Acetazolamide (AZA), the only drug generally approved for hydrocephalus, is effective in only 25-30% of patients, and its effect on basal fluid flow in choroid plexus (CP) is unknown. The drug reversibly inhibits Aquaporin 4 (AQP4), the most highly expressed ‘water pore’ in the brain, and it is postulated that it reduces CSF production by modulating the expression and/or function of AQP1 . In this study, we sought to elucidate the direct effect of AZA on basal fluid flow in CP.CP tissues were harvested from 10-day Sprague-Dawley rat pups. CP cultures were grown to confluence in Transwell permeable supports and tested using: a) Kir 7.1 immunocytochemistry to confirm CP cell morphology; b) Lucifer Yellow assay and trans-epithelial electrical resistance (TEER) to determine level of confluence; c) fluid assays using TRITC-labeled Dextran to assay the direction and extent of fluid flow through the monolayers; d) expression of AQP1 protein by immunoblotting and immunocytochemistry.Immunblotting and immunocytochemical analyses showed that AQP1 protein level decreases rapidly with 10 um AZA treatment. The reduction of AQP1 protein was transient as its level returned to baseline 12 hours after AZA exposure. Transwell fluid assay indicated that the early loss of AQP protein is correlated with decreased basal fluid flow in CP.The observed effect of AZA on AQP1 protein level suggests that AZA can directly modulate basal fluid flow in CP. The AZA induced effect in CP resulted in an initial fluid flow into the basolateral side of the transwells, with subsequent flow into the apical side of the CP membranes. Our results indicate that modulating AQP1 function via effectively targeted pharmaceuticals could potentially yield new treatments for hydrocephalus.


The patient's delay in the visit to a hospital seems to play an important role in prognosis in invasive cutaneous squamous cell carcinoma (SCC). This report explored prognostic factors of cutaneous SCC focusing on patient delay in hospital visit. Data of 117 Japanese patients who were treated for invasive cutaneous SCC in our facility between 2000 and 2010 were used for analysis. A multivariate Cox proportional-hazard modelling revealed that a pair of TNM stage  and poorer histological differentiation , and a pair of tumour size  and rapid growth  were a prognostic factor whereas patient delay in hospital visit was not. However, patient delay in hospital visit was correlated with larger tumour size. Cutaneous squamous cell carcinoma (SCC) is a malignant tumour of keratinocytes that tends to metastasise and leads to mortality. In cutaneous SCC, a number of clinical and histological factors have been found to be poor prognostic factors. For example, tumour size, gender, preceding lesion, histological findings such as degree of the differentiation, and tumours location have been reported as a prognostic factor of local recurrence, metastasis, and disease-specific death –5. In adA patient registry system from the Department of Dermatology at the Kyoto University Hospital was accessed to retrieve medical information on patients in whom invasive SCC of the skin was histopathologically diagnosed and treated from 2000 to 2010. During that period, one hundred and forty-nine patients were retrieved. Twenty cases were excluded because of tumour in situ, and two cases because of lack in clinical and pathological information. Ten cases with genital SCC were excluded because of different staging system. The remaining 117 Japanese patients who were treated for invasive cutaneous SCC were eligible for analysis. Followup was performed every 3 to 6 months by physical examination and blood works. A mean followup was 29.9 months. Variables were demographic characteristics, TNM stage of cutaneous squamous cancer  [Descriptive statistics were calculated to estimate the frequencies, means, and standard deviations of the study variables. Survival analyses were conducted from the date when a patient had noticed the tumour to the date of death with use of the Kaplan-Meier method. Data from patients who were alive at the study followup  were censored on that date. U test were used. Preliminary analysis was conducted exploring whether patient delay in hospital visit varies according to change of other prognostic factors. As distribution of patient delay in hospital visit was skewed right, Spearman rank correlation coefficients and Mann-Whitney P based on Wald statistics higher than 0.1 for variable removal from the model. As a pair of stage and tumour size showed multicollinearity, either of them was used for the initial multivariate model. The results were adjusted for age. The SPSS 12.0  was used for all statistical analyses. Missing values were excluded from each analysis. All given probabilities are two tailed and the significance levels were set at 5%. Then, a Cox proportional-hazards model was used for univariate and multivariate analysis to evaluate the effect of predictors on disease-specific survival . First, P = 0.59). A total of 117 patients were included into the analysis. The characteristics of the patients were shown in P = 0.04) and less rapid growth . Preliminary analysis revealed that patient delay in hospital visit was associated with other prognostic determinants. Longer patient delay was significantly correlated with larger tumour size (Spearman rho rank correlation coefficient = 0.20, Of 117, eighty-eight cases were considered in the Cox regression analysis, as 18 cases had missing values, and 11 cases were censored before the earliest event occurred. In univariate analysis tumour size, TNM stage, rapid growth, lymphovascular invasion, and the differentiation were identified as a significant determinant, while patient delay in hospital visit, age, and sex were not. Multivariate model revealed that a pair of TNM stage and poorer histological differentiation and a pair of tumour size and rapid growth were a prognostic factor . The current study addressed the question whether patient delay in hospital visit and other factors affect the prognosis of invasive cutaneous SCC using multivariate analysis. As expected, tumour size, TNM stage, differentiation of the tumour , 3, and Based on the literature , we firsIn contrast, the differentiation of tumour was shown to be an independent prognostic factor as has been expected. As differentiation of tumour is not considered when staging, careful followup is required for cases with cutaneous SCC of poor differentiation irrespective of stage. In this study, patients with genital SCCs were not considered because of different staging system and small sample size. Genital SCCs tended to be at more advanced stage and of poorer differentiation than SCCs in other site at the time of diagnosis . This phSeveral limitations of the study deserve mention. First, this study was conducted at a single medical facility and consisted of a relatively smaller sample, thereby limiting generalization of the results to patients of other hospitals. In addition, because the sample lacked diversity with respect to race, we were unable to assess the effect of that important factor on outcomes. However, our study reflects the prognostic factors of cutaneous SCC in Japanese population. In summary, our study showed that tumour size, TNM stage, differentiation of the tumour, and rapid growth were thought to be a prognostic factor, while prolonged patient delay seems to affect indirectly the prognosis by influencing larger tumour size.


We investigate a three-dimensional multiscale model of vascular tumour growth, which couples blood flow, angiogenesis, vascular remodelling, nutrient/growth factor transport, movement of, and interactions between, normal and tumour cells, and nutrient-dependent cell cycle dynamics within each cell. In particular, we determine how the domain size, aspect ratio and initial vascular network influence the tumour's growth dynamics and its long-time composition. We establish whether it is possible to extrapolate simulation results obtained for small domains to larger ones, by constructing a large simulation domain from a number of identical subdomains, each subsystem initially comprising two parallel parent vessels, with associated cells and diffusible substances. We find that the subsystem is not representative of the full domain and conclude that, for this initial vessel geometry, interactions between adjacent subsystems contribute to the overall growth dynamics. We then show that extrapolation of results from a small subdomain to a larger domain can only be made if the subdomain is sufficiently large and is initialised with a sufficiently complex vascular network. Motivated by these results, we perform simulations to investigate the tumour's response to therapy and show that the probability of tumour elimination in a larger domain can be extrapolated from simulation results on a smaller domain. Finally, we demonstrate how our model may be combined with experimental data, to predict the spatio-temporal evolution of a vascular tumour. Angiogenesis marks an important turning point in the growth of solid tumours. Avascular tumours rely on diffusive transport to supply them with the nutrients they need to grow and, as a result, they typically grow to a maximal size of several millimetres in diameter. Growth stops when the rate at which nutrient-starved cells in the tumour centre die balances the rate at which nutrient-rich cells on the tumour periphery proliferate. Under low oxygen, tumour cells secrete angiogenic growth factors that stimulate the surrounding vasculature to produce new capillary sprouts that migrate towards the tumour and the new vessels increase the supply of nutrients to the tissue, enabling the tumour to continue growing and to invade adjacent healthy tissue. At a later stage small clusters of tumour cells may enter the vasculature and be transported to remote locations in the body, where they may establish secondary tumours and metastases In more detail, the process of angiogenesis involves degradation of the extracellular matrix, endothelial cell migration and proliferation, capillary sprout anastomosis, vessel maturation, and adaptation of the vascular network in response to the blood flow Tumour growth and angiogenesis can be modelled using a variety of approaches  each with their own cell cycle and subcellular-signalling machinery. Nutrients are supplied by a dynamic vascular network, which is subject to remodelling and angiogenesis. The interactions between the different modules are depicted in https://computation.llnl.gov/casc/sundials/main.html) to integrate the subcellular ODEs, and SuperLU (http://crd.lbl.gov/~xiaoye/SuperLU/) to solve the linear systems for the flow calculation.Our model is formulated on a regular grid that subdivides the simulation domain into lattice sites. Each lattice site can be occupied by several biological cells whose movement on the lattice is governed by reinforced random walks, and whose proliferation is controlled by a subcellular cell cycle model. The vascular network consists of vessel segments connecting adjacent nodes on the lattice, with defined inflow and outflow nodes with prescribed pressures. We also specify the amount of haematocrit entering the system through the inlets. The vessel network evolves via (i) sprouting of tip cells with a probability that increases with the local VEGF concentration, (ii) tip cell movement is described by a reinforced random walk, and (iii) new connections forming via anastomosis. In addition, vessel segments with low wall shear stress may be pruned away. Elliptic reaction-diffusion equations for the distributions of oxygen and VEGF are implemented on the same spatial lattice using finite difference approximations, and include source and sink terms based on the location of vessels (which act as sources of oxygen and sinks of VEGF) and the different cell types (e.g. cells act as sinks for oxygen and hypoxic cells as sources of VEGF). The flowchart in http://www.povray.org) and OpenInventor (http://oss.sgi.com/projects/inventor/).It was necessary to implement several changes to the computational algorithm when extending the model from 2D to 3D. For example, in order to reduce the calculation time of the diffusion fields, the resulting linear system of equations is now solved with an iterative GMRES-solver, rather than the direct SuperLU-solver which was used in http://www-personal.umich.edu/~wagnerr/MersenneTwister.html). In order to assess the degree of stochasticity in our model simulations, we carried out multiple simulations using different seeds for the random number generator. In the next section we will study the variation in mean values of our stochastic simulations as, for example, domain size varies. Therefore it is necessary to check if these mean values really differ significantly or if they only represent different samples that belong to the same probability density. We estimate the confidence intervals of our mean values by applying a bootstrapping method—lternatively one could apply non-parametrical methods, such as the Wilcoxon-Mann-Whitney-Test Stochastic components of the model include endothelial tip cell emergence and random walks of different cell types, with trajectories generated from a sequence of random numbers , so that overall VEGF production is significantly higher in the tumour. This elicits a stronger angiogenic response in the tumour than in the normal tissue because the sprouting probability increases with the VEGF concentration (Equation (11) in Here we focus on establishing whether it is possible to extrapolate simulation results obtained for small subsystems to their associated larger ones. An overview of the numerical experiments that we perform is given in ond case , we consot occur .In It was not possible to reproduce the behaviour of the large domain by the small domain. In order to determine whether this result is due to our choice of boundary conditions we implemented periodic boundary conditions. Periodic boundary conditions allow vessel connections to “wrap around” the domain and mimic connections between subsystems in larger domains. As before simulations are carried out in three different settings. In the first case (where reflecting/zero-flux boundary conditions are imposed as discussed above), angiogenic sprouts are reflected at boundaries . In the second case, singly-periodic boundary conditions are applied: the domain is periodic in the ions see . In the tion see .In Guided by the results presented above, we now consider a subsystem that is initially characterised by multiple vessels with different pressures and flow orientations. In order to determine whether the multi-vessel subdomains are representative of larger domains, simulations with equivalent initial distributions of vessels but different domain sizes are performed. Averaged results from 10 simulations are presented in Further, in this case, the choice of boundary conditions  has a strong effect on the growth and composition of the tumour and the associated long-time volume fractions. The above results demonstrate the influence of domain size on vascular tumour growth. Therefore it is important to assess how the domain size affects predictions about the efficacy of cancer therapies. A key question is whether predictions about tumour response to therapy in larger domains can be extrapolated from simulation results on smaller domains. We investigated these issues by performing the following simulations: a small tumour is implanted in a poorly vascularised tissue with reduced oxygen delivery, such as would arise following exposure to a vascular-targeting agent  give a reasonable representation of the dynamics in the larger domain, but a single subdomain can generate quite different characteristics. Even with periodic boundary conditions, one vessel pair is not representative of two (or more). This is in part because, although periodic boundary conditions allow vessel connections to be made from a parent vessel to itself . Imposing periodicity in the axial direction of the initial vessels would demand that the vessel radii at both ends of the domain be equal, which is not usually the case. Pressure and flow calculations are also problematic: one has to keep track of the boundary across which vessels leave and re-enter the domain. The total pressure drop along each vessel is similar in a periodic alignment, whereas the absolute pressures differ. If a sprout from close to a low-pressure outflow leaves the domain in a downstream direction it re-enters at the opposite domain side and can connect to the original network close to a high-pressure inflow. This would lead to a very short vessel segment with a nearly maximal pressure drop, which could lead to extreme vessel dilation. The same principle holds for vessels that leave the domain in the upstream direction. One potential resolution to this problem would be to consider only the pressure drop Building on these results for a basic subdomain with two parallel countercurrent initial vessels, we showed that the choice of basic repeating unit can have a significant impact on the system's dynamics. In particular, for a unit of eight parallel vessels with equal pressure drops, but a variety of mean pressures, we found that a single unit was representative of larger domains. In these cases, the choice of boundary conditions had a strong influence on the system's dynamics. For doubly-periodic boundary conditions the tissue grows in an efficient manner, with a low vascular density—this is because the initial vessels are in close proximity and their mean pressures are sufficiently different Click here for additional data file.Video S2Simulation showing the dynamics of normal cells and the vascular system with two straight vessels as initial condition which correspond to (4.09 MB WMV)Click here for additional data file.Video S3Simulation showing tumour evolution with an experimentally-derived initial vascular network, illustrating the steps depicted in (5.59 MB AVI)Click here for additional data file.Video S4Simulation showing the development of a vascular system with increased endothelial cell chemotaxis, with the normal cells faded out. (Results in (1.12 MB WMV)Click here for additional data file.Video S5Simulation showing the evolution of a vascular system, with 16 straight initial vessels, embedded in a population of normal cells. Increased endothelial tip cell chemotaxis leads to the vascular system depicted in (2.29 MB WMV)Click here for additional data file.Video S6Simulation showing tumour cells growing in a normal vascular network and additionally inducing angiogenesis ((1.71 MB WMV)Click here for additional data file.Video S7Simulation showing the angiogenic response of the vascular system when a tumour is implanted. The normal vascular network is coloured in grey, whereas new angiogenic vessels are coloured red ((0.96 MB WMV)Click here for additional data file.Text S1Mathematical model, experimental methods and tables. A more detailed description of the mathematical model, experimental methods and the parameter values are given in this supporting information file.(0.25 MB PDF)Click here for additional data file.


Rationale. Prevention of wear-mediated osteolysis, the most common complication in total joint arthroplasty, is a great challenge for orthopedic surgery. Despite the diversity of current biomarkers of periprosthetic osteolysis , the major interferences and the great amount of sample necessary for analysis limit their use in clinical practice.Objective. The aim of this paper is to present three new electrophoretic methods using Hyrys-Hydrasys SEBIA system that have been used for the first time in Electrophoresis Laboratory of our hospital in the analysis of joint fluid for the prevention of periprosthetic osteolysis in revision arthroplasty.Methods and results. Analytical aspects of agarose gel electrophoresis of joint fluid proteins and lipoproteins as well as SDS-agarose gel electrophoresis of joint fluid proteins, their performances and clinical value are presented. The decreased level of albumin and increased level of alpha1 and alpha2 globulins were frequent changes detected on SEBIA electropherograms and good indicator for the presence of an inflammatory reaction generated by particle debris. In addition, a slightly increase of LDL mobility could provide good information about a high oxidative stress. Moreover, the Ig G assessed by using SDS-agarose gel electrophoresis could be a potential biomarker for an immunological reaction towards orthopedic implants.Discussion. Electrophoresis of joint fluid using Hyrys-Hydrasys SEBIA France system is a new analytical technique able to remove the most of current biomarkers disadvantages due to the determination of particular proteins  by using minimal amounts of joint fluid with minor interferences, minimal cost and rapid results.  AbbreviationsCTX, crosslinked C-telopeptide; IL- interleukins; Ig G, immunoglobulin G; LDL, low density lipoprotein; NTX, crosslinked N-telopeptide; PICP, procollagen I C – terminal extension peptide; SDS, sodium dodecyl sulphate Oxidative and nitrosative stress [3] as well as type IV hypersensitivity and other immunological mechanisms [4] are important co-players in this process.According to this etiology, current biomarkers of implant wear include markers of bone turnover  [2] and inflammatory reactions  [3] as well as products of wear process  [5]. Despite their specificity and sensitivity, the multitude of interferences  [6] and the less availability of analytical procedure, limit their use as routine tests. The analysis of particular proteins  in synovial fluid from a specific joint is of great interest because it could provide information about the biochemical changes from that specific joint. However, the great amounts of joint fluid necessary for the analysis of every protein mentioned, the viscosity, and the invasive character of arthrocentesis could limit their clinical value. Thus, the identification of the new analytical procedures with minor interferences, which require minimal amounts of joint fluid, is a great challenge in the prevention of orthopedic implant failure.Wear mediated osteolysis and consequent aseptic loosening is the most common cause of revision of total joint arthroplasty. The chronic inflammation induced by wear debris, macrophage activation and osteoclast mediated bone resorption are the main contributors to periprosthetic osteolysis [7]. The invasivity of arthrocentesis has been reduced by the sample collection only intraoperatively and before the incision of joint capsule, in accordance with the guidelines from scientific literature [8]. Electrophoresis of joint fluid in Hyrys-Hydrasys SEBIA France system could be a new prognostic tool for wear mediated osteolysis due to its advantages: the qualitative and quantitative analysis of a large number of protein fractions that may be identified by using a small quantity of joint fluid (0.3-0.5 ml), the minor interferences, and the possibility of fast introduction as routine test because of the procedure and instrument availability [7]. If some qualitative and/or quantitative abnormalities are detected, other electrophoretic analyses are required in order to extent the number of proteins with clinical value. Thus, joint fluid lipoproteins electrophoresis could be an additional tool for synovial lipoproteins profile and screening of oxidized LDL (low-density lipoprotein), biomarker strongly correlated with the presence and severity of inflammatory process and oxidative stress. In addition, SDS agarose gel can be a useful test for the identification of Ig G, albumin, haptoglobins, and other inflammatory, oxidative stress and immunological markers [7]. An association of these types of electrophoresis could considerably improve the clinical value, in particular in special cases (patients with rheumatoid arthritis and osteoporosis who underwent arthroplasty). The first step in electrophoretic analysis of joint fluid is agarose gel electrophoresis, a classic technique that is able to separate 5 or 6 protein fractions: albumin, alpha1, alpha2, beta  and gamma globulins [ Sample preparation and storage9]. For electrophoresis of proteins and lipoproteins, the viscosity of joint fluid was reduced by using Fluidil SEBIA (25μl Fluidil and 75μl joint fluid), a solution frequently used for dilution of viscous or turbid sera. For SDS-agarose gel, a dilution with saline solution is necessary in order to obtain about 0.2 g/dL proteins. No pre-treatment with Fluidil is necessary for this type of electrophoresis. The joint fluid samples harvested by arthrocentesis at revision surgery were centrifuged at 2000x g for 10 minutes at room temperature in order to remove cells and other debris. The supernatants aliquoted can be stored until 2 month at - 20ºC , and a good indicator for a high oxidative stress [7]. The quantification of LDL/oxidized LDL is the main advantage of this electrophoresis .  SDS- agarose gel electrophoresis of joint fluid proteins is a very useful additional tool for some biomarkers that cannot be separated using other types of SEBIA electrophoresis  , it would be expected that these biomarkers could provide information about the presence of a local inflammatory response, as well as antioxidant and antimicrobial defense capacity [7]. However, the main limit is that lactoferrin cannot be separated from transferrin (both with a molecular weight of 80 kDa) and measured individually. In addition, the level of these two markers could be often low or non-titratable. Further long-term studies should evaluate their role in the assessment of individual reactivity towards orthopedic implants and risk of prosthetic failure. No previous studies about the predictive value of lactoferrin and lysozyme for aseptic loosening have been published. Taking into account their role in inflammatory reactions due to metal debris, oxidative stress and antimicrobial defense [7]. No data about the previous use of electrophoresis of joint fluid as routine analysis has been published in scientific literature.Electrophoresis of joint fluid proteins using automated system Hyrys-Hydrasys SEBIA France is a new analytical technique that has been used for the first time in Electrophoretic Laboratory of our hospital [7]. Moreover, the presence of some discordances between the severity of wear mediated osteolysis assessed by electrophoresis and radiological techniques is a strong evidence that electrophoresis could play a key role in the prevention of aseptic loosening in patients undergoing joint arthroplasty [14]. The association of different types of SEBIA electrophoresis for joint fluid analysis appears to be a viable and robust alternative for the prognostic of periprosthetic osteolysis, with a high potential of fast introducing as routine test. The evaluation of many proteins in joint fluid  using a small quantity of sample with minor interferences provide many valuable information concerning the inflammatory response to particles debris, oxidative stress and immunologic reactivity towards implants as important mechanisms of periprosthetic osteolysis [3]. Because the electrophoresis of joint fluid in SEBIA France system has been used for only 3 years and the aseptic loosening could appear in a longer period, the predictive value of electrophoresis should be investigated by further studies.Although the sample collection is limited to the revision surgery, the role of joint fluid electrophoresis is considerably increased because it offers some information about the biochemical changes that have occurred during the primary implant, thus preventing the risk of aseptic loosening by choosing a suitable implant material, type of prosthesis and further therapy, in particular with antioxidant agents [7].  In conclusion, despite the main role of these proteins in periprosthetic osteolysis, none of these has absolute clinical value. For an accurate prognosis of aseptic loosening, the electrophoresis results must be correlated with the other biochemical markers and radiological data [


Blocking studies, conducted by pre-administration of 100-fold excess E3S, resulted in significantly lower  tumour uptake in both xenograft models, suggesting the involvement of an active carrier-mediated process. The expression of OATP1A2 was detected in tumour sections from both xenografts, with significantly higher expression (p = 0.002) in the MCF-7 xenografts. Overall, the higher tumour uptake and tumour-to-muscle ratio, alongside the higher expression of OATP1A2, in the MCF-7 xenograft model suggests the potential of E3S to serve as a novel ligand for targeting hormone dependent breast cancers.The current study investigates the potential of estrone-3-sulphate (E3S) as a ligand for targeting Organic Anion Transporting Polypeptides (OATP), a family of membrane associated uptake transporters, for detection and diagnosis of hormone dependent breast cancers. E3S, an OATP substrate, is a predominant source of tumour estradiol in post-menopausal patients. To assess the potential of E3S as a ligand, distribution of exogenous E3S was determined at the whole body, tumour and cellular levels in murine models of hormone-dependent (MCF-7) and independent (MDA-MB-231) breast cancers. The highest levels of tumour uptake were observed at 6 h post injection (p.i) with significant difference (p = 0.04) between the level in MCF-7 (13.9±3.1%ID/g) and MDA-MB-231 (10.4±1.1%ID/g) . The highest tumour-to-blood ratios  were observed at 48 p.i., and highest tumour-to-muscle ratios  were observed at 6 h p.i. Analogous to total tumour uptake,  Nearly 75% of estrogen dependent breast cancers are detected in post menopausal women with very low ovarian production of estradiol E3S has a log P value of 1.4 2 in Dulbecco’s modified Eagle medium (DMEM) containing 1% penicillin-streptomycin solution  and supplemented with 10% fetal bovine serum . Sub-confluent cells were harvested by trypsinization with trypsin-EDTA .MCF-7 and MDA-MB-231 cells were purchased from American Tissue Culture Collection . The cells were cultured at 37°C and 5% CO6) or MCF-7 (1.5×107) cells suspended in saline were injected into the right hind flank of each mouse and allowed to grow into tumour xenografts until they reached a diameter of 5–8 mm. For MCF-7 xenografts, a 17-β-estradiol pellet (0.25 mg/pellet)  with a 21-day release profile was intradermally implanted into each mouse using a 10 gauge Trocar needle (Innovative Research of America), one day prior to tumour cell inoculation. Following the completion of the 21-day period, an additional 4 days were allowed for clearance of plasma circulating estradiol, prior to initiation of the pharmacokinetics and biodistribution studies in MCF-7 tumour bearing mice. Plasma was collected from both xenograft models one day prior to initiating the pharmacokinetics and biodistribution studies and it was confirmed that the estradiol levels were undetectable in both xenograft models.Animal experiments were carried out in compliance with the Canadian Council on Animal Care regulations and were approved by the Animal Care Committee of the University of Toronto (AUP # 20008985). Six to eight week-old (20–25 g) female athymic CD-1 ovariectomized nude mice were purchased from Charles River . MDA-MB-231 (5×10∧2)/2, where length is the longest diameter and width is the shortest diameter perpendicular to length. The mean tumour volumes were 88.46±20.5 and 107.91±25.5 mm3 for the MCF-7 and MDA-MB-231 xenografts, respectively.Tumor volume was calculated as follows: (length×(width)3H]-E3S . The dose of E3S was determined following consideration of the reported circulating plasma concentrations of E3S (i.e. 1.65 to 3.84 pmol/mL) in vitro from Km values) 1/2α and t1/2β), volume of distribution at steady-state (Vss) and clearance (CL) were estimated by fitting the blood radioactivity vs. time curves to a two-compartment model with i.v. bolus input using Scientist® Ver. 3.0 software .Non-tumour bearing female athymic CD-1 ovariectomized nude mice (n = 4) were intravenously injected via the tail vein with E3S -trien-17-one hydrogen sulphate)  suspended in saline at a dose of 0.25 nmoles E3S/kg (approximately 87.6 ng/kg) body weight spiked with 0.5 µCi/mL [3H]-E3S) and sacrificed at 2 or 6 h post injection (p.i.) for biodistribution studies. Tumour bearing mice (MDA-MB-231 and MCF-7) (n = 5) were also injected with 0.25 nmoles/kg E3S (spiked with 0.5 µCi/mL [3H]-E3S) and sacrificed at 2, 6 or 48 h p.i. Excised tumours were divided into two halves, with one half used for biodistribution studies and the other for evaluation of ex vivo cellular uptake of E3S. The amount of [3H]-E3S in tissues was quantified using a previously described method with minor modifications Immediately following blood sampling mice were sacrificed under anaesthesia by cardiac puncture and the following tissues and organs were collected and weighed: liver, kidney, spleen and pancreas, bladder, uterus, lung, heart, intestine, stomach, tumour and surrounding muscles. Additionally, two groups of non-tumour bearing mice were injected with 0.25 nmoles/kg E3S (spiked with 0.5 µCi/mL [3H]-E3S). The mice were sacrificed 6 h p.i. with blood and tissues collected. Radioactivity and the corresponding E3S concentrations in samples were analyzed as described above.To determine the specificity of E3S uptake, additional groups of mice (n = 4) bearing MDA-MB-231 or MCF-7 xenografts were injected via the tail vein with 100-fold excess E3S (25 nmoles/kg body weight), two hours prior to the injection of 0.25 nmoles/kg E3S . The column was activated using methanol, followed by water. 100 µL of plasma was then loaded on to the activated column. This was followed by successive washing steps with water and hexane. The analyte was eluted from the column using methanol and then dried under a stream of N2. The sample was reconstituted in DMSO:Methanol 1∶1 v/v prior to HPLC analysis. SPE recovery was determined by spiking all samples prior to SPE extraction with 20 µL of each of the standards . The radioactivity in each eluate was determined by liquid scintillation counting. It was noted that only minor amounts of radioactivity (less than 1%) were present in the hexane and aqueous eluates. The methanol eluates were used for HPLC analysis.Plasma separated from whole blood collected from tumour bearing mice at 2, 6 or 48 h p.i., was used for HPLC analysis of E3S and its known metabolites. Known metabolites of E3S include estrone and estradiol 18 reversed-phase HPLC column  Analytes were eluted using an isocratic-gradient of 20 mM ammonium sulphate buffer (solvent A) and methanol (solvent B). The mobile phase consisted of solvent A/solvent B; 60/40% for 0–5 min, followed by a linear gradient of 60/40% to 25/75% for 5–45 and 25/75% for 45–50 min at a flow rate of 1.0 mL/min and UV detection at 280 nm. The HPLC eluate from the UV detector was collected every minute for over 50 min in eppendorf tubes and radioactivity in each sample was determined. The radioactivity elution profile for the fractions was plotted and compared with the standard HPLC chromatograms of E3S, estrone and estradiol. The area under the radioactive peaks corresponding to E3S, estrone and estradiol at 2, 6 or 48 h p.i. provided an estimate of the levels of E3S and the known metabolites in plasma at the respective time points.HPLC analysis of E3S and its known metabolites was performed using a Perkin Elmer HPLC system  equipped with a UV detector. Separation was performed using a Waters CImmunohistochemical staining of tumour sections was performed by the Pathology Research Program (PRP) at the University Health Network . Formalin-fixed paraffin-embedded sections (4 µm) of the MCF-7 (n = 6) and MDA-MB-231 (n = 6) xenografts were dewaxed and rehydrated. Antigen retrieval or unmasking was performed by the heat induced epitope retrieval (H.I.E.R) method followed by serum block for 10 min with 10% normal goat serum. Sections were drained and incubated with primary rabbit polyclonal antibodies for anti-OATP1A2  and anti-CD31  at 1∶1200 and 1∶1000 dilutions, respectively. These sections were then incubated with Alexa Fluor 488 goat anti-rabbit labelled secondary  and goat anti-rabbit Cy3 for 60 min for the anti-OATP1A2 and anti-CD31 stained sections, respectively. The slides were then air dried. Expressions of OATP1A2 and CD31 were also examined in human brain tissue sections and in bladder tumour sections, respectively, as positive controls. These sections were then stained with diaminobenzidine (DAB) chromogen and images were acquired using the ScanScope XT (Aperio technologies) at 20X maginification. Slides without any primary antibody staining were also imaged and these served as negative controls. The final images are shown at 10X magnification.For the OATP1A2 and microvessel densities analysis, microscope images identifying the presence of OATP1A2 and blood vessels (recognized by CD31) were acquired at 10x magnification using an Olympus BX50 upright fluorescence microscope  equipped with an EXFO fluorescence illumination source. Images of complete tumour sections were produced by stitching tiled images using MetaMorph software . Microvessel and OATP marker densities were measured as the total area of positive signal divided by the surface area of the tumour section analyzed using the “area fraction” tool of the ImageJ software. The unit for immnohistochemical expression was reported as % area Immunoblotting was performed as described previously t test for unpaired experimental values or one-way analysis of variance (ANOVA) for analysis of repeated measures, as appropriate. p<0.05 is considered statistically significant.All the results were obtained from groups of n≥3 and are presented as mean ± SD. Statistical analysis was performed using Graphpad InStat version 3.0 software . Statistical significance was assessed by two-tailed Student's 3H]-E3S in non-tumour bearing athymic CD1 ovariectomized mice. Each mouse was administered 0.25 nmoles/kg E3S spiked with 0.5 µCi/mL [3H]-E3S (represented as E3S/[3H]-E3S). E3S was found to exhibit a biphasic pharmacokinetic profile with short distribution and long elimination half-lives (t1/2α (h) = 0.04±0.01, t1/2β (h) = 17.0±3.0). The AUC, CL and Vss were 222.8±49.4%I.D./mL*h, 0.5±0.1 mL/h and 10.6±1.5 mL, respectively.3H-E3S was evaluated in non-tumour bearing mice as well as in hormone dependent (MCF-7) and non-hormone dependent (MDA-MB-231) xenograft mouse models. The biodistribution of E3S in non-tumour bearing mice was similar to that observed in the tumor bearing mice  or 48 h (11.8±1.4% ID/g) (p = 0.53) p.i. In contrast, the tumour uptake of E3S in the non-hormone dependent xenograft model (MDA-MB-231) increased from 4.8±1.8% ID/g at 2 h p.i. to 10.4±1.1% ID/g at 6 h p.i. (p = 0.01) and did not significantly change at 48 h (9.4±0.3% ID/g) (p = 0.20). There was a significant difference in tumour uptake between the two xenograft models at 2 h (p = 0.01), 6 h (p = 0.04) and 48 h p.i. (p = 0.02). The highest tumour-to-blood ratios were observed at 48 h p.i. as a result of clearance of E3S from the circulation  and 2-fold (p = 0.02) reduction in tumour uptake in MCF-7 and MDA-MB-231 xenografts, respectively. Similarly, there was a 1.7-fold (p = 0.01) and 2.0-fold (p = 0.003) reduction in kidney uptake in MCF-7 and MDA-MB-231 tumour bearing mice, respectively. A slight but insignificant decrease in liver uptake was also observed in both xenograft models.To determine if the high uptake observed in the tumour, kidneys and liver was mediated through an active carrier process, a blocking study was performed in which MCF-7 and MDA-MB-231 tumour bearing mice were injected via the tail vein with 100-fold excess E3S (25 nmole/kg) 2 h prior to administration of 0.25 nmole/kg E3S/-E3S.Biodistribution of E3S/[ Biodistribution of E3S/[3H]-E3S at 2 h, 6 h and 48 h p.i. in non-tumour bearing mice expressed as % injected dose per gram (%ID/g). (TIF)Click here for additional data file.Figure S2HPLC analyses of estrone-3-sulphate (E3S) and its metabolites, estrone and estradiol, in plasma at 48 h p.i. UV channel represents reversed-phase HPLC (see methods section for HPLC conditions) chromatograms of E3S, estrone and estradiol. For metabolite analyses plasma samples of mice injected with E3S/[3H]-E3S, were collected at different time points  and were spiked with 0.625 mg/mL of E3S, estrone and estradiol, prior to solid phase extraction. HPLC eluates were collected every min for 50 min and the radioactivity in the samples were counted and plotted. Peak area of the radiometric channel represents the contribution of the E3S and its metabolites in plasma.(TIF)Click here for additional data file.Figure S3Immunoblot and densitometric analysis of OATP1A2 transporters. Protein expression of OATP1A2 was investigated in xenograft tissues . To determine the specificity of the respective antibody used, a positive control cell line over expressing the OATP1A2 transporter (HEK293/OATP1A2: represented by lane 1) was included in the blot. Results of the densitometric analysis were expressed as mean ± SD of three separate tumours for each xenograft model. A significant difference in protein expression was observed between the two xenograft tissues. **p = 0.002 is considered to be statistically significant.(TIF)Click here for additional data file.Figure S4Functional and non-functional vessels (stained with CD31) in MCF-7 (A) and MDA-MB-231 (B) xenograft sections. No statistically significant difference was observed in the microvessel density between the MCF-7 and MDA-MB-231 tumour xenografts.(TIF)Click here for additional data file.Figure S5Positive and negative controls for OATP1A2 (A) and CD31 (B). Human brain tissue and bladder tumour tissue sections were used as positive controls to determine specificity of antibodies used for OATP1A2 and CD31 staining, respectively. The same tissues were stained with secondary antibody (without any primary antibody) to determine non-specific binding and these served as negative controls.(TIF)Click here for additional data file.Table S1Plasma analysis of estrone-3-sulphate (E3S) and its metabolites estrone and estradiol in methanol, hexane and water fractions collected, following solid phase extraction (SPE). The plasma samples were collected at 2 h, 6 h and 48 h post-injection (p.i.) from MCF-7 and MDA-MB-231 tumour bearing mice and were then subjected to SPE.(DOCX)Click here for additional data file.


The grant and funding organization in the Funding Statement were incorrectly given. The Funding Statement should read: "This work was supported by a grant from the National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIBR No. 2013-01-052). The funder had taken part in the role of conceive and design the experiment."


Intravenously (i.v.) administered nanomedicines have the potential for tumour targeting due to the enhanced permeability and retention (EPR) effect, but in vivo tumour models are rarely calibrated with respect to functional vascular permeability and/or mechanisms controlling intratumoural drug release. Here the effect of tumour type and tumour size on EPR-mediated tumour localisation and cathepsin B-mediated drug release was studied.N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer–doxorubicin (Dox) conjugate (FCE28068) (5 mg/kg Dox-equiv) were used as probes and tumour levels (and Dox release) measured at 1 h after i.v. administration in a panel of murine and human xenograft tumours.Evans Blue (10 mg/kg) and an Evans Blue and FCE28068 displayed similar tumour levels in the range of 2–18 % dose/g at 1 h for B16F10 and L1210. Approximately half of the tumour models evaluated exhibited tumour size-dependent accumulation of FCE28068; smaller tumours had the highest accumulation. Administration of free Dox (5 mg/kg) produced tumour levels of <2.5 % dose/g independent of tumour size. Whereas the degree of EPR-mediated targeting showed ~12-fold difference across the tumour models evaluated, Dox release from FCE28068 at 1 h displayed ~200-fold variation.Marked heterogeneity was seen in terms of EPR effect and Dox release rate, underlining the need to carefully calibrate tumour models used to benchmark nanomedicines against known relevant standard agents and for optimal development of strategies for late pre-clinical and clinical development.The online version of this article (doi:10.1007/s00280-013-2209-7) contains supplementary material, which is available to authorized users. The upper aqueous layer and tissue pellet were then carefully removed. The remaining organic phase was evaporated to dryness using a sample concentrator under constant nitrogen gas (N2) flow over 20 min. The samples obtained were re-dissolved in HPLC grade methanol (100 μL) by vortex-mixing (30 s) and centrifuged  to remove any remaining biological materials. Samples (100 μL) were then analysed by HPLC using a C18 μBondapak™ column eluted with propan-2-ol in water (29 % v/v) adjusted to pH 3.2 with orthophosphoric acid with a fluorimetric detector . Data were collected and analysed using a PowerChrom integrator and software programme (PowerChrom v 2.0.7). For each experiment, a Dox standard curve was prepared in parallel.Following i.v. administration of free Dox, drug in tumour samples was quantified using a previously described HPLC method , 21. ThrAs HPMA copolymer–Dox does not extract directly into the organic solvent, free Dox present in the tumour samples could be extracted and quantified as described above , 21. To t test for comparison of the mean of two small samples. Statistical differences of at least p < 0.05 were considered statistically significant. To allow comparison between “small” and “large” tumours, the three smallest and three largest tumours available were grouped according to the classifiers for “small” and “large” described in the figure legends where tumour size as a variable is examined.Data describing tumour accumulation of Evans Blue or HPMA copolymer–Dox were expressed as either (i) the  % administered dose per tumour  or (ii) the administered dose % dose/g tumour). Data shown are mean ± SE. Statistical significance was calculated using Student’s To assess evidence for the operation of the EPR effect, intratumour levels of Evans Blue, FCE28028 and Dox detected at 1 h following i.v. administration in mice bearing s.c. B16F10 or L1210 tumours are shown in Figs. In contrast to the results obtained in the B16F10 models, when FCE28068 was administered to mice bearing MAC 15A (8.2–12.6 % dose/g), MAC 26 adenocarcinoma (6.9–10.8 % dose/g) or Meta-7 lung (3.5–4.7 % dose/g) tumours . Although PAXF 546 showed a similar trend, it should be noted that the three smallest and the three largest tumours of PAXF 546 pancreatic carcinoma tumours did not give statistically significance different values. In contrast, the RXF 1220, COR L23, IMR 32, SK-N-SH and SK-N-DZ tumours  displayed size-independent accumulation of FCE28068. . Comparison of summary mean values obtained for FCE28068 accumulation in all the tumour xenografts  is shown in Fig. In vitro and in vivo pre-clinical models used to screen novel low molecular weight anticancer agents and biologics have evolved significantly to better mimic the clinical disease setting . WhIn the 1950s and 1960s, radio-iodinated albumin and albumin aggregates were used clinically to image tumours. Unknowingly, these imaging agents were already capitalising on the EPR effect for tumour selectivity , as do a111In-DTPA-labelled PEGylated liposomes injected i.v. to mice bearing KB human head and neck squamous cell xenografts also displayed tumour levels inversely correlated with tumour size: 15.1 ± 10.8 % dose/g in tumours of <0.1 g and 3.0 ± 1.3 % dose/g in tumours of >1 g [A significant observation in these experiments is a 12-fold variation in functional EPR, with many models displaying tumour size-dependent accumulation. The higher tumour localisation was noticeably in the smaller tumours, and this maybe a reflection of greater angiogenic activity in smaller tumours, reduced or absent tumour blood supply in hypoxic regions of larger necrotic tumours, an increase in tumour interstitial fluid pressure as the tumour grows, or a combination of all of these factors.  of >1 g . In this111In-DTPA-labelled PEGylated liposomes to image 17 patients with locally advanced cancers [99mTc-DTPA-labelled PEGylated liposomes that localised to lung and head and neck cancers [Whatever the mechanism, there is a growing body of evidence that nanosized vectors show greatest targeting to the smaller tumours and this could provide an important opportunity to localise to those micrometastases so difficult to diagnose and treat effectively. Use of  cancers  gave pos cancers , others  cancers .Overexpression of lysosomal thiol-dependent proteases in many human tumours has been well documented , and theThe wide variation in passive targeting (% dose/g) and Dox liberation observed here (1 h) highlights the need to calibrate in vivo tumour models in respect of these parameters before they are used to define pharmacokinetics and/or antitumour activity of nanomedicines. This would provide more clinically relevant models for optimisation of lead candidates and benchmarking performance against anticancer nanomedicines already in routine clinical use . Evans BFig. 1 FCE28068 levels in MEXF 276, PAXF 546, MAXF 449 and RXF 486 tumours at 1 h after i.v. administration. The data in panels (a) - (d) show individual tumours. (PPT 165 kb)Fig. 2 FCE28068 levels in RXF 1220, IMR 32, COR L23, SK-N-SH and SK-N-DZ tumours at 1 h after i.v. administration. The data in panels (a) - (e) show individual tumours. (PPT 178 kb)(PPT 146 kb)Fig. 3 Summary of tumour uptake of FCE28068 and the rate of DOX release in the MAC tumours . Below is the link to the electronic supplementary material.


Fibromyxoma is a rare odontogenic tumour which is benign, but locally aggressive. The etiology of these tumours is unknown, but because of its limitation to the teeth bearing areas and occasional presence of odontogenic epithelial fragments within the tumour which suggest that it is of odontogenic origin. It is a slow growing painless tumour that frequently occurs in second and third decades of life. Females are more commonly affected than males. The tumour can cause gradual expansion of the cortical plates and cause loosening and displacement of teeth, although root resorption may be rare. The surgical treatment of these tumours consists of complete enucleation or radical excision. The aim of this paper is to present the rarity of a fibromyxoma of the maxilla. Fibromyxoma is a benign tumour of ectomesenchymal origin with or without odontogenic epithelium . The terA 12-year-old female patient visited our unit with a chief complaint of swelling on left side of the face since 6 months. Extraoral examination revealed diffuse swelling over the left cheek involving lateral part of the nose and infraorbital region . Skin ovFibromyxoma is a rare aggressive intraosseous lesion derived from embryonic mesenchymal tissue associated with odontogenesis. The maxilla and anterior region of the mandible are rarely affected. When found in the maxilla it usually behaves more aggressively than that of the mandible. It involves the zygoma, maxillary sinus, and even the orbits. Evidence that may support the odontogenic origin of fibromyxoma of the jaws was contributed by Thoma in 1934. He stated that the greater portion of myxomas that occur in the jaws are derived from embryonic tissues of the dental papilla, the dental follicle, or the periodontal membrane , 8. MostRadiographically, the fibromyxomas present themselves as multilocular or unilocular radiolucency with well-defined borders. Most of them are multilocular. Radiological investigations reveal homogenous radiolucencies with different appearances like honey comb, soap bubble, or tennis racket , 9. DiffGrossly the tumour is greyish-yellow multinodular tissue. Its consistency varies. Some portions of the tumour may be sticky, gelatinous, or semisolid, and others may be firm. The surface of the tumour is shiny and glistening.Histologically the fusiform or stellate cells are elongated, having cytoplasmic processes stretching in various directions. It contains few cells that lie in a myxoid ground substance being elongated with spindle-shaped nuclei and having the appearance of fibrocytes. It is of diagnostic challenge to the examiners because it is difficult to differentiate from the other odontogenic tumours. Mitotic figures are rarely seen. The ultrastructural studies of an odontogenic myxoma demonstrate the presence of two basic functional types of cells: secretory and nonsecretory. The secretory cell type is the principal tumour cell and resembles the fibroblast . The tumour is not radiosensitive, and hence surgery is the only treatment of choice. The surgical treatment of the fibromyxoma involves enucleation and curettage, radical excision, en bloc resection. The avoidance of recurrence is strongly related to the complete resection of the lesion. Thomas has stated that recurrence of fibromyxoma is uncommon if enucleation is complete. Myxomas or fibromyxomas show a recurrence rate between 25% and 43%. Harder et al. stated that rate of recurrence varies widely as does the choice of treatment. Their study supports conservative surgery as the appropriate treatment for odontogenic myxomas, as they have found no evidence of malignant change or increasingly aggressive behaviour in any of the recurrences .Chen et al. proposed conservative treatment as the preferred method for excision with extensive resection reserved for large tumors. In the long bones the tumour is frequently malignant and tends to recur with great frequency after removal, but fibromyxomas of the jaws appear to have a better prognosis.In conclusion, fibromyxomas of the jaws are rare but not uncommon. The prime treatment considerations include the age of the patient and recurrence of the lesion. The tumour is not radiosensitive, and surgery is the only choice of treatment. In this young unmarried girl as the extent of lesion is confined to the maxillary sinus, we have opted for an intraoral conservative surgical approach.


The possibility of eradicating cancer by selective destruction of tumour blood vessels may represent an attractive therapeutic avenue, but most pharmaceutical agents investigated so far did not achieve complete cures and are not completely specific. Antibody conjugates now allow us to evaluate the impact of selective vascular shutdown on tumour viability and to study mechanisms of action.We synthesised a novel porphyrin-based photosensitiser suitable for conjugation to antibodies and assessed anticancer properties of its conjugate with L19, a clinical-stage human monoclonal antibody specific to the alternatively spliced EDB domain of fibronectin, a marker of tumour angiogenesis.in vivo localisation around tumour blood vessels and that its conjugate with a photosensitiser allows selective disruption of tumour vasculature upon irradiation, leading to complete and long-lasting cancer eradication. Furthermore, depletion experiments revealed that natural killer cells are essential for the induction of long-lasting complete responses.Here we show in two mouse model of cancer (F9 and A431) that L19 is capable of highly selective These results reinforce the concept that vascular shutdown can induce a curative avalanche of tumour cell death. Immuno-photodynamic therapy may be particularly indicated for squamous cell carcinoma of the skin, which we show to be strongly positive for markers of angiogenesis. Aggressive solid tumours  and haemInhibition of key mediators of tumour angiogenesis has culminated in the clinical application of Avastin, a humanised monoclonal antibody binding soluble VEGF-A, thus stopping interaction with its receptors  could have a more profound impact on the disease, causing an avalanche of tumour cell death as a result of oxygen and nutrient deprivation . Indeed,Therapeutic strategies based on selective occlusion of tumour vasculature are marked with an inherent paradox. Although, it is clear that the collapse of tumour vasculature can lead to massive tumour cell death, it is less obvious that all tumour cells  should die as a result of a vascular insult. In fact, when solid tumours are treated with combretastatins  conjugates, which selectively localise to the tumour neovasculature Photodynamic therapy (PDT) is a localised, non-invasive treatment modality for superficial malignancies that relies on the combination of non-toxic PSs and visible light in the presence of oxygen to generate cytotoxic reactive oxygen species. Photodynamic therapy causes localised cell death directly by induction of apoptosis and/or necrosis as well as indirectly by destruction of tumour vasculature and delayed stimulation of an immune response against the malignancy subjected to PDT  that absorb light and that may display a toxic action in their immediate surroundings either by generation of short-lived reactive oxygen species (such as singlet oxygen)  or by loThe results presented in this study show that aggressive tumours, implanted in the skin of nude mice, can be completely eradicated as a result of vascular damage mediated by immuno-PDT using SIP(L19)–PS conjugates. Interestingly, complete tumour ablation required the presence of natural killer (NK) cells, as only partial inhibition of tumour growth could be observed following NK cell depletion with a monoclonal antibody specific to asialo-GM1.These findings reinforce the concept that the selective ablation of tumour neovasculature can lead to long-lasting tumour eradication and may be of clinical significance for the treatment of squamous cell carcinoma (SCC).in situ forms of SCC, represent the most frequent type of cancer in the fair-skinned population. Their incidence is increasing world wide, with immunocompromised patients being particularly affected phenyl]-10,15,20-tris-(4-N-methylpyridimiumyl)porphyrin trichloride and was synthesised from commercially available materials and is described in detail in the The PS used for conjugation to SIP(L19) was 5-[4-. Six- to eight-week-old female Balb/c Nude CAnN.Cg-Foxn1nu/Crl mice were obtained from Charles River Laboratories . All animal experiments were performed under a project license granted by the Veterinäramt des Kantons Zürich (169/2008).WI-38 VA-13 fibroblasts (ATCC number CCL-75.1), F9 murine teratocarcinoma cells (ATCC number CRL-1720) and A431 human epidermoid carcinoma cells (ATCC number CRL-1555) were maintained in exponential growth in DMEM medium adjusted to contain 4 mThe cloning, expression and purification of SIP(L19), SIP(F8) and SIP(F16) antibodies have been described previously (−1 in DMSO) was added to purified SIP antibody  and incubated for 4 h at 30°C, gently shaking, avoiding light exposure. Small immune protein–PS conjugates were then purified from free reagent over a PD-10 column  and dialyzed overnight against PBS pH 7.4 at 4°C.Small immune protein–PS (SIP–PS) conjugates were prepared as follows: ∼20-fold molar excess of amine-reactive porphyrin (PS 10 mg ml−1 of SIP solution gives absorption of 1.4 U at 280 nm) and at 422 nm for the PS (ε=159 100 M−1 cm−1). In addition, mass spectrometric analysis was used to assess the labelling ratio. For MALDI-TOF/TOF MS analysis, the conjugates were mixed with a sinapinic matrix (10 mg ml−1 in 50% CAN and 0.05% TFA) at a 1:4 dilution and spotted onto a MALDI target plate. Analysis of samples was performed on a freshly calibrated AB4800 MALDI-TOF/TOF mass spectrometer . For data analysis, the Data Explorer software (version 4.8) of Applied Biosystems was used. A Superdex 200 size-exclusion column  was used to analyse the gel filtration profile of the antibodies before and after PS conjugation under native conditions using fast protein liquid chromatography .Small immune protein–PS conjugates were analysed by SDS–PAGE under reducing and non-reducing conditions using the Invitrogen PAGE system following the manufacturer's instructions. The gels were first imaged under a Cy5 filter lamp and then stained with Coomassie brilliant blue. The labelling ratio was estimated spectroscopically by measuring the absorbance at 280 nm for the SIP (assuming that a 1 mg mlin vitro photocytotoxicity assay WI-38 VA-13 fibroblasts were used by seeding 30 000 cells per well in a 96-well plate and incubating overnight at 37°C in 5% CO2. The next day medium was removed and cells were incubated with 50 μl of SIP or SIP–porphyrin conjugate in the appropriate dilutions (in PBS) for 1 h at 37°C. Cells were subsequently washed with PBS twice to remove unbound antibodies, and cells were covered with 50 μl of PBS. The cells were then irradiated using a KL 1500 electronic tungsten halogen lamp  equipped with a 620/60 filter  for a total light dose of 60 J cm−2. After light treatment, PBS was removed and 100 μl of fresh medium were added. The cells were then incubated at 37°C, 5% CO2 atmosphere over night. Controls include SIP–porphyrin conjugate without irradiation, PBS only with irradiation, unmodified SIP with irradiation and medium only without irradiation. The following day, cell viability was measured using the Cell Titer 96Aqueous One Solution Cell Proliferation Assay , following the manufacturer's instructions. The percentage of cell growth was calculated as a ratio of the counts between treated cells over the relative control (cells treated with SIP–porphyrin conjugate without light exposure).For the 6) were injected subcutaneously into the flank of Balb/c nude mice. When tumours were established and clearly palpable (∼50 mm3), mice were randomly distributed among the groups  and irradiated with a laser  24 and 48 h after intravenous injection in the tail lateral vein of 150 μg of SIP(L19)–PS. In addition to SIP(L19)–PS, control groups included mice receiving SIP(L19)–PS in the absence of light, mice treated with an irrelevant antibody–PS conjugate (SIP(F16)-PS) and mice treated with saline. For depletion of NK cells (−1)/xylazine (6 mg kg−1) before light irradiation. Differences between different groups were compared using Student's t-test. (B) A431cells (3 × 106) were injected subcutaneously into the flank of Balb/c nude mice. When tumours were established and clearly palpable (50–100 mm3), mice were irradiated once with a laser  24 h after a single intravenous injection in the tail lateral vein of 150 μg of SIP(L19)–PS conjugate. Mice were monitored daily and tumour growth was measured three times per week with a digital caliper using the following formula: volume=length × width × width × 0.5. Mice were killed when the tumour reached a volume >2000 mm3. In addition, mice were photographed using a digital reflex camera (Nikon D90).(A) F9 murine teratocarcinoma cells  are reported in the 5-(4-Carboxyphenyl)-10,15,20-tri-(4-pyridyl)porphyrin was used as starting material to introduce an activated ester that allows the conjugation of this PS to amino residues on proteins.N-hydroxysuccinimide ester, it was first converted to the corresponding acyl chloride by reaction with thionyl chloride –PS conjugate (LD50=3 nM), whereas no detectable cell killing was observed without irradiation.Small immune protein(L19)–PS is a non-internalising antibody conjugate. In line with previous observations  exclusively stains blood vessels in frozen sections of F9 tumours, as revealed by immunofluorescence analysis. By contrast, SIP(F16) does not recognise any antigen in the mouse  and therculature .−1. This is an order of magnitude less than doses typically required to cure tumours in mice using PDT based on similar PSs (One molecule of homodimeric SIP(L19) was coupled to an average of six PS molecules , resultiilar PSs .in vivo therapeutic activity of SIP(L19)–PS, we injected this conjugate in tumour-bearing mice (150 μg per mouse) and irradiated tumours with red light (two doses of 60 J cm−2). Mice treated with SIP(L19)–PS exhibited a strong anticancer response, with three out of four complete eradications that were long lasting , followed by 3 min irradiation (60 J cm−2) 24 h later. Although tumours in saline-injected mice were not affected by irradiation, A431 lesions in mice receiving SIP(L19)–PS were rapidly converted into black scabs (We further tested SIP(L19)–PS in mice bearing A431 tumours as a model of SCC of the skin. To mimic a clinical procedure for the treatment of SCC patients, we injected the antibody–PS conjugate in mice bearing subcutaneous A431 tumours in a single dose  to survive and re-grow . To inveNatural killer cells efficiently infiltrated tumours within 6 h after immuno-PDT treatment . In contTo investigate whether immuno-PDT strategies based on vascular targeting antibodies could be used for the ablation of skin lesions of SCC, we first performed an analysis by immunofluorescence samples of human SCCs and normal skin, using in addition to L19, the F8 and F16 human antibodies, specific to the alternatively spliced EDA and the A1 domain of tenascin-C, respectively , but arein vivo. This allows selectively damaging the tumour vasculature by irradiation with red light, leading to massive tumour cell death and to lasting cures. Depletion experiments indicated that the action of NK cells is essential for the induction of complete cancer eradication, killing cells neighbouring on normal tissue at the periphery of the tumour.In this article, we have shown that an antibody–PS conjugate specific to the alternatively spliced EDB domain of fibronectin, a marker of angiogenesis, has the ability to localise selectively around tumour blood vessels In the past, selective damaging of tumour neovasculature has been studied with antibody–toxin and antibody–tissue factor conjugates , ASA404 ex vivo fluorescence microscopy , actinic keratosis, head and neck carcinomas and the premalignant condition Barrett's oesophagus. Light penetration of tissues reaches a maximum of only several millimetres at wavelengths around 750–800 nm  PDT regimens. These findings suggest that the therapeutic efficacy of PDT in the clinical treatment of human skin cancers may be greatly enhanced by the selective delivery of PSs conjugated to vascular tumour-targeting antibodies. At the same time, we envisage a significant reduction of curative PS doses and of side effects in such targeted immuno-PDT regimens.Clinical trials are now needed to elucidate whether the promising results obtained with SIP(L19)–PS in the A431 tumour model are confirmed in patients with cutaneous SCC or other EDB-positive skin lesions. If so, then antibody–PS conjugates such as SIP(L19)–PS may greatly extend the applicability of immuno-PDT in the clinic, sparing many skin cancer patients the more invasive treatment modalities of surgery and radiotherapy.Independently of any practical application of immuno-PDT , engaging the Fc portion of the antibody with Fc-γ receptor 3a , leading to degranulation . The act


Internal gold fiducials are necessary for tracking the translational and rotational movements of target lesions during stereotactic radiosurgery. The fiducials are generally placed under image guidance in and around the lesions by interventional radiologists. Specific challenges are encountered during the procedure. This article discusses the basic principles and techniques as well as the specific complications. CyberKnife is the first FDA-approved device for full-body dynamic radiosurgery and was developed by Accuray in 2001. It deliv4Understandably, a mechanism is necessary to track the lesions’ translational and rotational movements so as to achieve high (sub-millimeter) accuracy and safety. Lesions can be tracked either using bones or internal fiducial markers.The fiducials are internal markers, made of 99% gold and can be radiographically visualized. They are available  as 0.8 ×With the patient on the treating table, two 45°- angled radiographs are obtained centering the beam at the lesion every few seconds. Identifying fiducials as distinct entities on these radiographs is necessary for tracking lesion movement during treatment and optimal conditions for this include a minimum of 2 cm distance between any two fiducials and a minimum of 15° degree angle within any group of three fiducials. To track rotational movement, a minimum of three fiducials placed in three orthogonal planes is necessary. Once in the body, there will be a small amount of migration of the fiducials by a few millimeters for the first few days but by one to two weeks, migration is assumed to be nil due to development of fibrosis around the fiducials. Also, if the fiducial is placed at the periphery of the lung or any other visceral organ, there is a possibility that it may move into the pleural and peritoneal cavities, respectively.et al. have reported the successful use of CT scan for this purpose and have concluded that it is an accurate and safe method.[Though CT scan is a popular guiding tool for percutaneous fiducial placement for CyberKinfe treatment, there are no major studies regarding this. In a recent retrospective study of 105 consecutive patients, Sotiropoulou e method. No majorAs in all percutaneous procedures, preparation starts with achieving a normal coagulation profile, ruling out active infection and obtaining informed consent. Three days of intravenous antibiotic (amikacin) along with oral ciprofloxacin and tinidazole for five days starting from day one are routinely given at our institute whenever small or large bowel transgression is contemplated. Under aseptic precautions and local anesthesia, an 18G Chiba or lumbar puncture needle is inserted under CT scan or USG guidance. The fiducial is advanced through the needle after confirming that the position of the needle tip is in or around the lesion. The final location of the needle tip is always confirmed by imaging to ensure that each fiducial is placed at a different site, within or near the lesion of interest. Confirmatory axial slices and topograms should be obtained Figures –4. PatieA total of 122 patients have undergone image-guided fiducial placement in our department for the purpose of stereotactic radiosurgery for various malignant conditions in the last 11 months. Percutaneous  and transrectal (27 TRUS-guided for prostate cancer) methods have been used and a total of about 450 gold fiducials have been placed. The target lesions were in the chest , the abdomen  and the pelvis .Three or more fiducials were tracked in most of the patients. In a minority of patients, the fiducials migrated and were not tracked by the CyberKnife. Three patients had one fiducial each migrating into the pleural cavity, five patients had one fiducial each migrating into the peritoneum and 14 patients had intra-organ migration in the lung or viscera. Intra-organ migration generally does not result in the loss of a fiducial, and since no migration is expected within the organ after 7-14 days, planning of treatment is generally done 14 days after fiducial placement. One patient had a significant pneumothorax, for which, a chest tube was inserted immediately after the procedure. One patient had a late pneumothorax on the fourth day after the procedure and was treated with a chest tube. Three patients had prophylactic chest tube placements for anticipated pneumothorax due to severely emphysematous lungs. Two patients had moderate hemothorax, which did not require active treatment. Small hematomas occurred in many patients, which did not require any active treatment and were self-limiting. No other major complications occurred in any of the patients.Though the basic principles, access routes and complications are similar to those of any other biopsy procedures performed by interventional radiologists, a few challenges are specific to fiducial placement. A thorough knowledge of fiducial placement principles is necessary. Migration of the fiducials is unpredictable and can cause loss of fiducials. Fiducial embolism, though extremely rare, is unique to this procedure and is particularly dangerous if placed in one of the larger pulmonary veins. Atelectasis of the lung following pneumothorax/hemothorax is a limitation while attempting peripheral placement of fiducials. When the bowel is to be traversed, systemic antibiotic treatment may be necessary. Informing the patient about the procedure and being prepared for possible complications is important.


Rapid fluid administration is often required for resuscitation when patients are admitted in emergency department with hypovolemic shock or excessive blood loss. Various methods have been described earlier to increase the fluid administration speed. Larger vein size, larger bore cannula, height of fluid, pressure over fluid bottle etc. are some of methods described in such situations.We here describe a novel method to administer intravenous fluid rapidly and this method can be utilized in emergency and trauma settings. In prehospital trauma and emergency settings, the immediate establishment of venous access and rapid fluid administration may be difficult in resuscitation of patients in hypovolemic shock due to massive blood loss. Rapid infusion systems (RIS) have been successfully used for delivering large amount of intravenous fluids at standard and rapid flow rates. Although RIS are undoubtedly the convenient and most effective way of delivering fluids in short span of time but it has its own limitations.We present a novel device which can be successfully used in prehospital trauma and emergency settings. The steps for designing this equipment are as follows Figure :1. A collapsible bag/Thin plastic intravenous fluid bottle is taken (A)® safeset  is inserted in the intravenous (IV) fluid bottle and the other end is connected to the patient's intravenous cannula (B).2. The sterile Intrafix3. The spike end of intravenous set is inserted above fluid level in fluid bottle(C)4. The sphygmomanometer inflation bulb is connected to latex end of intravenous tubing (that is inserted in fluid bottle) (D).5. The roller clamps of both IV tubing's are rolled ON.® safe set  IV infusion assembly is used for infusing fluids to the patient.By inflating the inflation bulb of the sphygmomanometer the air is pushed in the fluid bottle. This creates a vertical pressure head on the surface of the fluid column and along with gravitational force, it pushes fluid through the fluid bottle along the IV tubing, to the patient. The IntrafixIn the past, there were many methods used for rapid delivery of fluids. Amongst them various pressurization techniques like gravity-fed infusion, manual compression of fluid chamber, and compression of fluid chamber using flexible  and rigid pressure bags , were popularly used. The efficacies of these techniques to increase the flow rates were also evaluated. They found that manual push-pull technique was better than gravity fed infusion system ,2.PressuPresently, rapid infusion systems  are commonly used to administer blood and fluids at desired and rapid rates. These rapid infusion devices are large, bulky, expansive, and costly to operate. All of these devices are operated by large, heavy, non-portable, roller pump mechanism and require fresh sets of fluid administration each time. These devices cannot be used with typical peripheral IV cannula but require large-bore central-line or venous cut-down catheters which can be inserted only by experts. Although rapid infusion devices are a proven life saver but this technology is not commonly accessible most of the hospitals in developing countries because of the aforementioned reasons. Further, the modern RIS are bulky and costly that makes their use difficult and cumbersome in emergency and disaster scenarios.The rapid infusion device assembly we describe solves many problems present in prior art devices. It is small, portable, and if desired can be designed easily by any health care provider in any type of set-up. It is inexpensive, environment friendly and can be potentially available to patients even in small rural hospitals. This can be used with any IV tubing or other commonly available hospital equipment and can be used with central lines, venous cut-down catheters, or peripheral IV s that nurses or paramedics can insert. Therefore, it has a potential application for use in ambulances, in the fields, in emergency rooms, military applications and camp surgeries in disasters.® safeset  IV infusion assembly for infusing fluids instead of conventional IV tubing assembly, as this IV infusion set has a unique airtight hydrophilic filter membrane (pore size 15 μm) with an air barrier integrated into the drip chamber which allows only fluid to pass and thereby prevents the inflow of air into the tubing. This acts as a safety feature against air passing through the IV tubing thus preventing chances of air embolism. It also negates the need of pushing the air bubble up and out of the bottle and priming of the drip chamber while switching of the bottle with air in the infusion system. Thus further reduces the time of administration as compared to the conventional IV tubing, thus ideal for rapid infusion of fluids in emergency situations.We are routinely using this device successfully in our institution in ambulances and ER settings for immediate volume resuscitation without any complication of air embolism or hemolysis. We used IntrafixWe strongly recommend that rapid infusion of fluids using this assembly can be used as an effective alternative to traditional and standard methods especially in prehospital and peripheral setups for rapid volume resuscitation.The authors declare that they have no competing interests.nd author MS.DK designed this device along with 2MS wrote this manuscript with corrections by DK.Both authors read and approved the final manuscript.


Our aim in this study was to assess the dynamic changes in muscle tissue oxygen saturation  and before and after each VE.We conducted a prospective study of 42 fluid challenges in patients undergoing major abdominal surgery with evidence of hypovolaemia, defined as pulse pressure variation (PPV) >13% and SV variation (SVV) >12%. CI, SV, SVV (FloTrac/Vigileo) and PPV were measured before and after VE. Fluid responsiveness was defined as an increase of SV >15% after a 500-mL colloid infusion over 15 minutes. In all patients, the muscle StOP < 0.001) difference in SV changes between positive and negative responses to VE. There was a statistically significant difference in PPV and SVV values before VE in the positive and negative fluid responses . Data are presented as medians and 25th and 75th percentiles Before VE there was no significant difference in StO2 values relative to baseline , without a significant difference (P = 0.36) between positive and negative fluid challenges. Hypovolaemia was associated with a significant reduction (P = 0.004) in StO2 recovery slope, with a significant difference (P = 0.02) between positive and negative fluid challenges. The VE-induced increase in the StO2 recovery slope was 62 ± 49% (P < 0.001) for positive fluid challenges and 26 ± 34% (P = 0.04) for negative fluid challenges.No patients were preload-responsive after anaesthesia induction. Twenty-nine of forty-two fluid challenges (69%) were positive for VE, with a statistically significant (2 recovery slope. Restoring effective intravascular volume with fluid loading significantly improves the StO2 recovery slope, despite apparently ineffective changes in systemic haemodynamics.Hypovolaemia significantly affects the muscle StO Fluid loading is a first-line therapy when hypovolaemia is suspected in patients with evidence of hypoperfusion, and it is commonly used in operating rooms and ICUs. The maintenance of adequate oxygen delivery and tissue perfusion is considered a primary goal in volume replacement  while avAlthough a fluid challenge should correct macrohaemodynamics (stroke volume (SV) and cardiac output (CO)), the ideal volume replacement strategy should also improve microcirculation perfusion and tissue oxygenation. Hypovolaemia during major surgery or sepsis leads to inadequate perfusion of the microcirculation and insufficient oxygen availability to meet tissue oxygen needs . However2), especially the StO2 recovery slope, during a standardised vascular occlusion test (VOT) is assumed to reflect the recruitment of microvessels in response to a local hypoxic stimulus . In the positive fluid challenge, VE also induced significant changes in CO and MAP, but there were no evident differences induced by the negative fluid challenge (Table P = 0.10).Compared to baseline values, hypovolaemia was associated with a significant reduction in SV  vs. 86% (78% to 88%); P = 0.15]. Table 2 values during hypovolaemia relative to baseline  and no significant difference between positive and negative fluid challenges (P = 0.36). There was also no significant difference in desStO2 values before VE relative to baseline (P = 0.53) or between positive and negative fluid challenges /second vs. 3.57% (2.71% to 4.58%)/second; P = 0.004]. There was also a significant difference in recStO2 before VE in the positive and negative fluid challenges (P = 0.02) (Table 2 hyperaemia recovery area relative to baseline (P = 0.30) or between positive and negative fluid challenges (P = 0.29) (Table There was no significant difference in the mean StO 77% to 8% vs. 86% 77% to 8% vs. 86% 77% to 8% vs. 86% 77% to 8% vs. 86%2 after VE (P = 0.22) in the positive and negative fluid challenges (Table 2 of 50 ± 47% (P < 0.001) ; for negative fluid challenge, the VE-induced increase in recStO2 was 26 ± 34% (P = 0.04)  in VE-induced increase in recStO2 between positive and negative responses to VE. The coefficient of variability of recStO2 was 25% and 33% for the negative fluid challenge group and the positive fluid challenge group, respectively, with a statistically significant difference between groups (P = 0.0453). The results obtained with the mixed model showed a significant interaction between covariates (P = 0.039). Variance attributable to each random effect was 45.27% for patient, 49.60% for time and 5.13% for residual variability. The area under the ROC curve of recStO2 was 0.740 . The optimal cutoff value for recStO2 was 18% . There were no significant changes in desStO2 or StO2 hyperaemia area during VE and no differences in positive and negative fluid challenges  or between VE-induced changes in CO and the StO2 recovery slope , VE also leads to a significant improvement in the StO2 recovery slope.The main findings of our study are that (1) hypovolaemia is associated with significant alterations in NIRS variables measured at the thenar eminence and (2) restoring effective intravascular volume with fluid loading significantly improves the StOMonitoring the effects of fluid therapy at the bedside remains a cornerstone in the operating room and ICU. Although there are limitations ,25, prev2 recovery slope. Previous data have suggested that hypovolaemia leads to inadequate perfusion of the microcirculation, which results in insufficient oxygen availability [2 recovery slope is believed to reflect the microvascular blood flow response to a transient tissue hypoxia-induced oxygen deficit created by the ischaemic stimulus [2 target of 40%, as recommended by other investigators [2 recovery slopes were very close to those previously reported in healthy volunteers [2 values can be used to detect changes in peripheral tissue oxygenation resulting from a lower-body negative pressure model of simulated central hypovolaemia [2 values are insensitive to the assessment of tissue hypoperfusion [2 values in spite of an insufficient flow, support the findings of these previous studies.In our study, preload responsiveness was associated with a 25% decrease in the StOlability . The StOstimulus . Vasodilstimulus . We usedtigators ,33, and lunteers ,33. Prevvolaemia . A reducvolaemia . Nevertherfusion . Our res2 recovery slope with fluid loading. This suggests that restoration of intravascular volume in preload-dependent patients improves muscle tissue oxygenation and increases SV and CO. This finding may have important clinical implications, because the ultimate goal of resuscitation should be improvement of tissue oxygenation and perfusion. We hypothesise that this is due to improved microvessel recruitment during the fluid challenge, together with an increase venular blood compartment volume, despite the absence of macrocirculatory changes. It is unlikely that changes in the StO2 recovery slope with VE were due to changes in rheologic factors, because we found no significant differences in haemoglobin levels before and after VE. In addition, Creteur et al. [2 recovery slope remained low, or even decreased, in some of our patients after VE (Figure We also found a 50% increase in the StOr et al.  recently2 recovery slopes were associated reduced organ failure. Third, we placed the NIRS probe on the thenar eminence, a region with very little fat, and therefore there was little interference with the spectroscopic measurements. Although measurement at this site have very low variance [2 recovery slope [Our study has several limitations that need to be addressed. First, we studied only surgical patients. Although major surgery is associated with significant impairment in both microvascular flow and tissue oxygenation , our datvariance  and evenvariance . Althougry slope , the eff2 recovery slope based on NIRS measurements at the thenar eminence. In addition, our findings suggest that VE can improve tissue oxygenation. Taken together, our results suggest that measurements of StO2 using a standardised VOT could be a useful complementary tool along with the dynamic indices to improve fluid optimisation. Further studies are warranted to validate this hypothesis.In conclusion, with respect to our study conditions, we found that preload dependence is associated with significant changes in the StO• When hypovolaemia is suspected, fluid loading should restore systemic haemodynamics, but the ultimate goal of fluid resuscitation should be improvement of tissue perfusion and oxygenation.2 recovery slope based on NIRS measurements, suggesting the coexistence of microcirculatory abnormalities.• Preload dependence is associated with significant alterations in the StO2 recovery slope.• VE induces positive effects at the level of muscle tissue oxygenation as measured by the elevation of the StO2 recovery slope better assesses the effects of hypovolaemia and fluid loading on muscle tissue oxygenation than other NIRS-derived variables.• The StO2: StO2 desaturation slope; NIRS: near-infrared spectroscopy; PPV: pulse pressure variation; recStO2: StO2 recovery slope; StO2: tissue oxygen saturation; SV: stroke volume; SVV: stroke volume variation; VE: volume expansion.CI: cardiac index; CO: cardiac output; desStOThe authors declare that they have no competing interests.EF and ER conceived and designed the original study. SC and AP were responsible for patient enrolment and participated in data acquisition. EF, ER, BV, JD and JEB drafted the manuscript. All authors read and approved the final manuscript.