have included risk factors in statistical tumour growth models. In a study with a multi-state model setting, Chiu et al.  estimateBefore carrying out our analysis, we checked/evaluated our modelling procedure by carrying out a simulation study. Simulations were performed in order to ensure that our computer programme was able to retrieve “true” parameter values. The simulations were carried out by using an approach similar to the one described by us before , but thiThe estimates in this study regarding the overall tumour growth and the overall symptomatic sensitivity  were similar to the results in our earlier study . There aIn this work, we assume an exponential growth model with a gamma distribution for the inverse tumour growth rate. This made it possible to make the extension of including covariates in the submodels. Analytically tractable solutions would not be possible within a likelihood framework if other models, such as logistic growth, were used. However, in Fig. From Fig. P value = 6.3 × 10−4; Tables P value = 4.0 × 10−3) and a per-standard deviation odds ratio of 0.75. These estimates differ presumably because of the negative correlation between PMD and the other variables and underscore the importance of specifying each submodel as well as possible.With BMI and TBA included in our tumour growth model, our point estimate of the effect of PMD on screening sensitivity was −2.39 . The effects of body size on tumour growth and time to symptomatic detection work in the same direction, increasing the size of the tumour, and BMI and TBA are strongly correlated variables. It is quite possible that with a somewhat larger sample size we would have sufficient power to show that body size is significantly associated, separately, with both tumour growth and time to symptomatic detection. However, the estimated direction of the effect for BMI on tumour growth is as we would expect, and other studies have already found significant associations between BMI and Ki-67 among women older than 40 years [Although it is clear that including both BMI and TBA significantly improves the fit of our tumour growth model compared with a model with neither of these factors, we were not able to show that, when TBA is included in the model for symptomatic detection, BMI is significantly associated with tumour growth . Since our study is the first to test such an association, it is not possible to make a direct comparison with other studies. However, there is some indirect support for our result in other studies. Using a multivariate logistic regression analysis, Boyd et al. [We obtained somewhat stronger evidence that, when including BMI in the model for tumour growth, breast size affects symptomatic detection  also have high estrogen levels because of this exogenous source of estrogens. Therefore, it has been suggested that any relationship between tumour progression and BMI could be weaker among women receiving HRT . A more In future studies, we aim to extend the model to include more breast cancer risk factors/covariates. For example, some measure of HRT could be included in the tumour growth model. It has also been discussed whether mammographic density affects tumour growth, although, to date, little evidence has been presented for this association . It may One of the main reasons for developing the types of models described here is to understand more about the relationships between risk factors and the biology of breast cancer. As Vilaprinyo et al.  point ouSophisticated tumour growth modelling is needed to unravel the mechanisms behind the association between a large body size and larger breast cancer tumours at diagnosis. We develop a novel continuous tumour growth model with submodels for tumour growth, time to symptomatic detection, and screening sensitivity and further describe approaches for including covariates in these submodels. Using our growth model, we found that a large breast size is associated with a delayed symptomatic detection in postmenopausal women.


Hyperpolarised 13C1-pyruvate was injected into the femoral vein of anaesthetised rats and slice-localised 13C magnetic resonance (MR) spectra acquired. Spectral integral versus time curves for pyruvate and lactate were fitted to a precursor-product model to estimate the rate constant for tumour conversion of pyruvate to lactate (plk). Mean arterial blood pressure (MABP) and oxygen tension (ArtpO2) were monitored. Pyruvate and lactate concentrations were measured in freeze-clamped tumours.Hypoxic inspired gas was used to manipulate rat P22 fibrosarcoma oxygen tension (pO2 and tumour pO2 decreased significantly during hypoxia. plk increased significantly (p < 0.01) from 0.029 ± 0.002 s−1 to 0.049 ± 0.006 s−1 (mean ± SEM) when animals breathing air were switched to hypoxic conditions, whereas pyruvate and lactate concentrations were minimally affected by hypoxia. Both ArtpO2 and MABP influenced the estimate of plk, with a strong negative correlation between plk and the product of ArtpO2 and MABP under hypoxia.MABP, ArtpOplk, responds significantly to a rapid reduction in tumour oxygenation.The rate constant for pyruvate to lactate conversion,  In the glycolytic pathway, hyperpolarised 13C1-pyruvate (PA) has been the most studied substrate for cancer metabolism because of its high, rapid hyperpolarisation and relatively long T1 relaxation time +. Altered metabolism is a common feature of cancers and many tumour cells metabolise glucose to lactate under aerobic as well as anaerobic conditions, a phenomenon known as aerobic glycolysis or the Warburg effect Hyperpolarisation increases n = 36) was used to determine local tumour oxygen tensions (pO2) via probes incorporating an oxygen-sensitive fluorophore (OxyLite™). These measurements were used to establish a suitable inspired gas mixture for inducing tumour hypoxia in subsequent MRS experiments (see n = 18) was used for MRS experiments, in which the rate constant for conversion of intravenously administered PA into lactate (plk) in tumour tissue was estimated under normoxic and hypoxic conditions. A third group of animals (n = 28) was used for ex vivo measurement of pyruvate and lactate concentrations in tumour tissue following the same gas challenges as used for MRS.All experiments were conducted in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986 and following published guidelines ents see . A seconWhen tumours reached 18 ± 6 mm mean diameter (mean ± SD), rats  were anaesthetised with isoflurane and a femoral vein, a femoral artery and a tail vein cannulated. Following surgery, rats were heparinised (1500 Units/kg i.v.) and anaesthesia maintained for imaging by propofol  infused via the tail vein cannula at a rate of ∼40 mg/kg/h. Rectal temperature was maintained at 37 °C throughout surgery and imaging, using a homoeothermic blanket system .13C1-pyruvic acid  was hyperpolarised (denoted PA) using a HyperSense polariser as described previously 2 was manipulated by supplying the inspired gas as normal air or hypoxia  via either tracheal intubation or face-mask at 1 L/min commencing ∼3 min prior to PA infusion and maintained throughout the MRS experiment.13C/1H 20 mm surface coil (Bruker), positioned ∼1–2 mm above the tumour to minimise respiration artefact, was used for MR acquisitions (see 13C-spectroscopy. 13C-MRS data acquisition commenced with automated transfer of PA from the polariser and infusion into the rat 2) using an ABL80 Flex blood gas analyser .A 13C1-pyruvate and 13C1-lactate peaks were integrated from phase-adjusted spectra for each acquired slice and time point . me point c. Signal+/NADH. See Supplemental Material for details.Concentrations of pyruvate and lactate in P22 fibrosarcomas were measured in a control, untreated group and groups where the surgery, anaesthesia regime, timing of pyruvate infusions (not hyperpolarised) and gas breathing conditions used in the MR experiments were replicated. Rats were sacrificed ∼6 min after the final pyruvate infusion, where given. Tumours were rapidly excised, freeze-clamped in liquid nitrogen and stored at −80 °C before biochemical analysis. Pyruvate and lactate concentrations (performed at least in triplicate) were assayed spectrophotometrically, based on the levels of the co-factors, NADt-tests, as appropriate, were used for testing the significance of differences between two groups. ANOVA with Bonferroni post hoc testing was used for multiple group comparisons. Linear regression analysis was used for correlations between physiological parameters – ArtpO2 and MABP. A p value of <0.05 was considered statistically significant.Analysis was carried out using MATLAB software. Paired or unpaired two-tailed Student’s 2 in P22 fibrosarcomas was 18.0 ± 1.7 mmHg (mean ± SEM), with no effect of tumour size in the range used  and decreased further until response reached a plateau after 3 min  from 0.029 ± 0.002 s−1 under air-breathing conditions (1st PA infusion) to 0.049 ± 0.006 s−1 under hypoxic conditions (2nd PA infusion)  but not in the normoxia group  negatively correlated with plk in the hypoxia group during the hypoxic challenge, where MABP * ArtpO2 values were below approximately 5000 mmHg2   c. No cor00 mmHg2 c.p < 0.05; Results from freeze-clamped tumour tissue  showed that administration of exogenous pyruvate did not significantly increase tumoural pyruvate concentrations, though the variance between individual values within each group was increased a. Lactatplk) for conversion of pyruvate to lactate in tumour tissue, using hyperpolarised pyruvate (PA) and 13C MRS. We emphasise that plk is a fractional rate constant (s−1) and that the rate of conversion of pyruvate to lactate (μmol s−1 (g tissue)−1) is given by the product of plk and the tissue pyruvate concentration (μmol (g tissue)−1). No significant changes in the concentration of pyruvate were observed in any of the treatment groups after the administration of exogenous pyruvate, suggesting rapid basal turnover of pyruvate. The increase in plk observed under hypoxic conditions therefore indicates an increase in the rate of conversion of pyruvate to lactate.We have detected a significant hypoxia-induced increase in the fractional rate constant (+ needed for glycolysis to proceed. Many other mechanisms, such as inactivation of the mitochondrial enzyme pyruvate dehydrogenase by hypoxia-induced reactive oxygen species Allosteric inhibition of phosphofructokinase by ATP and citrate is the primary mechanism for the Pasteur effect. The inverse of this effect under hypoxia results in increased pyruvate production via glycolysis and its subsequent reduction to lactate, thus regenerating the NADplk observed under hypoxia, biochemical analysis of freeze-clamped tumours only showed a significantly higher concentration of tumour lactate in hypoxic animals compared to air-breathing animals, when the latter received no exogenous pyruvate (control untreated group, plk are a more sensitive index of induced hypoxia than changes in lactate concentration or the L/P ratio, even though pyruvate and lactate undergo rapid inter-conversion Despite the increase in plk alongside only borderline increases in lactate concentration under hypoxia could also be explained by 13C spin exchange 13C-lactate, metabolised from PA, enters the tumour lactate pool, where its concentration is much lower than the endogenous 12C-lactate concentration. Therefore, there is a higher probability that 12C-lactate, rather than 13C-lactate, is subsequently metabolised or exported, retaining 13C spins within the lactate pool. Temporarily higher lactate levels in tumours, under hypoxic conditions, would increase the likelihood of 13C label being retained within the large lactate pool, resulting in high estimations of plk. In order to fully define the ultimate fates of pyruvate in tumours, simultaneous steady-state concentrations of metabolites and quantitative fluxes between different metabolic steps would need to be measured, as recently described for cancer cells in culture A significant increase in tumour plk to be higher for the second PA infusion in the normoxia group was most likely related to the measured decrease in MABP between the first and second acquisition; an expected effect of general anaesthesia (plk. This is to be expected since MABP reflects tumour perfusion pressure and we previously showed that MABP correlates directly with blood flow in the P22 tumour 2. In the hypoxia group, this effect would be exacerbated by the reduction in oxygen delivery caused by the decrease in ArtpO2. Indeed, the product of MABP and ArtpO2 was strongly influential on plk in the hypoxia group, where ArtPO2 values dropped significantly. Unsurprisingly, this was not the case in the normoxia group, where ArtpO2 values were at physiological levels. General anaesthesia could have affected our results in other ways. For instance, propofol has been found to decrease mitochondrial function in certain cell/tissue types The tendency for esthesia . Exogeno2 and MABP at the time of PA infusion (We found no correlation between ArtpOinfusion , althougplk (2 (plk for the 1st PA infusion in the hypoxia group was lower than plk for the equivalent acquisition in the normoxia group, under the same nominal air-breathing conditions (2 to monitor the physiological status of animals throughout the duration of in vivo dDNP experiments.There was considerable inter-tumour heterogeneity in both tumour kpl  and pO2 kpl (2  but whetnditions . This dinditions , which s13C-pyruvate 13C T1s of pyruvate and lactate equally would be informative, as differences would affect the plk values obtained. Furthermore, our current procedure provides only a single estimate of plk per tumour. In order to probe the well-known heterogeneity of the tumour microenvironment, an imaging based method such as spectral-spatial EPI One potential limitation of the dDNP technique is that it requires infusion of supra-physiological levels of plk, responds significantly to a rapid reduction in tumour oxygenation. A full quantitative analysis of pyruvate kinetics, including the acquisition of arterial input functions, would also enable pyruvate clearance from blood to tissue to be estimated. This provides the opportunity to investigate tumour vascular function, in addition to oxygenation-related metabolism changes, from a single PA infusion. Further studies incorporating simultaneous measurements of tumour pO2 and plk are warranted and necessary to determine whether there is any relationship between absolute tumour pO2 and pl.kWe have shown that the rate constant for pyruvate to lactate conversion, 13C MRS is starting to be assessed as a way to monitor treatment efficacy. Our results emphasise the need to take oxygenation changes into account when interpreting dDNP data from such studies.Since tumours are highly dependent on pyruvate metabolism to lactate for progression, measurement of hyperpolarised pyruvate metabolism in tumours by The authors have no conflicts of interest to declare.


There is limited information on the impact on perioperative fluid intervention on complications and length of hospital stay following pancreaticoduodenectomy. Therefore, we conducted a detailed analysis of fluid intervention in patients undergoing pancreaticoduodenectomy at a university teaching hospital to test the hypothesis that a restrictive intravenous fluid regime and/or a neutral or negative cumulative fluid balance, would impact on perioperative complications and length of hospital stay.We retrospectively obtained demographic, operative details, detailed fluid prescription, complications and outcomes data for 150 consecutive patients undergoing pancreaticoduodenectomy in a university teaching hospital. Prognostic predictors for length of hospital stay and complications were determined.One hundred and fifty consecutive patients undergoing pancreaticoduodenectomy were evaluated between 2006 and 2012. The majority of patients were, middle-aged, overweight and ASA class III. Postoperative complications were frequent and occurred in 86 patients (57%). The majority of complications were graded as Clavien-Dindo Class 2 and 3. Postoperative pancreatic fistula occurred in 13 patients (9%), and delayed gastric emptying occurred in 25 patients (17%). Other postoperative surgical complications included sepsis (22%), bile leak (4%), and postoperative bleeding (2%). Serious medical complications included pulmonary edema (6%), myocardial infarction (8%), cardiac arrhythmias (13%), respiratory failure (8%), and renal failure (7%). Patients with complications received a higher median volume of intravenous therapy and had higher cumulative positive fluid balances. Postoperative length of stay was significantly longer in patients with complications . After adjustment for covariates, a fluid balance of less than 1 litre on postoperative day 1 and surgeon caseloads were associated with the development of complications.In the context of pancreaticoduodenectomy, restrictive perioperative fluid intervention and negative cumulative fluid balance were associated with fewer complications and shorter length of hospital stay. These findings provide good opportunities to evaluate strategies aimed at improving perioperative care. It is recognized that liberal fluid administration is common practice after major hepatobiliary and pancreatic surgery . HoweverAfter Austin Health Human Research Ethics Committee approval, we conducted a retrospective analysis of consecutive patients undergoing open PD at a university teaching hospital with expertise in hepatobiliary-pancreatic surgery including liver transplantation. A total of 150 consecutive patients between January 2006 and November 2012 were included. Patients undergoing total, distal or completion pancreatectomy were excluded from analysis. Data was extracted from a prospectively managed electronic hospital database. Comprehensive cross checks using computerized medical records were reviewed by three independent investigators who checked that a thorough and accurate tally of fluid administration, losses and complications were recorded. All biochemical, hematologiocal, laboratory and radiological results were reviewed. Any complication coded by the hospital in the health information database was cross checked with clinical records to ensure that the complication was correctly reported and coded. Complications were recorded as unexpected events occurring during surgery or the postoperative period, with pancreatic leak and delayed gastric emptying graded and classified according to the International Study Group of Pancreatic Surgery -6. ComplU test. Multivariate analysis was undertaken using a backward stepwise logistic regression model to identify factors associated with postoperative discharge by day 14, including all factors where the P value was less than 0.1 on univariate analysis. A cut off of 14 days represented the median length of stay for patients undergoing uncomplicated PD at our institution [We defined a restrictive fluid regime as: intraoperative fluid therapy ≤ 4 litres, day 1 fluids ≤ 3 litres, day 2 fluids ≤ 2 litres, and day 3 fluids ≤ 1.5 litres or a neutral or negative cumulative fluid balance at the same time points. A liberal fluid regime was defined as intraoperative fluid therapy > 5 litres, day 1 fluids > 4 litres, day 2 fluids > 3 litres, and day 3 fluids > 2 litres or a positive cumulative fluid balance at the same time points. This definition was selected to be consistent with the “REstrictive Versus LIbEral Fluid Therapy in Major Abdominal Surgery: RELIEF Study” protocol . A stati2) and ASA class III (76%) , middle-aged (mean: 66 years old), overweight (mean: BMI 26.1 kg/m%) Table .The operative details and pathologies of patients undergoing PD with and without complications are summarised in Table Table We performed a retrospective study of detailed fluid intervention, complications and length of hospital stay in patients undergoing PD. We found that, as hypothesized, restrictive fluid intervention and a neutral/negative cumulative fluid balance were associated with reduced complications and shorter length of hospital stay. Importantly, we found that improvement in PD outcomes occurred with increased surgical caseload, and surgeon experience remained an important determinant of overall morbidity.The demographic and clinical features of our patients are consistent with other studies of similar types of surgery ,10-13. LSimilar to other studies we found the surgical case load to have a significant impact on perioperative morbidity following PD -19. PancThere are several limitations of our study. Although this is the largest study examining the association of detailed fluid intervention, fluid balance and surgical outcomes in patients undergoing PD, only 150 patient records were reviewed. Data was collected from a hospital maintained database, which limited our ability to recover any missing or unclear data. Similar to a previous study . Our stuWhilst the findings of this study suggest that positive fluid balance is associated with postoperative complications and increased length of hospital stay in the setting of PD, this does not imply causality. Increased perioperative fluid intervention may occur as a consequence of complications. Sepsis, peritonitis, renal and cardiac failure, and pancreatic leak all can cause fluid retention and/or edema. Fluid balance may therefore simply be a marker of illness rather than the cause. It is also plausible that both of these mechanisms co-exist. Whilst there is emerging evidence that early goal directed therapy and fast track programs improve surgical outcomes and reduce postoperative hospital stay ,22, therTherefore, these results demonstrate that a high surgical caseload and a restrictive perioperative fluid intervention regime, with negative cumulative fluid balance were associated with fewer complications and shorter length of hospital stay. These findings, specific to patients undergoing PD, are of particular interest as the higher rates of complications and prolonged length of stay provide good opportunities to evaluate strategies aimed at improving perioperative care for this group.The authors declare that they have no competing interests.LW and MN conceived the study, participated in the final planning and study design, performed data collection and all statistical analyses; they were responsible for the final writing of the manuscript. DW, BP, COT, ST were responsible for the collection of anesthesia data, cross checking of all anesthesia records with medical records, and calculation and checking of all fluid balances. They also assisted with data entry into a database and the writing of the manuscript. DK and RB were the responsible for the collection of data in the intensive care and high dependency units, entering of data into the master database, interpretation of data, and assistance with the writing of the manuscript. CC, LM and MN were responsible for the collection of all surgical outcomes and lengths of hospital stay. They cross checked surgical records with hospital medical records. In addition they assisted with the writing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2253/14/35/prepub


A small group of cell surface receptors are proteoglycans, possessing a core protein with one or more covalently attached glycosaminoglycan chains. They are virtually ubiquitous and their chains are major sites at which protein ligands of many types interact. These proteoglycans can signal and regulate important cell processes, such as adhesion, migration, proliferation, and differentiation. Since many protein ligands, such as growth factors, morphogens, and cytokines, are also implicated in tumour progression, it is increasingly apparent that cell surface proteoglycans impact tumour cell behaviour. Here, we review some recent advances, emphasising that many tumour-related functions of proteoglycans are revealed only after their modification in processes subsequent to synthesis and export to the cell surface. These include enzymes that modify heparan sulphate structure, recycling of whole or fragmented proteoglycans into exosomes that can be paracrine effectors or biomarkers, and lateral interactions between some proteoglycans and calcium channels that impact the actin cytoskeleton. Decorin, on the other hand, with roles in collagen fibril formation and regulation of innate immunity, has only one chondroitin or dermatan sulphate chain3. Not surprisingly, since proteoglycans can be intracellular, cell surface, or extracellular matrix components, they are increasingly studied in the context of tumour growth, the tumour and stem cell niche, and invasion, metastasis, and tumour-host interactions9.Proteoglycans are present in all cellular and tissue compartments. Moreover, in mammals they are expressed by virtually all cells. By definition, proteoglycans consist of a core protein to which one or more glycosaminoglycan chains are covalently attached. While the number of proteoglycan core proteins in the mammalian genome is not large, their form and functions are highly variable. Aggrecan, a major constituent of cartilage matrix, for example, may have >100 chondroitin sulphate chains, which are key to its function in the maintenance of a hydrated, compression-resisting matrix12. The former are linked to the membrane through a glycosylphosphatidylinositol anchor, while the syndecans are transmembrane, with a highly conserved short cytoplasmic domain. Usually the core proteins carry two to five heparan sulphate chains, but syndecans may sometimes also, or alternately, carry chondroitin or dermatan sulphate chains5. The synthesis of heparan sulphate chains is a complex Golgi apparatus-localised process; while all of the transferases and other modifying enzymes involved in their synthesis are known, their regulation is not13. The importance of heparan sulphate synthesis lies in the fact that this glycosaminoglycan has an ability to interact with a wide array of binding partners that include cytokines, chemokines, growth factors, extracellular matrix macromolecules, enzymes, and lipoproteins15. Heparan sulphate chains have regions of high modification (i.e. high levels of sulphation) interspersed with regions of low, or no, sulphation15. This most complex of all post-translational modifications is under scrutiny, since most protein binding partners of heparan sulphate engage with highly sulphated domains16, so the control of its synthesis and how this may change with transformation are important issues. Moreover, mature heparan sulphate chains can be further modified by a single mammalian heparanase enzyme and by two sulphatases that selectively remove the sulphates of some glucosamine residues19. Heparan sulphate editing is now a topic of great interest in tumour biology and some recent developments are summarised below.On the surfaces of most mammalian cells are representatives of two major families of heparan sulphate proteoglycans (HSPGs), the glypicans and syndecansin cis as co-receptors with other receptors, e.g., tyrosine kinase growth factor receptors and integrins20. The notion was that the heparan sulphate chains provided binding sites for ligands that could then be concentrated for high-affinity receptor binding and subsequent signalling. It now seems clear that there are more intricate interactions at the cell surface that involve independent roles for the cell surface HSPGs. Some of the latest insights into cell surface HSPG functions with relevance to tumour biology are briefly reviewed here. Recent information on the roles of other classes of extracellular matrix proteoglycans in cancer can be found elsewhere21.For many years, it was assumed that cell surface HSPGs had few independent functions but were mostly actingvia the ability of heparan sulphate to fine-tune molecular interactions that regulate cell behaviour22. Over the last decade, it has become increasingly apparent that enzymes can edit heparan sulphate structure, thereby precisely modulating its function and regulating cell behaviour. These enzymes include the endoglucuronidase heparanase, which cleaves and shortens heparan sulphate chains of proteoglycans that as a consequence possess new non-reducing termini, and the extracellular sulphatases Sulf-1 and -2 that selectively remove 6-O sulphates. Both of these enzyme activities are proving to be powerful regulators of tumour behaviour.There is abundant evidence that heparan sulphates, owing to their diversity in structure and location, play important roles in regulating the growth and progression of cancer. Much of this regulation occurset al. demonstrate that heparanase-mediated trimming of syndecan-1 heparan sulphate chains and upregulation of matrix metalloproteinase-9 (MMP-9) expression results in enhanced shedding of syndecan-1 from the cell surface. Shedding exposes a juxtamembrane site on the syndecan-1 core protein that binds to both very late antigen-4 (VLA-4 [integrin α4β1]) and vascular endothelial growth factor receptor-2 (VEGFR2). This coupling of VLA-4 to VEGFR2 activates the latter, thereby initiating downstream signalling that displaces the cytoskeletal adaptor protein paxillin from VLA-4, in turn facilitating the activation of Rac GTPase and polarised cell migration23. This mechanism is in play on both endothelial cells and tumour cells and demonstrates how heparanase, in concert with syndecan-1, drives angiogenesis, tumour cell invasion, and subsequent metastasis.Heparanase is associated with aggressive tumour behaviour including enhanced growth, angiogenesis, and metastasis. Although a number of studies in many tumour types have supported these conclusions, a unifying mechanistic explanation of precisely how heparanase promotes angiogenesis and metastasis was lacking until recently. In a paper just published in Oncogenesis, Jung24. Elevated heparanase expression by myeloma cells enhances their resistance to both bortezomib and melphalan and this resistance is reversedin vivo when mice are treated with the heparanase inhibitor Roneparstat25. Furthermore, heparanase was shown to be present at a high level on tumour cells that survive extensive chemotherapy in myeloma patients, lending further support to the notion that heparanase promotes resistance to therapy25. Together, these findings raise the exciting possibility that the efficacy of anti-cancer drugs may be enhanced when combined with the use of heparanase inhibitors. This is of particular interest, as there are currently four anti-heparanase drugs in clinical trials in cancer patients19. These drugs are all heparin mimetics that are thought to inhibit heparanase activity by blocking the enzyme’s active site. However, recent solving of the crystal structure of heparanase provides an opportunity for the discovery of small molecule inhibitors of enzyme activity that should exhibit improved specificity over the heparin mimetics26. Heparanase-neutralising antibodies have also recently shown promise in attenuating the growth and metastasis of lymphoma and myeloma tumours in mice27.Evidence is also emerging that heparanase plays a key role in promoting chemoresistance. In breast cancer cell lines expressing a high level of heparanase, inhibition of the enzyme sensitised the cells to killing by lapatinib30.While heparanase may have important roles in supporting tumour angiogenesis, it is important to recognise that it is not the only mechanism. Many angiogenesis-promoting growth factors, such as VEGF, fibroblast growth factors (FGFs), cytokines, and chemokines, have high affinity for heparan sulphate. It is therefore likely that vascular remodelling is a consequence of multiple interactions involving cell surface HSPGsO sulphates from heparan sulphate chains, the impact of these two extracellular sulphatases on tumour growth and progression remains controversial. By altering the composition of heparan sulphates, the Sulfs regulate the signalling capacity of heparin-binding growth factors such as Wnts, FGF, EGF, and VEGF, among others19. Predictably, this has important consequences for tumour behaviour. What is surprising is that despite their seemingly identical function, there are data to support the conclusion that Sulf-1 suppresses tumour growth while Sulf-2 promotes tumour growth32. However, such a generalisation appears to be misleading because there is evidence that in some instances Sulf-1 promotes, while Sulf-2 inhibits, tumour growth. Together, these findings strongly suggest that there are factors beyond the catalytic activity of the Sulfs that determine their ultimate impact on tumour behaviour34  binds to all syndecans through one of its two PDZ domains37, and this was shown to be important for the endosomal and trafficking events that lead to exosome formation38. The other PDZ domain of syntenin had high affinity for the membrane phospholipid phosphatidylinositol 4,5-bisphosphate . Syntenin also interacts through its C-terminal domain with Bro1/ALG-2-interacting protein (ALIX39), a central player in exosome formation. In turn, ALIX links to a multiprotein endosomal sorting complex required for transport (ESCRT), with additional roles for the GTPase Arf6 and phospholipase D240. Exosomes are now recognised as important signalling vesicles, containing a number of proteins, lipids, and even nucleic acids such as RNAs and miRNAs. They are produced by most cells, including tumour cells, and interest in them from the tumour perspective focuses on whether they can be detectable biomarkers in fluids and their potential roles in regulating the tumour environment  family. Indeed, elimination of the TRPC7 channel  reverts the S4KO cells to wild-type in terms of adhesion, cytoskeleton, and junction formation58. This was accompanied by reductions in cytosolic calcium that were shown to be increased in the null cells compared to matching wild-type cells. Further work with epithelial cells and, moreover, genetic experiments withCaenorhabditis elegans (which possesses a single syndecan) show that this regulation of TRPC type channels by syndecans may be a highly conserved and important role for this proteoglycan family58.The four mammalian syndecans all interact with the actin cytoskeleton62. A good example is breast cancer, where high levels of syndecan-1 expression, particularly in the tumour stroma, are an indicator of poor prognosis64. In other studies, syndecan-2 upregulation has been shown to alter the adhesion and invasiveness of MDA-MB231 breast carcinoma cells and colon carcinoma cells66. The difficulty with many studies is understanding whether syndecan expression merely correlates with or is functionally related to tumour progression. In some cases, however, the situation is clearer. A wealth of evidence now suggests that syndecan-1 expression in myeloma is related directly to disease severity and progression68. Moreover, it is not only syndecans that may influence tumour progression. Evidence has accumulated rapidly over the past few years showing a relationship between glypican-3 expression and the progression of hepatocellular carcinoma71. This HSPG is expressed in foetal liver, but levels subside in postnatal life73. However, in a large majority of cases, glypican-3 is re-expressed in hepatocellular carcinoma74. The excitement about this HSPG revolves around the possibility that it may serve as a prognostic marker, but also a target for immunotherapy71. Early clinical trials have been reported, but clearly there is a long way to go. On a molecular level, it has been suggested that glypican-3 can bind both Wnt and Frizzled, the signalling receptor for Wnts, through its heparan sulphate chains71. However, the situation is complex, since glypican-3 in normal tissue may be a growth inhibitor. Rare core protein mutations giving rise to the Simpson-Golabi-Behmel syndrome are characterised by overgrowth and many dysmorphisms in patients and a corresponding murine model75. In hepatocellular carcinoma, however, there is also upregulation of Sulf-2. It now appears that selective removal of 6-O-sulphate residues from the glypican’s heparan sulphate chains leads to Wnt activation, possibly through its enhanced mobility, leading to Frizzled binding and signalling76. It is also possible that the heparan sulphate chains may bind hepatocyte growth factor and members of the FGF family78.The work with syndecans and channels has so far not embraced tumour cells. Since calcium is a potent regulator of the actin cytoskeleton, it may now be attractive to re-examine some of the previous observations on HSPGs and tumour cells. The literature is replete with studies showing that syndecans are often mis-expressed in solid tumours and in some cases relate to prognosis80, which is rich in proteoglycans, and the emerging roles of proteoglycans in stem cell differentiation81 are areas for future development. Moreover, it is not only HSPGs that present as targets in tumours. The chondroitin sulphate proteoglycan 4  is recognised as a cell surface marker of pericytes in the vasculature but is also present more widely, for example on neuronal and oligodendrocyte precursors82. It is also an emerging target for immunotherapy in a variety of tumour types, including melanoma, triple negative breast cancer, glioblastoma, mesothelioma, and sarcomas84.Recent developments have highlighted that both the heparan sulphate chains and the core proteins of cell surface HSPGs are highly and functionally relevant to tumour progression. Moreover, the increasingly recognised importance of the tumour cell nicheThe potential for cell surface proteoglycans to be targets for intervention are complicated by their multiple roles and ubiquity. It is perhaps likely that tumour cells, stromal/other host tissue, and the immune system utilise these proteoglycans and their downstream signalling in specific ways to regulate behaviour. Targeting will require detailed understanding, and therefore we can predict that new insights into the functions of proteoglycans will impact tumour biology for many years to come.


Pervasive presence of location-sharing services made it possible for researchers to gain an unprecedented access to the direct records of human activity in space and time. This article analyses geo-located Twitter messages in order to uncover global patterns of human mobility. Based on a dataset of almost a billion tweets recorded in 2012, we estimate the volume of international travelers by country of residence. Mobility profiles of different nations were examined based on such characteristics as mobility rate, radius of gyration, diversity of destinations, and inflow–outflow balance. Temporal patterns disclose the universally valid seasons of increased international mobility and the particular character of international travels of different nations. Our analysis of the community structure of the Twitter mobility network reveals spatially cohesive regions that follow the regional division of the world. We validate our result using global tourism statistics and mobility models provided by other authors and argue that Twitter is exceptionally useful for understanding and quantifying global mobility patterns. Reliable and effective monitoring of worldwide mobility patterns plays an important role in studies exploring migration flows  through a comparison of the Twitter data with worldwide tourism statistics and (ii) with commonly used models of human mobility.Our study relies on one full year of geo-located tweetsThe database had to be cleaned before the analysis to prevent the contamination of mobility statistics by errors and artificial tweeting noise. First, we examined all the consecutive locations of a single user and excluded those that implied a user relocating with a speed over 1000 km/h, i.e., faster than a passenger plane. Further, we filtered out such activities on Twitter as web advertising , web gaming , or web reporting . Those services can generate significant volumes of data, which do not reflect human physical presence in either the reported place or time. To correct for this artificial tweeting noise, we checked the popularity of a message’s source, assuming that those with only few users can probably be classified as an artificial activity. As the threshold, we used a cumulative popularity among 95% of users, constructing the ranking separately for each country. All tweeting sources falling below the threshold were discarded from further analysis. In total, the refinement procedure preserved 98% of users and 95% of tweets from the initial database.An essential first step in our cross-country mobility analysis was an explicit assignment of each user to a country of residence. This made our work different from most of the other Twitter studies that usually did not attempt to uncover users’ origin and characterized a study area using only the total volume of tweets observed in this area  A and B. Human mobility can be analyzed at different levels of granularity. In this study, we considered a user as being “mobile” if over the whole year the user had been tweeting from at least one country other than her or his country of residence. This applied to 1M users or around 8% of all those who used geo-located Twitter in 2012. where n is the number of tweeting locations, āi represents the location of a particular tweet (a pair of x–y coordinates), and ācm is a user’s center of mass. Low values of the radius indicate a tendency to travel locally, while higher values indicate more long-distance travels. The average values computed for users from different countries are shown in Next, we examined how spatially spread or concentrated the mobility of users is in a certain nation. This was captured through an average radius of gyration of the users. The radius of gyration measures the spread of user’s locations around her or his usual location. Here, we defined a usual location as the center of mass rather than a home location, as the latter was defined too broadly, at the level of a particular country. For each user, the radius of gyration was computed thus: The mobility profile of each country can be analyzed from two perspectives, as being either the origin or the destination of international travel. By building the directional country-to-country network of human travels, we were able to quantify both the inflow and outflow of visitors. Human mobility is subject to temporal variations. In order to uncover patterns occurring at the global level, as well as at the country level, we measured how many Twitter users were active outside of their country of residence for each day of 2012. The first pattern that emerged from the analysis was the weekly scheme of check-ins abroad . The tenWhen specific countries were considered, we discovered a variety of deviations from the aforementioned global pattern. Several of them were easy to interpret and were shared by more than one country. For instance, there was a substantial group of European nations with the biggest peak over one of the summer months and a few smaller ones, most probably connected to extended weekends, e.g., at the beginning of May . The second group covered popular summer destinations such as Spain, Italy, Croatia, or Greece , with a Next, we analyzed the topology of the country-to-country mobility network created by travelers within the Twitter community. As it has already been proven by many other studies, partitioning of a raw network of human communication interactions, e.g., based on mobile phone data Blondel , can leaTaking advantage of our methodology of assigning users to their country of residence and focusing on mobile users, we built a worldwide country-to-country network of tweet flows. Each country was considered as a node, and the edges of the network were weighted with the number of Twitter users traveling between a pair of nodes. The network was directional, as the connections were built from the country of residence to countries where a user appeared as a visitor. To deal with the sparseness of the network and different levels of Twitter representativeness, we filtered out all countries with the outgoing Twitter population smaller than 500 users and countries where the Twitter penetration was below 0.05%. The flows were normalized by the Twitter penetration rate in the country of a user’s origin in order to estimate the real mobility flux rather than just a number of Twitter users. The top 30 flows between different countries are presented in The network partitioning procedure was based on the well-known modularity optimization approach Newman  and usesAfter obtaining the initial split of the network, the partitioning procedure was applied iteratively to the subnetworks inside each community, much as Sobolevsky, Szell, et al.  did. As R2 of 0.69 and 0.88), which confirms the validity of the estimated mobility figures.For global mobility, it is difficult to find a bias-free human mobility dataset that would enable direct validation of results obtained with Twitter. An interesting comparison could be made, for instance, with the register of flight connections, although this could be hampered by the possibility of confounding a segment of an indirect travel for direct travel from one’s home country to an intended destination. In practice, such a comparison is also obviously prevented by the difficulty of accessing such data. In this study, we relied on tourism statistics provided by the World Economic Forum WEF  at the cr is a displacement of certain length and β = 1.62.We further validated the results indirectly by demonstrating that mobility measures derived from Twitter activity exhibit similar statistical properties as those obtained using other datasets. First, we computed the distance between each pair of consecutive user locations (tweets) and plotted the frequencies of computed displacement on a log-log scale. Similarly to other studies, we found that the distribution is well approximated by the power law A: (2) Fβ = 1.75), bank note dispersal , and Foursquare check-ins . We also plotted the frequency of previously computed users’ radiuses of gyration  was slightly lower.Given the limited access to global mobility data suitable for a direct comparison with Twitter-based human travels, we further tested our data against a commonly accepted mobility model – the classic gravity approach – as yet another way of indirect verification of uncovered patterns. Many studies proved the gravity law to provide a good basis for modeling the intensity of interactions between locations, depending on their weights and distance, in the context of not only mobility  is characteristic of West European and other developed countries. Traveling distance was additionally affected by the geographic isolation of a country, such as Australia or New Zealand. Through the analysis of temporal patterns, we found a globally universal season of increased mobility at the end of the year. Although the summer mobility was increased for a wide range of countries, it varied in terms of intensity and duration, and in some cases there was no increase at all. Additionally, we discovered patterns driven either by cultural conditioning or by special events occurring in a country. In many cases, the results well confirmed logical expectations, which we treat as an indicator of the legitimacy of Twitter as a global and objective register of human mobility.Furthermore, we demonstrated that the communities detected using a Twitter mobility network formed spatially cohesive regions reflecting the regional division of the world. This finding is important from several standpoints. First, it is in agreement with the results obtained by Blondel, Krings, and Thomas , Ratti eR2 around 0.7) shows a fairly good correspondence, given the wider scope of mobility captured through Twitter and a significantly different method of data acquisition. Further, we confirmed that Twitter data exhibit similar statistical properties as other mobility datasets. For instance, measures such as radius of gyration and probability of displacement are well estimated with the power law distribution (similarly as in Cheng et al. Further, we validated, to a certain extent, geo-located Twitter as a proxy of global mobility behavior. We demonstrated that the number of visitors estimated for different countries based on Twitter data is in line with the official statistics on international tourism. The correlation (We believe the analysis presented in this article proves the potential of geo-located Twitter as an objective, freely accessible source of data for global mobility studies. Further research will focus on exploring the applicability of Twitter activity for finer spatial scales.


This paper analyses the impact of jitter when applied to route discovery in reactive (on-demand) routing protocols. In multi-hop non-synchronized wireless networks, jitter—a small, random variation in the timing of message emission—is commonly employed, as a means to avoid collisions of simultaneous transmissions by adjacent routers over the same channel. In a reactive routing protocol for sensor and ad hoc networks, jitter is recommended during the route discovery process, specifically, during the network-wide flooding of route request messages, in order to avoid collisions. Commonly, a simple uniform jitter is recommended. Alas, this is not without drawbacks: when applying uniform jitter to the route discovery process, an effect called delay inversion is observed. This paper, first, studies and quantifies this delay inversion effect. Second, this paper proposes an adaptive jitter mechanism, designed to alleviate the delay inversion effect and thereby to reduce the route discovery overhead and (ultimately) allow the routing protocol to find more optimal paths, as compared to uniform jitter. This paper presents both analytical and simulation studies, showing that the proposed adaptive jitter can effectively decrease the cost of route discovery and increase the path quality. For wireless routing protocols , flooding is for acquiring or disseminating topological information. In its most basic form, flooding consists of requiring every router in the network to retransmit each received packet exactly once over all of its interfaces. This mechanism leads to the well-known broadcast storm problem , a conseHowever, the elimination of redundant retransmissions is not sufficient for completely avoiding packet collisions. Even if no redundant transmissions are present, wireless flooding in self-organized, decentralized networks leads to packet collisions caused by the simultaneous transmission of adjacent routers over the same wireless channel. These collisions constitute an important source of packet losses in wireless flooding.Different approaches have been explored to address this issue and to minimize its impact in wireless multi-hop networks. Classic MAC (Medium Access Control) collision avoidance mechanisms ,6 are noAccording to the IETF (Internet Engineering Task Force), the problem of packet collisions in a MANET (Mobile Ad hoc Network) can be further alleviated by introducing jitter  in the network layer. In RFC 5148 , the use1.1.et al. , the message copy sent through the longer path (in terms of the number of hops), p1, may reach the destination faster than the message copy over p2; and this, with a non-negligible probability. Now, if a random delay on retransmission—jitter—is applied at each hop and if that jitter is selected from a uniform random distribution , where α ∈  and mαJ is a minimum jitter value. Note that α = 0 corresponds to the uniform jitter distribution specified in RFC 5148  (α ≠ 0). The trade-off between randomness and path length deterministic dependence can be controlled by way of parameter α ∈ : the closer α is to one, the more deterministic becomes the total delay of an RREQ packet with respect to the path length.Window jitter reduces the randomness and increases the (deterministic) dependency of the total RREQ delay to the length mαJ. Intuitively , this makes it less likely that a larger number of hops is traversed by RREQ packets in a shorter time. The implicit assumption is that “shorter” paths (in the number of hops) are preferable to “longer” paths, considered worse for routing (hop count metric).Under the window jitter distribution, each additional hop in the path traversed by an RREQ packet causes at least an additional delay of 3.3.The window jitter principle can be naturally extended to non-trivial link metrics, for instance based on the probability of successful transmission (Expected Transmission Count ) or the mJ, mJ]. This reduces the probability of delay inversion or, equivalently, increases the probability that an RREQ packet is forwarded faster by routers receiving it on better links.Given a link quality indicator LQ ∈  , jitter values are selected uniformly within the interval . Let UP and UD denote the probability of inversion with uniform jitter and the inter-path delay difference with uniform jitter, respectively. Then, the probability of inversion UP has the expression detailed in Proposition 1.In the case of uniform jitter, UP, in two paths of n and m, has the following expression:The probability of inversion with uniform jitter, n and m, i.e., the probability that a path of m hops performs faster forwarding than a path of length n. m, for cases in which n< m. Both the theoretical values and the results from a discrete-event simulation  are displayed. m − n as the absolute length of these paths (in number of hops) increases. Note that both π1 : {m = ct.} and π2 : {Δ = m − n= ct.}, respectively.n = 2, m = 3), this probability is Expression mJ, meaning that it cannot be addressed by modifying the jitter interval length; and (ii) from m − n, but also on the absolute values of path lengths n and m: as paths become longer, more random jitter values are assigned to an RREQ message, and it is more likely that delay inversions occur.Two aspects can be highlighted from this analysis: (i) from mnJ, where n is the path hop length) and a fixed lower bound set to zero. Recall that, while a single uniform jitter value within  has mean n uniform jitter values converges in law  to a Gaussian distribution with mean n hops) thus lead to more variant total jitter distributions.The fact that delay inversion is more frequent in long paths (in terms of number of hops) is due to the fact that the range in which total jitter values are possible  has a linearly growing upper bound + notation stands for the positive part , fori.e., z, and zero otherwise.n m, Therefore, the probability of delay inversion with paths of The expression ofcorresponds to:whereWP = 0 otherwise .n and m. In the analysis, the hop count metric is considered, i.e., the routes with less hops are more preferred. α is set to PW = 0 to PW = 1  is significantly more steep with the modified  distribution of jitter values than with the distribution of RFC 5148 , the probability of auto-collision in the one-hop neighborhood of S corresponds to n, mJ, L). Although the specific values for the probability of auto-collision depend on the chosen values, and, in particular, the relation between L and mJ, there are some general trends that can be identified. The use of window jitter leads to more frequent auto-collisions at the first hop, due to the effective reduction of the interval of retransmissions , which implies a more deterministic retransmissions schedule by one-hop neighbors of S. This effect, however, is specific to the first hop and becomes less relevant in multi-hop flooding, as the network has a bigger diameter and routes grow longer, as shown in further experiments (Sections 5 and 6).4.2.2.When using jitter in a flooding operation, the flooded packets must be stored in buffers in the intermediate routers for the time imposed by the jitter interval. The average occupancy of intermediate routers' buffers is obviously related to the distribution of jitter values.t = t0, for each of the different jittering distributions studied. Assuming a Poisson process with rate λ for packet arrival, the number of packets that arrive (on average) within  ms .Standard RFC 5148 jitter, mJ = 200 ms. Jitter is selected within  ms .Standard RFC 5148 jitter, m=J 100 ms. Jitter is selected within  ms .Window jitter, m=J 150 ms. Jitter is selected within  ms .Window jitter, Different jitter settings are compared:For each of the five settings, the shortest-path and shortest-delay strategies for the RREQ forwarding scheme are evaluated.6.2.This section describes the most relevant results, observed in the performed simulations of various jitter distributions  under shortest-path RREQ forwarding (Section 6.2.1) and shortest-delay RREQ forwarding (Section 6.2.2) configurations.6.2.1.The first observation that can be highlighted from the shortest-path RREQ forwarding results is that the use of “standard” jitter does not have an significant impact on the RREQ flooding performance, when compared with the no-jitter setting; neither in terms of collisions, control overhead or data path length. Differences can be observed, in contrast, between window jitter and “standard” and no jitter.i.e., overhead) by reducing the cases of delay inversion, as shown in i.e., the time between RREQ transmission and reception of the corresponding RREP) with respect to the network density in the shortest-path RREQ forwarding strategy, depicted in Regarding the evolution of route discovery delay transmissions being the remedy commonly employed for alleviating these losses. The performance of the flooding operation has, as this paper has presented, a direct impact on the quality of the routes discovered. A similar conclusion was found in , in whicFor the purpose of route discovery, the performance of a flooding operation can be evaluated through three main criteria: (1) the resulting quality (path lengths) of discovered routes; (2) the delay incurred when discovering a new route; and (3) the number of collisions caused by route request flooding. Collisions and route quality have a direct impact on the network load: packet collisions may lead to retransmissions, longer paths imply that more transmissions happen than would otherwise be required between a source and a destination. Alas, with respect to jitter, it is not possible to optimize a flooding operation according to all criteria: an infinitely large jitter interval might, for example, eliminate collisions, but would incur unacceptable delays before routes were established. This section summarizes the trade-offs achieved by the different mechanisms proposed and evaluated in this paper.i.e., without jitter) in wireless multi-hop networks entails an unacceptable level of packet collisions. The use of “standard jitter”  reduces the amount of flooding collisions, at the expense of: (1) slowing down the flooding process; and (2) causing the “delay inversion” effect, as described in Section 3. According to the analysis of Section 4, delay inversion does not depend on the length of jittering time interval (mJ) and becomes more relevant in long paths; that is, in big-diameter networks.It is well established that pure flooding (RFC 5148  distribumαJ). This dramatically reduces the probability that delay inversion occurs, and thereby an improvement of the quality of discovered routes (in the shortest-delay mode) or a reduction in control overhead and packet collisions (in the shortest-path mode). It also increases the number of collisions in the first hop of flooding of the flooding process , as jitter selection becomes more deterministic. Further evaluation of the performance of RREQ flooding, with “standard” jitter and window jitter, via graph and network simulations (Sections 5 and 6), provide empirical evidence of the route quality improvement with window jitter and shows that the number of collisions along the explored paths is also reduced with this mechanism. Additional collisions in the first hop are thus largely compensated for by the reduction in the number of transmissions farther away; an effect that becomes relevant as the network density increases. Window jitter introduces a minimum non-zero pre-forwarding delay , adaptive jitter presents clear advantages with respect to the two static configurations (standard and window jitter). In adaptive jitter, pre-forwarding delays depend on the quality of the link through which the packet was received. Not surprisingly, the performance is then better than in static jitter configurations, both in terms of control overhead and route quality. Under the shortest-path mode, route quality is similar for all settings, but adaptive jitter reduces the number of RREQs and RREPs up to 30%. There is still a trade-off between route quality and flooding delay, meaning that adaptive jitter entails additional delays; such a delay becomes negligible as the network density grows.8.Jitter is used for message flooding in wireless multi-hop networks, in order to avoid data losses. It has been shown to be an efficient remedy in proactive routing protocols, such as OLSR , and hasHowever, when applied to the route discovery process in reactive routing protocols, using the jitter mechanism prescribed by RFC 5148  has sevei.e., jitter according to [i.e., immediate retransmission without jittering). This is not only in terms of incurred delay, but also in terms of avoided collisions, control overhead and quality of discovered routes. Comparisons have been performed by way of both probabilistic analysis and experimental analysis through graph and network (NS2)-based simulations.Not using jitter is not an option: route discovery relies on the flooding of RREQs, and with intermediate routers immediately retransmitting a received RREQ, collisions will still occur and (RREQ) packets will be lost. Therefore, this paper explores ways to employ jitter on RREQ flooding for reactive routing protocols, while minimizing the impact of delay inversion. In particular, window jitter and adaptive jitter, are compared to “standard” jitter (rding to ); and, oThe proposed “adaptive jitter” is a simple modification to “standard” jitter, yet despite this simplicity, is able to, very substantially, reduce the probability of experiencing the effect of delay inversion, manifested by sub-optimal routes resulting from the route discovery process. In its most general version, it simply consists of sending flooded messages sooner across better links, which entails a higher probability of the route discovery process resulting in optimal routes. This paper also details the impact of jitter parameters and distribution, on flooding and route discovery performance. Depending on the characteristics of the network, the information available to the routers and the specific route discovery mode, different trade-offs between the main involved parameters  can be achieved with the proposed jitter distributions.The results, pretended in this paper, indicate that window jitter  is particularly beneficial for reducing overhead in dense networks, in which packet collisions are more frequent and, therefore, delay inversion more likely to happen, due to the the diversity of available paths. These jitter mechanisms thus are interesting for large resource constrained networks, such as in battery-powered mesh and sensor networks, where extraneous transmissions and long paths are particularly harmful to the network lifetime. Future work should see to confirm these results, both through additional analysis, more complex network simulations and real-world experiments. In particular, future work should study the impact of router mobility and wireless channel unreliability on the applicability of window and adaptive jitter.


PD-L1 expression on neutrophils contributes to the impaired immune response in infectious disease, but the detailed role of PD-L1 expression on neutrophils in HCC remains unclear.We investigated the phenotype and morphology of neutrophils infiltrated in tumour tissues from both patients with HCC and hepatoma-bearing mice.+ neutrophils-to-PD-1+ T cells was higher in peritumoural tissue and better predicted the disease-free survival of patients with HCC. We further confirmed a higher frequency of PD-L1+ neutrophils and PD-1+ T cells in hepatoma-bearing mice. Functionally, the PD-L1+ neutrophils from patients with HCC effectively suppressed the proliferation and activation of T cells, which could be partially reversed by the blockade of PD-L1.We found that neutrophils dominantly infiltrated in the peritumoural region. The neutrophil-to-T cell ratio (NLR) was higher in peritumoural tissue than that in the intratumoural tissue and was negatively correlated with the overall survival of patients with HCC. Infiltrating neutrophils displayed a phenotype of higher frequency of programmed cell death ligand 1 (PD-L1) positive neutrophils. The ratio of PD-L1Our results indicate that the tumour microenvironment induces impaired antitumour immunity via the modulation of PD-L1 expression on tumour infiltrating neutrophils.The online version of this article (doi:10.1186/s13046-015-0256-0) contains supplementary material, which is available to authorized users. Hepatocellular carcinoma (HCC) remains one of the most malignant cancers, as the incidence and mortality of this cancer continues to increase each year . The proTumour-associated inflammation has been considered one of the hallmarks of cancer progression . EvidencAmong a number of mechanisms that mediate T cell anergy and tumour immune evasion in patients with HCC, Program death ligand 1 (PD-L1) and Program death (PD-1) have emerged as central players , 15. It Neutrophils have been considered non-professional antigen presenting cells (APCs). Recent studies have demonstrated the expression of co-inhibitory molecules, such as PD-L1, following in vitro exposure to cytokines  or after+ neutrophils in the peritumoural region of patients with HCC. A lower level of neutrophil infiltration in peritumoural tissue predicted a better overall survival in patients with HCC. A decreased PD-L1+neutrophil/PD-1+ T cell ratio in peritumoural tissues was closely correlated with prolonged recurrence-free survival (RFS) of patients with HCC. Furthermore, tumour-derived factors, including GM-CSF and TNF-α, contributed to PD-L1 expression on neutrophils in patients with HCC. The levels of tumour-derived soluble factors, including GM-CSF, TNF-α, G-CSF and MCP-1, are higher than those in the tumour site. Taken together, we propose an alternative explanation that higher level of neutrophils in peritumoural tissues was negatively correlated with the prognosis in hepatocellular carcinoma.In this study, we revealed an accumulation of PD-L1n = 32) were patients at Beijing hospital who had received a physical examination. All samples were collected in accordance with local ethical guidelines. The ethics committee of Peking University People’s Hospital approved the protocol of all written informed consent. Conventional clinicopathologic variables and treatment modalities that were given after resection are detailed in Additional file All 149 patients with HCC from Guilin Medical College Affiliated Hospital were enrolled according the following criteria: a) the pathology of each patient was confirmed; b) patients did not exhibit signs of distant metastasis and had not received anticancer therapy prior to surgery. The tumour stage was determined according to the 6th edition tumour-node-metastasis (TNM) staging system . Data we5 per mouse in 100 μl sterile saline to obtain tumour-bearing mice. Mice in the control group received an equivalent volume of sterile saline.With regards to the animal experiment, 6–8-week-old female BALB/c mice were used. All animal protocols were approved by the Review Board of Peking University People’s Hospital. All animals were provided by China Academy of Military Medical Science and bred in specific pathogen-free (SPF) animal facilities in our hospital. H22 cells (hepatoma cell line) were injected in BALB/c background mice subcutaneously at 4 × 10For in vitro experiments, human HCC cell line of HepG2 and MHCC-97H were used. LX-2 cell, which was established for hepatic stellate cells, was used to mimic the liver microenvironment.+ T cells and CD8+ T cells were enriched by a magnetic cell separation system . The purity was above 95 % and the viability was above 93 %, as evaluated by flow cytometry and 0.4 % trypan blue (Gibco BRL) staining, respectively.Peripheral blood- and tissue-infiltrated cells were collected and isolated as previously described. Tissue-infiltrated lymphocytes were obtained by density gradient centrifugation with 40 % Percoll and 80 % Percoll . For functional analysis, human neutrophils were isolated and then purified according to CD16 and CD66b coexpression by the BD Aria II Cell Sorting System . CD4Biopsy samples were fixed in 10 % formalin and paraffin-embedded. Then, the samples were cut into 5-μm-thick sections. Immunohistochemistry was performed according to the appropriate protocols as previously described . The folFor flow cytometry, the following antibodies for the analysis of human samples were used: FITC-conjugated anti-CD16, PE-conjugated anti-PD-L1, PerCP-conjugated anti- CD45, APC-conjugated anti-CD11b, APC-conjugated anti-CD14, FITC-conjugated anti-CD3, PE-conjugated anti-CD8, PerCP-conjugated anti-CD4 and APC-conjugated anti-PD-1. All human antibodies were purchased from BD Bioscience . Antibodies used in the mouse experiments were purchased from eBioscience  as follows: FITC-conjugated anti-Gr-1, PE-conjugated anti-PD-L1, PerCP-conjugated anti-CD11b, APC-conjugated anti-CD48, APC-conjugated anti-CD8, FITC-conjugated anti-CD3, PE-conjugated anti-PD-1, and PerCP-conjugated anti-CD4. Data were analysed with FlowJo5.6.7 .Cytokines in the culture supernatant and tissue lysates were evaluated with the Luminex200 System . The detection kit includes the following cytokines: G-CSF, GM-CSF, IL-6, IL-1β, IL-8, IL10, IL-17, TNF-α, IFN-γ, TGF-β, MCP-1, SDF-1 and MIP-1α. Briefly, hepatoma cell lines were maintained at 70 % cell confluence in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % FBS. After 48 h, the supernatant was collected and centrifuged to deplete contaminated cells. In some experiments, the tumour cells were pretreated with LX-2 supernatant for 24 h. Tumour tissues were preserved at −80 °C until use. We used 500 μl of tissue lysis buffer for 30 mg of tumour tissue, peritumoural tissue and adjacent non-tumour tissue. Lysis buffer was applied for the analysis of cytokines.Tumour culture supernatant (TSN) was obtained as described above. Briefly, hepatoma cell lines were maintained at 70 % cell confluence in DMEM supplemented with 10 % FBS. After 48 h, the supernatant was collected and centrifuged to remove the contaminated cells. Purified neutrophils from healthy donors were then exposed to culture medium with TSN  from hepatoma cell lines or cytokines including TNF-α (200 U/ml) and GM-CSF (20 ng/ml). Neutrophils cultured in medium alone served as the control group.Total RNA of the neutrophils was extracted with Trizol reagent . cDNA was synthesized with High Capacity RNA-to-cDNA Kit . qPCR was performed with a LightCycler 480  and SYBR Select Master Mix . The specific primer sequences for human PD-L1 and GAPDH  are listed as follows: PD-L1, forward 5’-TGGCATTTGCTGAACGCATTT-3’, reverse 5’-TGCAGCCAGGTCTAATTGTTTT-3’ and GAPDH, forward 5’- ACAACTTTGGTATCGTGGAAGG-3’, reverse 5’- GCCATCACGCCACAGTTTC -3’, respectively. All of the above studies were performed according to the manufacturer’s protocol.t-Test. Correlations between parameters were assessed with the Pearson correlation analysis and linear regression analysis. Univariate and multivariate analysis were performed with the Kaplan–Meier method as well as the Cox proportional hazards regression model and were compared with the log-rank test. Values of P < 0.05 were considered statistically significant between the different groups. The in vitro studies were performed independently in at least three separate experiments.All statistical analysis was performed with SPSS 16.0 . Differences in the immunostaining of the cells between the subgroups were determined by the paired samples + neutrophils as well as CD3+ T cells  in the peritumoural tissue (pNLR) and in the intratumoural tissue (iNLR), and found that the pNLR was significantly higher than the iNLR . Furthermore, a Pearson correlation coefficient analysis confirmed the hypothesis that the number of infiltrated neutrophils was negatively correlated with the number of lymphocytes in the peritumoural regions  but not in the intratumoural regions .To investigate the distribution of neutrophils and T lymphocytes within the tissue, we performed immunohistochemical staining for CD66 and CD3 in tumour samples from 149 patients with HCC. Strikingly, compared with the tumour nests, the peritumoural tissues contained a higher number of CD66blls Fig. . This su+ neutrophils in peritumoural tissue, >305 cells/field under the microscope was considered high (n =120). The univariate analysis revealed that an increase in the number of intratumoural neutrophils was not significantly associated with patients’ postoperative survival . In contrast, the patients with increased peritumoural CD66b+ neutrophils showed a decreased overall survival rate . Next, after we performed immunohistochemistry for the double-labelling of CD66b and CD3, we divided the 69 patients with HCC into two groups according to the median value of the NLR. The postoperative survival time of individuals in the lower CD66b+/CD3+ ratio group was significantly longer than that of individuals in the high NLR group . We found that both high pNLR  and high iNLR  were significantly correlated with poor overall survival (OS). In addition, the pNLR may be better than iNLR for the prediction of OS in patients with HCC.Based on the above observations, we tested the correlation between the infiltration of neutrophils in different regions and the clinical prognosis of patients with HCC. We followed 149 patients with HCC for 90 months. According to the median value of the number of CD66b+ T and CD8+ T cells . And the frequency of PD-1-positve cells was also significantly increased on CD4+ and CD8+ T cells that were isolated from intratumoural and peritumoural tissues , the surface expression of PD-L1 on circulating neutrophils was higher in patients with HCC Fig. . We obsells Fig. . Moreoven = 65 for peritumoural, n = 64 for intratumoural). PD-L1/PD-1 pathway is usually associated with tumour progression and early recurrence. We calculated the number of PD-L1+ neutrophils and the number of PD-1+ T cells. The PD-L1+ neutrophil/PD-1+ T cell ratio in both peritumoural and intratumoural tissues could predict the postoperative recurrence-free survival (ROS) .The above findings were further confirmed by the immunohistochemical staining of tumour tissues and adjacent peritumoural tissues from a separate cohort of patients with HCC  from the hepatoma cell lines of HepG2 and MHCC-97H or from LX-2-pretreated hepatoma cells (HepG2/LX-2 TSNs) for the indicated time points. The results showed that PD-L1 expression on neutrophils was increased in a time-dependent manner in response to TSNs or HepG2/LX-2 TSN treatment. In addition, neutrophils that were exposed to HepG2/LX-2 TSN exhibited a greater expression of PD-L1 than those neutrophils that were exposed to TSNs or control culture medium Fig. .Fig. 5PDBased on the above results, the cytokines in the tumour supernatant and in the tissue lysates from tumours, peritumoural and adjacent non-tumour tissues were analysed with a Luminex 200 system as described in the materials and methods section. We observed higher levels of several cytokines, including G-CSF, IL-1β, GM-CSF, IL-6, IFN-γ, TNF-α, TGF-β, RANTES, MCP-1, SDF-1 and MIP-1α, in TSNs compared with control culture medium  in peritumoural tissue were correlated with prolonged patient survival after surgical treatment. These findings suggest that patients with a high level of peritumoural neutrophil infiltration and pNLR require a much closer follow-up after surgery. We reported here that the co-inhibitory molecule PD-L1 was overexpressed on neutrophils from patients with HCC. The tumour microenvironment contributes to the up-regulation of PD-L1 expression on neutrophils both in vitro and in vivo. Although PD-L1 expression has been observed on neutrophils in patients with infectious diseases, such as patients who are infected with tuberculosis  and HIV P value than the number of infiltrating neutrophils, which indicates the regulatory function of neutrophils on adaptive immunity in the development of HCC. Despite the different conclusions of these studies, our study was in accordance with those from the two groups that found that neutrophils predominantly infiltrated the peritumoural tissues rather than the tumour site itself. This suggests that a high level of peritumoural-infiltrating neutrophils should not be ignored in the management of HCC.The prediction of survival is critical to the management of HCC. Our data suggest that the level of peritumoural neutrophil infiltration as well as the pNLR value could be used for the prediction of patient survival after surgery. Data from clinical studies have indicated the correlation between the presence of neutrophils and poor prognosis. An increased level of neutrophils in peritumoural tissue promotes angiogenesis at the edge of the tumour via the production of MMP-9 , which i+ neutrophil/CD8+ T cell ratio has been shown to be associated with prolonged RFS and OS in patients with HCC [+ neutrophil/CD3+ T cell ratio in peritumoural tissue was significantly higher and a better predictor of patient survival than that in the tumour itself. The peritumoural site is a barrier to the migration and dissemination of tumour cells in the earlier stages of cancer development. On the contrary, the peritumoural site is often the favourable “special zone” for the dissemination of tumour cells due to angiogenesis and the immunosuppressive micro milieu; this milieu is characterised by the infiltration of multiple types of stromal cells including lymphocytes, TANs, TAMs, MDSCs, TAFs and vascular endothelial cells. The local tumour microenvironment contributes largely to the phenotypic and functional modification of neutrophils. A morphological analysis of the peritumoural marginal region has shown that this area is always rich in tumour-associated fibroblasts (data not shown) and immune cells, including neutrophils. Neutrophils accumulate in the tumour site due to the tumour microenvironment-derived cytokines and chemokines. For example, IL-17- producing T cells recruit neutrophils that then accumulate in the peritumoural region via the expression of chemokines by endothelial cells [A number of studies have indicated a prognostic value of a higher preoperative NLR in patients with HCC , 13, 31.al cells , 11. Tumal cells . The pre+ tumour-infiltrating neutrophils in HCC, especially along the edges of the tumours tissue. This confirmed the idea that stromal cells in peritumoural tissues contribute largely to the overexpression of PD-L1 on neutrophils.Tumour-supportive neutrophils are rich in tumour-promoting products such as arginase, MMPs and VEGF. Cytokines and chemokines are an efficient impetus for the migration of neutrophils. For example, neutrophil infiltration is closely related to the presence of TGF-β in the tumour site  or to thOur study provides a possible explanation for the previous reports with regards to the prognostic implications of greater numbers of neutrophils and increased NLR. In addition, we demonstrated the critical role of the tumour milieu in the modulation of the phenotype and function of tumour-infiltrated neutrophils. Although neutrophils might be considered one of the targets of tumour therapy, future immune therapy strategies may harness the combinatorial targets.


Neuroendocrine tumours occur throughout the body but are rare in the head and neck region and particularly rare in the middle ear. Clinical findings are often nonspecific and therefore pose a diagnostic challenge. Furthermore, the nomenclature of neuroendocrine tumours of the middle ear is historically controversial. Herein a case is presented of a middle ear adenoma in a 33-year-old patient who presented with otalgia, hearing loss, and facial nerve palsy. A brief discussion is included regarding the histopathological features of middle ear adenomas and seeks to clarify the correct nomenclature for these tumours. Neuroendocrine tumours of the middle ear, although rare, are a significant clinical entity and a diagnostic challenge. Middle ear neuroendocrine tumours were first reported in 1976 by Derlacki and Barney as an “adenomatous tumour” of the middle ear  in orderWe describe the unique histopathological findings of a neuroendocrine adenoma of the middle ear in a 33-year-old male who presented to our institute with acute otalgia, hearing loss, and facial nerve palsy.A 33-year-old male presented to our emergency department with an acute onset of right sided facial nerve paralysis associated with a 4-day history of severe otalgia and intermittent tinnitus. He had a known background of a stable middle ear mass for which he was observed for 3 years at a different institution. He was otherwise fit and healthy with no comorbid illnesses or regular medications. Examination findings were consistent with an inflammatory middle ear effusion with a bulging tympanic membrane associated with right sided facial nerve palsy. Tuning fork tests demonstrated a conductive hearing loss in the affected ear. All other cranial nerves were intact. The patient was admitted under ENT for intravenous antibiotics and steroids. Within 72 hours of antibiotics administration, the otalgia subsided with improving facial nerve movement as well. He was discharged on oral antibiotics with follow-up in our outpatient department. With resolution of his middle ear infection, a pale red-pink mass was visible behind the tympanic membrane.Further imaging demonstrated a middle ear mass with partial disruption of the facial canal. A subsequent angiogram demonstrated an avascular middle ear mass. Mixed hearing loss was demonstrated in the right ear on audiogram. The patient was electively taken to the operating theatre for a middle ear exploration and mastoidectomy with the intention of confirming the nature of the tumour and performing a subtotal removal. Intraoperative frozen section raised the possibility of a neuroendocrine tumour. The patient was successfully discharged on postoperative day 1. At 2 months follow-up postoperative hearing was preserved with normal facial nerve function and no evidence of recurrence.Examination of histological sections stained with haematoxylin and eosin revealed small fragments of mucosa with underlying pieces of vital bone. The mucosa was surfaced by nonkeratinising stratified squamous epithelium which showed no epithelial dysplasia with no evidence of surface origin of the tumour. The underlying lamina propria was extensively infiltrated by an unencapsulated tumour. The tumour cells were arranged in small irregular nests and trabeculae with surrounding fibrosis. Occasional glandular structures were also identified. The individual tumour cells had scanty granular eosinophilic cytoplasm with hyperchromatic nuclei. Mitoses were not observed. There was no evidence of necrosis within the tumour. The tumour cells demonstrated positive staining for cytokeratin (AE1/AE3) with keratin 7 (CK7) staining the luminal cells in the glandular structures. The neuroendocrine immunohistochemical markers  were positive. No sustentacular cells were identified (S-100 negative) and the tumour had a low proliferation index of 3-4% (Ki-67).The so-called “carcinoid” tumour of the middle ear has previously been described by many names including neuroendocrine adenoma of the middle ear, adenomatous tumour of the middle ear, and adenocarcinoid tumours. We have opted for the term neuroendocrine adenoma of the middle ear as proposed by Thompson  based upDerlacki and Barney initially described the histological appearance of the tumour in 3 patients in their twenties as nests of cells with glandular epithelial appearance. The first case of “carcinoid tumour” in the middle ear which appears in the literature is described by Murphy et al. in 1980 who described neuroendocrine differentiation resembling an adenomatous tumour .With regard to nomenclature, we favour using the term neuroendocrine adenoma of the middle ear (NAME) as proposed by Thompson and Mills for this benign tumour over the term carcinoid tumour which by definition represents a well-differentiated neuroendocrine carcinoma with metastatic potential , 5. The Histologically NAME presents an unencapsulated tumour with similar cytomorphologic and staining patterns but different architectural growth patterns. They usually exhibit a dual cell population with growth patterns ranging from anastomosing cords, trabeculae to solid sheets, and glandular structures . The celNeuroendocrine adenomas of the middle ear occur most commonly in the 20–30-year age group. Nevertheless, adolescent cases have been described as well –9. The mDiagnosis is difficult and often not complete until histopathological examination has been undertaken. This is demonstrated in the literature with a number of cases initially managed surgically with a suspected diagnosis of cholesteatoma  or paragAlthough the tumours are usually unencapsulated and may on occasion exhibit an “infiltrative growth pattern” and very rarely metastasise, they are considered benign since the other features of malignancy which would include cytologic pleomorphism, mitotic activity, and necrosis as well as perineural and lymphovascular invasion are not observed.Patients should be followed up closely due to the possibility of recurrence and metastatic disease. Serial clinical examination should suffice with imaging indicated with suspicion of recurrence. Knerer et al. reported one case of recurrence for which a subtotal petrosectomy was indicated . More reThe majority of cases of middle ear neuroendocrine tumours reported in the literature were treated with radical mastoidectomy including resection of the ossicular chain where required. Further surgical treatment is indicated in the event of metastasis and has been reported in the literature with selective neck dissection. Parotid metastasis has been successfully managed with superficial parotidectomy. Adjuvant radiotherapy has been used in some cases in the literature; however, due to the paucity of available data, outcomes are difficult to measure and the impact of any radiotherapy administered is difficult to assess. In conclusion, although technically difficult, surgical resection of the tumour as well as the ossicles is the treatment of choice for NAME with excellent overall prognosis, a recurrence rate of only about 15% and only few documented cases of metastatic disease.