Fractionated radiotherapy, chemotherapy, and combined therapies are reviewed, providing a comprehensive overview of the modelling literature for current modellers and radiobiologists to ignite the interest of other computational scientists and health professionals of the ever evolving and clinically relevant field of tumour modelling.Mathematical and stochastic computer ( Modern treatment delivery methods for external beam radiotherapy employ techniques such as intensity modulated radiation therapy (IMRT) and image guided radiation therapy (IGRT) to deliver high radiation doses to the tumour planning target volume (PTV) or multiple PTV's with ever increasing accuracy and precision. However, to further improve outcomes for individual patients it will be necessary to explore in more detail the radiobiological processes that occur in human tumours and to predict optimal treatment plans for the individual patient. There is an increasing need to incorporate cellular behaviour and characteristics into individualised treatment planning and delivery. Undoubtedly, the future of cancer research rests on a multidisciplinary approach.The pursuit of an enhanced treatment regimen is conventionally performed through well-designed, randomised clinical trials. Clinical trials are indispensable prerequisites to establish novel therapeutic principles. Nevertheless, trials are lengthy processes which involve several influential factors for a decisive outcome: trial design, patient selection and followup, complex data analysis, and interpretation. Furthermore, trials cannot explore the sensitivity of the outcome to input parameters and covariates. in silico models) encompassing mathematical, physical, and engineering concepts representing the biological world. Models are an efficient way to complement the results of clinical trials. Beside animal models and cell lines, which are often used for preclinical studies, there are computer models . Mutually, the latter offer feedback to in silico models in support of further developments and optimisation (dashed arrow).The vo chain  is gainifirst principles approach and have “grown” the virtual tumour starting from a single cell . The tumour growth process is in itself a large area of research, allowing for investigations into the cell kinetics of malignant tissue. This line of study also provides modellers with ideas about how to simulate cell propagation during treatment, which is crucial because many treatments are delivered over multiple weeks or even months. As many of the models discussed are of the latter variety (modelling tumour growth as well as treatment), the proceeding section of this paper provides an overview of the key analytical and stochastic tumour growth models in the literature.When modelling the processes involved in the damage and elimination of tumour cells on the microscopic level, development of an algorithm to propagate a virtual tumour mass is first required. One approach has been to generate a full-sized macroscopic tumour mass with a predefined volume, cell type distribution, and so forth, based on average tumour statistics or a particular tumour of clinical interest; however, other modellers have used a The goals of tumour modelling vary among researchers and may focus on one key biological/radiobiological mechanism or explore mechanisms and ranges of parameter values. Many models aim to identify mechanisms and parameter values at the cellular level which are responsible for macroscopic tumour outcomes, for example, the cell kinetic properties affecting the tumour growth or shrinkage rates. Modelling can add to the understanding of cellular proliferative hierarchy and the differentiation processes, evolution of genetic mutations, tumour morphology, tumour pressure gradients, angiogenesis and oxygen distributions, diffusion of nutrients, and so forth. The modelling methodology employed to simulate a biological system may be analytical or stochastic in nature. Analytical  methodology is more traditional, utilising deterministic equation-based methods, often in the form of a set of equations incorporating multiple parameters. Stochastic methods use random number generation and probability distribution functions to simulate cell propagation and evolution of the tumour, for example, Monte Carlo (MC) methodology. Monte Carlo methodology enables a system to be modelled from first principles. It is a form of computation using random sampling and iteration to simulate the evolution of a physical or biological system and involves the use of probability distribution functions for decision making, for example, for the allocation of parameter values from a range of possible values. This technique is useful for modelling systems with a large number of coupled degrees of freedom, which are difficult to solve using equation-based methods.Tumours are complex entities, divers and heterogeneous, yet all share the ability to proliferate beyond the constraints limiting the growth in normal tissue. The growth of tumours is best represented by an exponential increase of cell number in time. Exponential growth is the simplest mode of growth assuming no cell loss or infertility. By growing exponentially, the tumour volume increases by a constant fraction in equal time intervals. Many human tumours during their growth show exponential behavior; however, there are tumours going through irregular or decelerating growth . A more It is generally accepted that human cancers grow in an exponential or Gompertzian manner. This assumption is based on analysis of the growth of transplantable animal tumours and on averages of tumour growth in human populations. Although not valid for all individual tumours, exponential growth may accurately describe averages of human tumour growth . HoweverModel parameterisation greatly depends on the available pool of biological, biochemical, or biophysical quantities used within the equations or model algorithm. When it comes to tumour growth, modelling of both kinetic and dynamic properties are envisaged. Cellular composition, volume doubling time, growth factor, cell loss factor, labelling index, cell cycle phase-related properties , and oxygenation status are some of the most commonly implemented modelling parameters. Also depending on the objective of the model, treatment-related factors are often found among input parameters.One of the earliest models of tumour growth and cell cycle simulation using the Monte Carlo approach is CELLSIM . CELLSIMin silico. These models consist of a regular grid of cells, each cell having assigned a certain state. New generations of cells are created based on predefined rules determining the state of each cell. For tumour growth simulations, cellular automata models became popular due to their ease of use. Qi et al. [Cellular automata models, also known as tessellation automata or cellular structure models, are one of the first types of cellular growth models developed i et al.  developeSeveral early tumour growth models have also considered the Gompertzian distribution to describe the shape of the tumour growth curve. Gyllenberg and Webb  explaineA stochastic model of tumour growth and invasion looking into the relationship between the histological pattern of tumours and their functional properties was developed by Smolle and Stettner . The modThe implementation of growth regulation and control mechanisms into three-dimensional models  of epithelial cell populations was the focus of an individual cell-based model aiming to simulate the growth pattern and behaviour of a number of different epithelial cell populations from undifferentiated stem cells up to tumour cells . The mec8 to 1010 cells, simulating tumour oxygenation is generally considered a requirement for most tumour types in order for model growth curves to match well with in vivo data. Lack of oxygen in tissue, hypoxia, is commonly defined by a pO2  threshold of 10 mm Hg, although clinical trials may use 2.5 or 5.0 mm Hg thresholds when reporting experimental results such as the Hypoxic Fraction (HF) of cells in the tumour. It is now commonly known that low tumour oxygenation results in radioresistance and is a major contributor to treatment failure due to tumour recurrence [When modelling clinical-sized tumours comprising up to 10currence . ConsequSince the initial experimental and subsequent mathematical modelling of tissue hypoxia by Gray et al.  based onThe 1980s saw the first real-time stochastic applications of tumour computer models, which paved the path for the complex Monte Carlo simulations of the current era. The tumour models reported on during the last decade tend to involve more detailed cell line specific kinetics and also include one or more treatment modalities with the option of individual patient data input, for example, spatial PET imaging hypoxia information, the review of which will be encompassed in the proceeding sections of this paper. However, a group concentrating on tumour growth based on the work of Anderson et al. , 53 provPredicting the outcomes of fractionated radiotherapy using models was initially developed utilising theories such as the Power Law equation of the Nominal Standard Dose theory of fractionated cell kill  and variUse of the standard LQ equation became extremely popular in the mid 1980s and was soon extended by many authors by the addition of various modification factors for modelling effects such as the “time-factor”  for rapiAlongside the development and widespread clinical implementation of the LQ model, other models were emerging in the last two decades of the 20th century. These models were initially mathematical based; however, a subset of modellers soon branched out into the utilisation of stochastic modelling techniques to describe the action and impact of ionising radiation on living tissue. As the models became more sophisticated, so too did the requirement not only to describe the effects of radiation more accurately, but also to model on smaller and smaller scales. Imaging modalities were beginning to collect data on the “mm” to cellular scales depending on the modality used, creating huge data sets when modelling tumours of macroscopic volumes.Macroscopic tumour modelling is most relevant for comparisons to human data and eventual translation into clinical use, consequently the modelling of the tumour vascular system became of interest. Inclusion of oxygenation parameters in models was driven by the increasing realisation of the importance of tumour hypoxia as reported from large clinical trials for certain tumour sites, for example, head and neck –68. ConsCELLSIM and later the 2D CELLGROW [Due to the advances and increasing usability of personal digital computers in the 1980s, the field of tumour and treatment modelling was boosted, particularly in the US and in European countries such as Germany and Norway. One of the first stochastic tumour growth and/or radiotherapy models of avascular tumours was called α and β parameter values for hypoxic tissue.In the following decade, a mathematical model describing the changes in tumour growth during development and LQ-based cell kill emerged , simulat2 values. In the next few years a stochastic model of tumour growth was developed, simulating cells with ranges of proliferative capacity , and investigating the kinetics of accelerated repopulation and treatments including radiotherapy and chemotherapy [Another mathematical model was reported in the late 1990s from Wouters and group , 69, whiotherapy , 5, 70. In 2002, two mathematical tumour treatment models incorporating stochastic parameter distribution were reported on, one aiming to model and clinically verify accurate microvascular density and heterogeneity in a 2D tumour cross section with cylindrical vessels , 19 and Søvik and colleagues  have devThe modelling work of Daşu et al. , 71, 72 Finally, there have been a number of stochastic vascularised tumour growth and/or radiotherapy treatment models produced in the past decade. Of those reviewed herein, one is a purely temporal model, while the others are spatial-temporal. In general, most modern models simulate cell growth on an individual cell basis; however, there is a trend in some models to average cellular properties and model “tumour voxels” or “geometrical” cells. This is often performed because of the large tumour volumes being applied and owing to the direct input of imaging  data into the models. This direct input is used in some model as an alternative to simulating the tumour growth process, and rather the tumour is created at full size based on clinical data, ready for treatment simulation. There are advantages and disadvantages to this technique, including the lack of understanding/research into microscopic aspects of the tumour, that is, cellular kinetics and how kinetic parameters change with tumour volume, but also the benefits of individualising the model for a particular patient and therefore providing more direct outcomes to compare with clinical data. in vitro tumour spheroids for small lung cell carcinoma. Since 2004, the group has published models of in vivo tumour systems [3. A comparison between six radiotherapy fractionation schedules has been performed by varying parameters such as the cell loss factor, OER, OERβ, cell cycle time and mutated versus wild type p53 status. Results show that accelerated schedules are superior to conventionally fractionated ones and that wild type tumours (higher α/β) respond well compared to mutated tumours.The Greek modelling group led by Stamatakos have published many papers regarding their models of growth and radiotherapy for lung and brain tumours. Initial work began with a 3D discrete time step model  , with an accurate time delay of tumour shrinkage predicted.A stochastic tumour simulation model  using ceAs mentioned in a number of the preceding model outlines, the majority of modern models use the standard LQ theory as a basis for stochastic cell kill; however, some models attempt to readdress cell survival theory by means of new equation sets. Such an example of this is the model by Hanin and Zaider  who haveIn-silico tumour growth and/or treatment models have various degrees of complexity, with numerous assumptions applied, as is inherent in all modelling applications. Commonly, the goal of the modeller is to determine the optimal treatment strategy, for example, dose fraction sizes and timing, to achieve tumour control or total cell kill for a specific tumour type. The goal should also be to achieve optimised tumour cell kill within minimal normal tissue toxicity levels, ideally on an individual patient basis. This goal, however, is very challenging for reasons including: data gathering from reliable in vivo experiments, the large and varied patient sets in clinical trials and the extremely complex biological and chemical processes involved in carcinogenesis and tumour evolution. Tumour radiotherapy modelling requires not only accurate radiation damage models but also the implementation of how the tumour cells respond to sublethal or lethal damage, which may change with dose received, tumour volume, oxygenation levels and numerous other cell line specific and individual tumour-based factors. Nevertheless, the development of models has and will continue to assist radiobiologists and clinicians in predicting tumour behaviour and understanding microscopic mechanisms and impact upon macroscopic and measurable tumour parameters, and as such the research should continue and be encouraged.in silico radiotherapy models.A selection of key tumour models from the literature are tabulated below regarding avascular tumour radiotherapy models  and vascWhile the vast majority of tumour growth and treatment response models (such as the ones presented above) are simulated on a cellular level, models on a smaller scale are a hot topic of current investigation. Monte Carlo models targeting the cell nucleus and cytoplasm have been developed using the ever-expending GEANT4 MC simulation toolkit. Barberet et al. have modelled different cell geometries found in a typical cell population in order to evaluate the absorbed energy from alpha particles and their response to different irradiation protocols . The picChemotherapy agents act via many different pathways and they vastly differ in biochemical structure, molecular mode of action, pharmacology, clearance, and side-effects. Furthermore, in chemotherapy there is no formalism equivalent to the linear quadratic model used in radiotherapy which would describe, in a simplistic yet practical way, cell survival. While quantitative modelling has great potential, it requires knowledge of the numerical values of multiple parameters in order to characterize the chemotherapy dose regimens. This information is rarely available; therefore, the modelling of combination-chemotherapy regimens (drug cocktails), or of large classes of chemotherapy agents, can induce errors and inaccuracies in the simulation process.The pool of chemotherapy-induced cell kill models is vast. Similarly to radiotherapy, chemotherapy models are divided between analytical and stochastic and they target various aspects of drug kinetics/dynamics and tumour response. In the paragraphs below, the main chemotherapy model categories are presented by listing the most representative papers from each group. Besides the most common cell kill models that investigate the correlation between tumour kill and drug concentrations/exposure times (area under the time-concentration curve), there are models studying the effect of various drugs on tumour cell kill along the cell cycle as well as compartment models focusing on drug pharmacokinetics. In addition, there are models which tackle the problem of drug resistance and repopulation during chemotherapy—two major factors which can lead to treatment failure. While models of drug resistance are more commonly reported in the literature, tumour repopulation during chemotherapy is usually a neglected factor when it comes to modelling.One of the first reviews on chemotherapy modelling was published by Aroesty et al. . The maiin vitro was reported by Hill in 1910 [Despite the obstacles imposed by chemotherapy modelling, models of drug pharmacokinetics and pharmacodynamics have been developed in the past, using either analytical or probabilistic methods . In fact in 1910 . Hill's There are several models studying the effect of various drug concentrations and exposure times on tumour control. Gardner  proposedNumerous studies have used the “area under the time-concentration curve” (AUC) as an approach to model chemotherapy. The area under the curve is a commonly used measure of total drug exposure and is obtained by plotting the concentration of the agent as a function of time and obtaining AUC by integration. While for some drugs  the effect is proportional to the AUC , for othin vitro data regarding time dependency of drug potency, slope of the concentration-effect curves, and relative degree of drug resistance. Levasseur et al. [The AUC models are usually based on r et al.  have creThere are convenient ways to describe drug pharmacokinetics inside the body, that is, the way plasma concentration of a drug changes over time. Depending on their properties  drugs follow multicompartmental behaviour  . The com1 which leads into the DNA synthesis phase S, followed by another growth phase G2 which then leads to mitosis, M. The options are to divide the cell cycle into two  or three compartments . This division into compartments facilitates the modelling of cell cycle-specific drugs, that is, different classes of chemotherapeutic agents . This type of cell cycle kinetics modelling was introduced by Swierniak et al. in the 1980s [On a cellular level, compartment models are designed for investigations into cell-cycle kinetics. In these models, the compartments are represented by the phases of the cell cycle, starting with the growth phase Ghe 1980s  and latehe 1980s , 96. Chemo-related factors such as drug diffusion, uptake/binding, clearance and their effect on cell cycle progression are individually incorporated in various spatio-temporal models –99. A moAnother multi-compartment model to predict cellular response of different cell lines to mitotic arrest when exposed to paclitaxel was developed by Basse et al.  with staOn similar principles of compartment modelling, the pharmacodynamics of Cisplatin have been simulated  by consiModels of drug resistance started to be developed in the late 1970s, with the Goldie Coldman [One of the major challenges of chemotherapy is associated with drug resistance caused by mutations in cancer cells.  Coldman  model onBirkhead et al.  have desMathematical models of gene amplification were developed by Kimmel and Axelrod  to studyn drugs, a cell must accumulate n mutations. Drug-induced cell death is regulated by the extent of drug resistance, whereby cells resistant to all drugs are not killed by the drug while cells susceptible to certain drugs are labelled with a drug-induced death rate dependent on treatment intensity. The model concluded that the success of the treatment is independent on turnover rate  for one-drug treatments but dependent on turnover rate for multiple-drug treatments. A more specific Monte Carlo model, looking into tumour resistance to Cisplatin was developed by Marcu et al. [Ample modeling of drug resistance was undertaken by Komarova  and Komau et al.  by modelModelling of crossresistance in cyclic chemotherapy treatment was tackled by Katouli and Komarova  showing tumour repopulation during chemotherapy is usually a neglected factor. As shown by Davis and Tannock [While repopulation during radiotherapy is an extensively studied aspect associated with treatment failure,  Tannock  the impa Tannock . CellulaModels are useful tools to simulate novel treatment schedules designed to improve treatment outcome by means of higher therapeutic ratio. Such a novel regimen for Cisplatin was suggested by Marcu and Bezak  in a neoMechanistic mathematical models developed to improve the design of chemotherapy regimen were summarised by Gardner  and Gardin vitro experiments, the number of unknown parameters originating from multidrug interaction turns modelling into an exigent task. The literature on chemomodelling shows that optimum treatment strategies are hard to derive mainly because of lack of quantitative knowledge of the biological parameters of cancer chemotherapy. While quantitative data for a specific drug can be obtained from specific The literature is scarce on combined chemoradiotherapy models, a possible reason being the lack of quantitative experimental data on radiation-drug interactions. Studies which model combined treatment techniques aim their focus towards factors such as (1) timing between agents, (2) optimal combined schedules, (3) the extent of agent interaction , (4) dose-effect and (5) acquired resistance to treatment, to name just a few. Goldie and coworkers  simulateCisplatin and radiotherapy for advanced head and neck cancers was stochastically modelled by Marcu et al.  looking in silico, in order to select an optimal chemotherapeutical protocol. The theoretical model simulates the dynamic evolution of tumour population density under dual coupling periodic interventions (treatment). The model outcome is in fine agreement with Loewe's additivity model, a universal reference model for drug interaction, showing that the curative effect of the combined treatment is strongly dependent on the intensity, nature (sub/supra-additive) and the timing of the individual treatments. This study could serve as a useful preclinical pharmacokinetic assessment of drug-drug or drug-immunotherapy interaction.Besides radiotherapy and chemotherapy, immunotherapy has become an important aspect of today's cancer management. Based on logistic tumour growth law, the combination of chemotherapy with immunotherapy was recently simulated by Hu and colleagues  to addrein vivo tumour measurement and/or imaging , may include levels of specific protein synthesis and activity, a full description of tumour oxygenation from 0 to approximately 40 mm Hg, intrinsic radiosensitivity levels and cell kinetic behaviour of all cell types involved including all primary tumour cell types, vessel , surrounding normal cell information, patient condition variable such as immune system responses in the form of DNA damage repair efficiency.To further increase the power of complex tumour modelling into the future, greater depth of knowledge of tumour biology on an individual tumour basis will be required. This data is likely to be at micro(cellular) level as well as genetic and chemical level within the tumour. The data required for model input, necessitating the need for variations between patients;the dynamic nature of the parameters desired;time/expense due to complexity of testing procedures, for example, protein analysis;understanding the mechanisms of microscopic processes within the tumour which are often interrelated.Specific difficulties of data gathering for tumours models are listed in While overcoming the difficulties imposed by these parameters can be a complicated task due to clinical confines in obtaining quantifiable data, there is always an option to undertake computational sensitivity studies of specific parameters within biologically plausible limits. in silico tumour modelling is challenging, but wide open for expansion through the dedication of radiobiology, medical physics, and computational science researchers. Assumptions, estimations, idealisations, and trial-end-error situations will constantly be part of the modelling process. Nevertheless, despite its limitations, mathematical and computational modelling is already playing an integral role in several aspects of cancer management and it is expected to gain more ground in the near future by complementing other preclinical studies. The future of


In the Funding Statement, the number of the grant from the Czech Ministry of Health is incorrect. The correct number is: NT 12288-5/2011.


We studied the effects of enzyme inhibitors on the uptake of 6-[18F]fluoro-d-glucose ([18F]FDG) and scanned with positron emission tomography/computed tomography (PET/CT). For [18F]FDOPA studies, tumour-bearing mice and sham-operated controls were pretreated with enzyme inhibitors of aromatic amino acid decarboxylase (AADC), catechol-O-methyl transferase (COMT), monoamine oxidase A (MAO-A) or a combination of COMT and MAO-A. Mice were injected with [18F]FDOPA and scanned with PET/CT. The absolute [18F]FDOPA uptake was determined from selected tissues using a gamma counter. The intratumoural biodistribution of [18F]FDOPA was recorded by autoradiography. The main [18F]FDOPA metabolites present in the pancreata were determined with radio-high-performance liquid chromatography.Mice bearing orthotopic BxPC3 pancreatic adenocarcinoma were injected with 2-deoxy-2-[18F]FDG uptake was high in pancreatic tumours, while [18F]FDOPA uptake was highest in the healthy pancreas and significantly lower in tumours. [18F]FDOPA uptake in the pancreas was lowest with vehicle pretreatment and highest with pretreatment with the inhibitor of AADC. When mice received COMT + MAO-A inhibitors, the uptake was high in the healthy pancreas but low in the tumour-bearing pancreas.[18F]FDG and [18F]FDOPA is suitable for imaging pancreatic tumours. Unequal pancreatic uptake after the employed enzyme inhibitors is due to the blockade of metabolism and therefore increased availability of [18F]FDOPA metabolites, in which uptake differs from that of [18F]FDOPA. Pretreatment with COMT + MAO-A inhibitors improved the differentiation of pancreas from the surrounding tissue and healthy pancreas from tumour. Similar advantage was not achieved using AADC enzyme inhibitor, carbidopa.Combined use of [ Due to late diagnosis and lack of effective treatment, pancreatic adenocarcinoma has the worst prognosis of all of the gastrointestinal cancers . Surgery18F]fluoro-d-glucose ([18F]FDG). [18F]FDG is taken up into cells by glucose transporters, where it subsequently undergoes phosphorylation by hexokinase-1 into [18F]FDG-6-phosphate. This tracer is efficiently taken up by a variety of tumour cells and reflects increased glucose metabolism [18F]FDG has been useful in the evaluation of indeterminate pancreatic masses, staging of pancreatic carcinoma, detection of metastatic disease and differentiation of viable tumours from post-therapeutic processes like necrosis or scar tissue [18F]FDG in pancreatic cancer is limited since inflammatory processes such as pancreatitis and abscesses take up [18F]FDG as well. Chronic pancreatitis is recognised as the most common reason for false-positive [18F]FDG-PET tumour findings in the pancreas Fluoro-l-3,4-dihydroxyphenylalanine ([18F]FDOPA), a fluorinated analogue of l-DOPA, an intermediate product in dopamine synthesis, is the most commonly used PET tracer in studies of hyperinsulinaemia and neuroendocrine tumours [18F]FDOPA is focused on the neuroendocrine nature of pancreatic cells FDOPA is extensively metabolised to 6-[18F]fluoro-l-3-methoxy-4-hydroxyphenylalanine ([18F]3-OMFD) by catechol-O-methyltransferase (COMT) and by AADC to 6-[18F]fluoro-l-3,4-dihydroxyphenylethylamine , which is rapidly sulphated by phenolsulfotransferase to fluorodopamine sulphate [18F]FDA is also transformed by monoamine oxidase (MAO). The two isoforms of MAO, MAO-A and MAO-B, deaminate dopamine to produce 6-[18F]fluoro-l-3,4-dihydroxyphenylacetic acid ([18F]FDOPAC) before it is converted into 6-[18F]fluorohomovanillic acid ([18F]FHVA) by COMT [18F]FDA is also converted into 6-[18F]fluoro-l-3-methoxytyramine by COMT before being oxidised to [18F]FHVA by MAO , autoradiography and chromatography in an orthotopic xenograft mouse model of pancreatic adenocarcinoma.The aim of this study was to improve the detection of pancreatic adenocarcinoma using PET. We used F2 as the labelling reagent [18F]FDOPA was 5.8 GBq/μmol. Radiochemical purity exceeded 98% in every production batch [[c method . [18F]FD reagent ,28. The on batch .18F]FDOPA, animals were pretreated with enzyme inhibitors 10 min before injection of [18F]FDOPA. Sham-operated and tumour-bearing animals were divided into groups according to weight. Inhibitors and study groups are described in Table n = 6 + 6, sham-operated and BxPC3), Ro-41-0960 , clorgyline  or a combination of Ro-41-0960 and clorgyline (n = 6 + 5). All enzyme inhibitors were purchased from Sigma-Aldrich Chemicals. Animals treated with 0.9% NaCl were used as controls (n = 6 + 5). Mice were either sacrificed for ex vivo analysis 10 min after injection of [18F]FDOPA or imaged using small animal PET/CT .In order to minimise the peripheral metabolism of 8FFDOPA, 18F]FDG into pancreatic tumours (n = 14) was evaluated using small animal PET/CT. [18F]FDG (dose 9.0 ± 2.3 MBq) was injected into the tail veins of the animals under isoflurane anaesthesia. CT was used for attenuation correction of the PET data and anatomical reference following a 20-min static scan, which was acquired in list mode at 60 min after injection. The next day, selected mice (n = 4) were subjected to CT and a 20-min dynamic PET scan using [18F]FDOPA tracer. The injected dose was 6.8 ± 0.1 MBq, and the injected mass was 16 ± 10 ng/g, as calculated from the known specific radioactivity at the time of injection. Images were reconstructed using a 2D filtered backprojection with a 0.5-mm ramp filter. Data were collected for 20 min after injection of [18F]FDOPA, and the corresponding time-activity curves were calculated. Images were analysed using the Inveon Research Workplace software (v. 3.0). Volumes of interest were drawn manually on the pancreas, tumours and the left cardiac ventricle (blood). The [18F]FDOPA uptake in tumours was expressed as time-activity curves of the pancreas and tumours normalised to blood radioactivity.Six weeks after orthotopic inoculation of the BxPC3 cells, the uptake of FDOPA. The HPLC method consisted of a μBondapak C18 column  which was eluted with a solution of 50 mM sodium acetate, 20 mM citric acid, 1.0 mM sodium 1-octanesulfonate, 1.0 mM di-n-butylamine and 1.35 mM sodium EDTA in water/methanol (90:10 v/v). Authentic standards of [18F]3-OMFD, [18F]FDA, [18F]FMT, [18F]FDOPAC and [18F]FHVA FHVA ,32 were t test. Radioactivity (ratios to non-target tissues and autoradiography) measurements were analysed using a two-way analysis of variance. Models included the main effects of pretreatment  and group . In further pairwise comparisons between pretreatments, the Tukey adjustment method was used. Interaction between pretreatment and group was also tested. Due to positively skewed distributions, log-transformed values were used in statistical analyses. SAS System for Windows was used in statistical computations ; p values less than 0.05 were considered as statistically significant.The mean weight of the sham-operated pancreas and pancreas with a tumour was compared with a two-sample 3 at 35 days after tumour cell inoculation and 760 ± 1,300 mm3 at 42 days after tumour cell inoculation. The mean weight of the sham-operated pancreas was 0.18 ± 0.03 g, while the weight of the pancreas with a tumour at 35 and 42 days after inoculation was 0.27 ± 0.08 g (p < 0.001 vs. sham-operated) and 0.73 ± 0.97 g (p < 0.001 vs. sham-operated), respectively. As expected, the weight of the pancreata increased in tumour-bearing mice compared with the sham-operated mice.No differences were detected between the weights of sham-operated and tumour-bearing mice (data not shown). No signs of cachexia were detected, which indicates that the tumours were not very large (data not shown). The tumour occurrence was 100%, and the mean tumour volume was 50 ± 40 mmex vivo distribution of [18F]FDOPA-derived activity was expressed as percentage of injected dose per gram of tissue . However, these differences were not dependent on the used pretreatment . Carbidopa pretreatment increased the uptake in the pancreata in both sham-operated and tumour-bearing mice compared with vehicle treatment (group adjusted p = 0.037 in pancreas-to-muscle and p < 0.0001 in pancreas-to-liver ratios), but the difference between the sham-operated and tumour-bearing mice was not high enough to separate the healthy pancreas from the tumour-bearing pancreas  were calculated based on measured radioactivities (% ID/g), and they are presented in Figure 18F]FDG PET/CT 6 weeks after tumour cell inoculation. Orthotopic pancreatic tumours exhibited increased glucose uptake  and COMT + MAO-A inhibitor-treated (p = 0.002) animals. In carbidopa-treated mice, no significant difference was detected between sham-operated and tumour-bearing animals (p = 0.733). These observations were in accordance with ex vivo biodistribution data , we identified [18F]3-OMFD (Rt = 7 min), [18F]FDOPAC (Rt = 8 min), [18F]FDA (Rt = 12 min), [18F]FHVA (Rt = 13 min) and [18F]FMT (Rt = 23 min). In the pancreata of vehicle-treated mice, three main metabolites were identified: [18F]3-OMFD, [18F]FHVA and [18F]FMT. Following the carbidopa pretreatment, the main metabolite in the samples was [18F]3-OMFD, while after MAO-A pretreatment, [18F]3-OMFD and [18F]FMT were identified. [18F]FDA was the major metabolite identified following pretreatment with COMT + MAO-A inhibitors. After the COMT pretreatment alone, the main metabolite was [18F]FDOPAC.Metabolites were analysed in pancreatic samples of sham-operated and tumour-bearing mice using HPLC. HPLC radiochromatograms from a radiometabolite study are shown in Figure 18F]FDG, which is the most widely used radiopharmaceutical for PET [18F]FDG in the duodenum may cause false-positive results, and imaging of the upper abdomen in general is influenced by respiratory and gastrointestinal tract motion [18F]FDG uptake and leading to false-positive tumour-related findings [18F]FDOPA is a commonly used PET tracer for investigating the activity of the dopaminergic system in neurological disorders. [18F]FDOPA PET has also been successfully used to visualise neuroendocrine, carcinoid and glomus tumours as well as pheochromocytomas and medullary thyroid cancers [18F]FDOPA also in the imaging of pancreatic adenocarcinoma and to assess the possible benefits of clinically available, widely used enzyme inhibitors of catecholamine neurotransmitters.At present, the treatment of pancreatic adenocarcinoma is difficult because the location of the tumour lesion is usually unknown. In clinical practice, pancreatic cancer is imaged using [ for PET . Howevert motion . Inflammfindings . [18F]FD cancers ,35-37. T18F]FDG. [18F]FDG revealed increased glucose metabolism in the upper left abdomen of the tumour-bearing mice, and tumour lesions were determined based on the anatomical location  differences in 18F-uptake ratios between sham-operated and tumour-bearing mice, which were pretreated with vehicle or COMT and MAO-A inhibitors prior to [18F]FDOPA injection. This observation can be explained by a change in [18F]FDOPA metabolism compared with that following carbidopa pretreatment. This was confirmed with tumour autoradiography. Although uptake was highest in carbidopa-treated pancreata, the difference between sham-operated and tumour-bearing pancreata was largest in animals pretreated with COMT + MAO-A inhibitors.18F]FDOPA uptake in the pancreas and therefore facilitates the localisation of the pancreas, but it also impairs the observer's ability to separate the healthy pancreas from the tumour because, in addition to exocrine pancreatic cells, tumour cells are also able to uptake [18F]3-OMFD, which is the main metabolite after carbidopa administration. COMT + MAO-A inhibitors increased the 18F radioactivity uptake in pancreatic tissue, while the uptake in tumours is poor due to the formation of [18F]FDA as main metabolite. Therefore, COMT + MAO-A inhibition improved the separation of the healthy pancreas from the tumour. However, the difference between the healthy pancreas and tumour was not clear in all cases after COMT + MAO-A administration, and the benefit over vehicle pretreatment remained modest. According to our observations, [18F]FDOPA combined with [18F]FDG imaging is a useful tool for detecting pancreatic adenocarcinoma, either alone or with COMT + MAO-A pretreatment, but not with carbidopa pretreatment. Since deducing the exact location of the tumour lesion is essential for a successful treatment, our data may have an important clinical value.Our study indicates that pretreatment of mice with carbidopa increases FDA: 6-[18F]fluoro-l-3,4-dihydroxyphenylethylamine; [18F]FDG: 2-deoxy-2-[18F]fluoro-d-glucose; [18F]FDOPA: 6-[18F]fluoro-l-3,4-dihydroxyphenylalanine; [18F]FDOPAC: 6-[18F]fluoro-l-3,4-dihydroxyphenylacetic acid; [18F]FHVA: 6-[18F]fluorohomovanillic acid; [18F]3-OMFD: 6-[18F]fluoro-l-3-methoxy-4-hydroxyphenylalanine; ID: Injected dose; MAO: Monoamine oxidase; min: Minute; NaCl: Sodium chloride; PET/CT: Positron emission tomography/computed tomography; PSL: Photostimulated luminescence per unit area; Rt: Retention timeAADC: Aromatic amino acid decarboxylase; AD: Aldehyde dehydrogenase; Bq: Becquerel; COMT: Catechol-The authors declare that they have no conflict of interest.in vivo experiments, analysed autoradiography, gamma counter and PET/CT data and drafted the manuscript. LH did the HPLC quality control. TJG scanned the subjects. SF and OS prepared tracers. TV made statistical analyses. MHS did HPLC analyses and supervised all other analyses. All authors edited the manuscript. All authors read and approved the final manuscript.MHS, JT and OS designed the study. JT made


Hepatocellular carcinoma is the most common primary tumour of the liver. The most common extrahepatic metastatic sites are the lung, lymph nodes, bones and adrenal glands. All forms of HCC demonstrate a tendency for vascular invasion, producing extensive intrahepatic metastases and, occasionally, portal vein or inferior vena cava extension with spread into the right atrium in extreme cases. Tumour spread of abdominal diseases via hepatic ligaments has also been previously reported. We report a rare case of hepatocellular carcinoma with extension into the falciform ligament, overlying rectus sheath and adjacent diaphragm with concomitant infiltration into the recanalised paraumbilical vein. A 73 year-old male presented to our centre with a painful swelling in the epigastric region which progressively increased in size over 1 month, alongside loss of appetite and fever with chills. He was an alcoholic, diabetic and also suffered from ischaemic heart disease. There was no history of hepatitis or prior blood transfusion. Physical examination revealed a firm, well-defined erythematous but smooth-surfaced epigastric mass measuring 8×7 cm. Spider naevi was noted on the legs and jaundice in the left sclera (right eye was blind).9 /L ; haemoglobin 103.0 g/L ; haematocrit 0.34 ; total protein 54 g/L ; albumin 24 g/L ; total bilirubin 8 μmol/L ; ALT 77 IU/L ; AST 83 IU/L ; α-fetoprotein 9 IU/L; Hepatitis B and C profile was negative and coagulation profile was normal.Pertinent laboratory values were as follows: white blood cell count 10.1×10An ultrasound examination revealed an ill-defined hypoechoic mass measuring 5.7×7.4 cm in the left lobe of the liver. The biliary ducts, gallbladder, pancreas and spleen were normal. Vascular channels ie. inferior vena cava, portal and hepatic veins were also patent. A provisional diagnosis of liver tumour was made. Subsequently a CT scan revealed an exophytic mass arising from an atrophic left lobe of the liver with surrounding inflammatory changes and extension into the overlying anterior abdominal wall  with maxLiver biopsy revealed a well-differentiated hepatocellular carcinoma. Patient underwent a wedge resection of segment 2. Intraoperative findings revealed an 8 cm nodular exophytic tumour arising from segment 2 of the liver with extension to the anterior abdominal wall and undersurface of the left hemidiafragm. The surface of the liver was nodular in appearance. Patient also underwent a cholecystectomy and excision of the infiltrated left diaphragm.At histopathology the specimen showed a tumour composed of nodules of malignant cells in sheets with moderate vascularity. The cells appeared hepatoid and displayed large irregular hyperchromatic vesicular nuclei and prominent nucleoli. Fibrous septa separated the tumour nodules and there was a thin capsule around the tumour. The adjacent hepatic tissue showed portal tract infiltration by chronic inflammatory cells. Special stains showed that immunohistochemistry for HBV was negative. The diaphragmatic specimen consisting of skeletal fibres showed nodules of malignant cells similar to that described in the liver specimen. There was no gallbladder invasion.The most important risk factor for the development of hepatocellular carcinoma (HCC) appears to be chronic hepatitis B or C viral infection . Liver cMany different treatment options are available depending on the staging of this aggressive tumour . The comKim et al. and Mori et al. ,6 have pRadiologically, disease processes, such as fluid collections within and obliterating the perihepatic-ligamentous fat or focal collections near the ligamentous attachments suggests ligamentous spread. Concommitantly there may be continous spread, also suggesting ligamentum continuity on CT scans or sonograms . LigamenWe therefore postulate the extension into the anterior abdominal wall may have occurred by direct extension of the exophytic tumour component into the paraumbilical vein within the round ligament and rectus via the falciform ligament. In addition, this patient had no history of having undergone a liver biopsy prior to discovery of the tumour mass, excluding tumour seeding along a previous biopsy tract.Although the HPE specimen analysis did not reveal evidence of fibrosis or cirrhosis of the liver parenchyma adjacent to the tumour, a thin capsule was found around the tumour and the nodular surface of the liver was confirmed macroscopically during surgery.A curative surgical approach with wedge resection of segment 2 of the liver was performed as the tumour was found to be confined to segment 2 with no evidence of metastases. The patient was subsequently followed up clinically at 3-monthly intervals with 6-monthly follow-up CT scans. A year after the operation, the patient developed tumour recurrence at the operative scar site on the anterior abdominal wall. He underwent a single cycle of radiotheraphy and subsequently chemotheraphy; however the tumour failed to respond favourably. The tumour demonstrated a slow growth pattern and currently measures approximately 3×4 cm. The patient is currently being managed conservatively and is otherwise well.


A novel hybrid continuum-discrete model to simulate tumour growth on a cellular scale is proposed. The lattice-based spatiotemporal model consists of reaction-diffusion equations that describe interactions between cancer cells and their microenvironment. The fundamental ingredients that are typically considered are the nutrient concentration, the extracellular matrix (ECM), and matrix degrading enzymes (MDEs). The in vivo processes are very complex and occur on different levels. This in turn leads to huge computational costs. The main contribution of the present work is therefore to describe the processes on the basis of simplified mathematical approaches, which, at the same time, depict realistic results to understand the biological processes. In this work, we discuss if we have to simulate the MDE or if the degraded matrix can be estimated directly with respect to the cancer cell distribution. Additionally, we compare the results for modelling tumour growth using the common and our simplified approach, thereby demonstrating the advantages of the proposed method. Therefore, we introduce variations of the positioning of the nutrient delivering blood vessels and use different initializations of the ECM. We conclude that the novel method, which does not explicitly model the matrix degradingenzymes, provides means for a straightforward and fast implementation for modelling tumour growth. Cancer is one of the most common diseases in adulthood and has gained more and more attention in research of different scientific disciplines in recent years. We focus on malignant brain tumours such as gliomas, but the models presented in this paper are generic and can also be applied on different tumour entities and other solid tumours through variation of the corresponding parameters.Generally, the models of tumour growth are described either on a macroscopic , 2, micrαf and thus also the remodelling parameter βf. To validate the findings, we introduce different arrangements of the nutrient delivering blood vessels. Further, we compare the novel proposed model with common ones by simulating different initial conditions for the ECM. In this work, we do not consider cell-cell adhesion of cancerous cells not only due to the decreased expression of neural cell adhesion molecules (NCAM) in aggressive gliomas, such as the glioblastoma (GBM) × which corresponds to 4 mm × 4 mm with boundary Γ : = ∂Ω. A 400 × 400 grid over Ω with a space step of h = 0.01 mm forms the basis for the discrete method since each square of the grid corresponds approximately to the area of a tumour cell, that is 6.25 × 10−6 cm2 . The grcm2  density f is given by: The hybrid model (cf. ) is compΩ × , where T defines the end of a given time interval, Dc and Du denotes the diffusion coefficients of the tumour cell and nutrients, respectively. Furthermore, χ is the chemotaxis coefficient and ρ the haptotaxis coefficient. Uptake and decay of particular substances due to tumour growth are αu and αf (uptake rate for nutrients and ECM), βf represents the remodelling parameter for the ECM.m is defined in Ω ×  as follows: To compare our results with common ones, we outline also the approach for modelling the extracellular matrix effected by enzymes. The complete system including the matrix degrading enzymes' (MDEs) concentration Dm denote the diffusion coefficients of the enzymes, αm the decay coefficient, and βm represents the production constant for MDEs.Due to the diffusive behaviour of the MDEs, the values are different for the uptake and the remodelling of the extracellular matrix comparing to . Hence, Ω. As for the different initial conditions for the ECM we refer to cini represents the initial tumour with 441 cells in the middle of the domain. This equation denotes the availability of nutrients in the cerebrospinal fluid (CSF), which is a supplier of essential nutrients to the brain :N and ΓD are the Neumann boundary and the Dirichlet boundary, respectively, with ΓN ∪ ΓD = Γ. We set ud = 1 since the concentration of nutrients, such as glucose and oxygen is highest in the capillaries.For computing the nutrients , 2b), a, a2b), at = mk, x = ih, and y = jh , we use forward differences at time point tm and second order central differences for the spatial derivative at point xi,j. The resulting equation for the 5-point stencil governing the chemotactic-haptotactic migration of a tumour cell in  = 0.8 is taken to allow for the assumption that the density of the ECM is high at the beginning, but smaller than 1, which would be equivalent to the maximal density and the value chosen in [For the tumour cell equations  and 2a)2a), resp cell in  and (2a) the ECM . On the hosen in . The chohosen in , where tc = 1 when a tumour cell is occupying the current location and take c = 0 otherwise. Intuitively, we use the highest value for the density, since the grid point is occupied by the cell. In this way we have a binary description of the tumour cells.For solving the ECM equations , 2c) we we2c) weαf and βf used in the newly proposed model, while the remaining parameters are taken from literature .The MDEs are diffusing, so that the concentration at positions where tumour cells are located is not so high as for the tumour cells, where we have the highest value value in  which isαf is smaller than βf will be 0.015. On that account and because 0 would mean no remodelling effects, the parameter is varying in the range  for not breaking the stability requirement 2a) descrtrast to  the necrtrast to . Having  one day , in our  one day .The grid location onto which the daughter cells are placed depends upon the cells occupying the neighbourhood of the mother cell. One daughter cell will always replace the mother cell, which is common to do so , 33. InsIn case there is no free space in the neighbourhood, the tumour cell becomes quiescent until free space is available or the cell becomes necrotic due to insufficient nutrient , 34, 35.Invasive glioma cells are resistant against apoptosis , 37 in f The choice of the local neighbourhood of an individual cell is crucial for the update of its state for lattice-based models –41. To aAnother problem can arise from the strategy for updating the state of a tumour cell inside the lattice. If we would run sequentially to look at every tumour cell one by one, the first cell has often more possibilities for migration (in case of division: to place the daughter cells) than the one being located very next to it. Consequently, cells are not updated in a left-to-right or top-to-bottom manner but randomly .u = 0.5. With hypoxia the tumour growth will transfer into the vascular stage through the process of angiogenesis [The distribution of the extracellular matrix, the progression of nutrients, and the tumour cell arrangement over time are shown in Figures ogenesis .In a first step, a homogeneous ECM see also  and two Computer simulations using two parallel blood vessels and a constantly initialized extracellular matrix is shown in The simulated tumour depicts a distribution that can typically be observed in vivo A large necrotic core surrounded by a rim of quiescent cells and an outer rim of strongly diffusive glioma cells. As a further step and to show the importance of the novel method, we compare results of the proposed approach with results of common ones. To this end, computer simulations using system , 1b), , ) and 2, 2b), , , 2d)) h) h2d)) h, , For the tumour cell distribution a quite similar size of the bulk can be observed, however, there are differences concerning the invasive properties of the cells. For the tumour progression modelled with the proposed approach , , ) the invAt last, we compare the tumour distribution assuming the typical Dirichlet boundary conditions for the nutrients, that is, we assume blood vessels located at all boundaries of the domain. Furthermore, a randomly initialised ECM Figures  is assumHowever, we have simulated the lastly described model cf.  one hund A hybrid approach has been developed that uses a coupled continuum-discrete model to simulate tumour growth. This method is devoted to the modelling of cellular processes of tumour cells, which is itself part of a complex system. The availability of efficient approaches therefore is an essential prerequisite for modelling tumour growth. To this end, a novel lattice-based approach has been developed that does not only provide a significant simplification compared to previous models, but also is computationally efficient  and, morThe degradation of the ECM in the area covered by the tumour can clearly be seen in all simulation results. This behaviour confirms our hypothesis of the direct interaction between tumour cells and the extracellular matrix. As an important feature the novel method provides the best results using a constant matrix, where the invasive behaviour of tumour cells can clearly be observed, in accordance to in vivo and in vitro behaviours , 43.Furthermore, the proposed model requires about 2.5 less computation time compared to the common model in case the ECM is heterogeneous . This reComparing the results for modelling haptotactic-chemotactic cancer growth using the common choice of the environment and the novel introduced approach, a rather similar size of the tumour can be observed, in case a heterogeneous ECM is used. Faced with the requirement of modelling much more complex processes than the degradation of the ECM through the MDE (in order to adequately model tumour growth) the proposed model provides a reasonable tradeoff between complexity and accuracy.Prospectively, it will also be essential to extend the devised model by cell-cell interactions such as pressure on neighbouring cells caused by mitosis or cell migration. We expect, that this pressure will make the thickness of the proliferating rim more distinct . For a m


Ever since the discovery of precisely timed events of cortical neurons , their rIn this work we theoretically investigate to what extent common synaptic afferents and synchronized inputs each contribute to closely time-locked spiking activity of pairs of neurons . We emplWe find that at fixed input covariance, already weakly synchronous inputs boost the synchrony in the outgoing spiking activity compared to shared input alone Fig. , sharpenhttp://www.nest-initiative.org).Partially supported by BMBF Grant 01IB001A (brain@work), the Helmholtz Alliance on Systems Biology, the Next-Generation Supercomputer Project of MEXT, EU Grant 15879 (FACETS), EU Grant 269921 . All network simulations carried out with NEST (


We hypothesized that DPOFA may increase fluid absorption across basolateral surface of the RPE.Retinal detachment is a disorder of the eye in which sensory retina separates from the retinal pigment epithelium (RPE) due to accumulation of fluid in subretinal space. Pharmacological stimulation of fluid reabsorption from subretinal space to choroid across the RPE has been suggested as a treatment strategy for retinal detachment. DPOFA, (R)-(+)-oxy]acetic acid, is an abandoned drug capable of inhibiting Cl-/HCO3- exchangers was conducted in bovine and human RPE to confirm that RPE from two species expresses the same repertoire of Cl-/HCO3- exchanger isoforms. The degree of amino acid homology between orthologous human and bovine RPE-specific isoforms was calculated after performing protein alignments. Transport of fluid across bovine RPE-choroid explants mounted in the Ussing chamber was used to assess the ability of DPOFA to modulate fluid absorption across the RPE.Reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA for six different transporters that may act as ClUsing RT-PCR we showed that three isoforms  are strongly expressed in human and bovine RPE preparations. Amino acid comparisons conducted for RPE-specific isoforms support the use of bovine RPE-choroid explants as an adequate experimental system for assessing fluid absorption activity for DPOFA. Our data is consistent with the fact that DPOFA stimulates fluid absorption across the RPE in bovine RPE-choroid explants.DPOFA seems to stimulate transport of water across the RPE in bovine RPE-choroid explants. Additional experiments are required to establish dose-dependent effect of DPOFA on fluid absorption in the bovine RPE-choroid experimental system. Control RT-PCR reactions conducted with water in place of cDNA confirmed specificity of PCR amplification . The degree of amino acid homology between orthologous human and bovine Cl-/HCO3- isoforms expressed in the RPE was calculated after performing protein alignments using ClustalW2 algorithm. The bovine-human homology for SLC4A2 and SLC4A3 isoforms is above 95% which is in the range of amino acid similarity between RPE-specific human transporters and Cl-/HCO3-exchangers from three other "responsive" for DPOFA species, cat, guinea pig, and rabbit (data not shown). Lack of sequence information for the SLC26A6 transporter from cat, guinea pig, and rabbit precluded us from performing similar analysis for this isoform. Overall, amino acid comparisons conducted for SLC4A2 and SLC4A3 isoforms are consistent with the idea that Bos taurus, along with human, cat, guinea pig and rabbit may constitute a responsive for DPOFA species further supporting the use of bovine RPE-choroid explants as an adequate experimental system for assessing fluid absorption activity for DPOFA. Attempting to define a dose-dependent effect of the test compound on fluid absorption, we conducted a series of experiments comparing DPOFA concentrations in the 1-20 μM range with the effect of a compound vehicle over the period of 30 minutes. While no statistically significant difference between compound doses could be established, we were able to detect statistically significant increase in fluid absorption after drug treatment versus vehicle control during the first 20 minutes after drug or vehicle addition when data for all DPOFA concentrations were combined. When added in 1-20 μM concentrations, DPOFA significantly (p = 0.013) increased water absorption within first 20 minutes by 3.22 ± 1.4 μl/cm2.h while vehicle control decreased absorption by 2.1 ± 1.39 μl/cm2.h  are strongly expressed in both RPE preparations -(+)-oxy]acetic acid; RPE: retinal pigment epithelium; HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).Columbia Technology Ventures, the technology transfer branch of Columbia University, filed a provisional patent application covering the use of DPOFA for treatment of retinal detachment.PI conducted fluid transport experiments in bovine RPE-choroid and analyzed fluid absorption data; QQ performed RT-PCR experiments; KP selected the compound and arranged for its synthesis, participated in the analysis of the fluid absorption data, analyzed RT-PCR results, performed amino acid sequence comparisons, and drafted 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/1471-2415/11/33/prepub


Salary support for A.P.P. Zhang was provided by training grant 5T32 AI007244 from the National Institute of Allergy and Infectious Disease (NIAID).


The renal clearance of infused crystalloid fluid is very low during anaesthesia and surgery, but experiments in conscious sheep indicate that the renal fluid clearance might approach a normal rate when the adrenergic balance is modified.1-receptor blocker) or 0.01 μg/kg/min of phenylephrine  over 3 hours. The distribution and elimination of infused fluid were studied by volume kinetic analysis based on urinary excretion and blood hemoglobin level.Sixty females  undergoing laparoscopic gynecological surgery were randomized to control group and received only the conventional anesthetic drugs and 20 ml/kg of lactated Ringer's over 30 mins. The others were also given an infusion of 50 μg/kg/min of esmolol (betaP < 0.03). When surgery had started the corresponding values were 34, 65 and 61 ml (P < 0.04). At 3 hours, averages of 9%, 20%, and 25% of the infused volume had been excreted in the three groups (P < 0.01). The kinetic analyses indicated that both treatments slowed down the distribution of fluid from the plasma to the interstitial fluid space, thereby preventing hypovolemia.Both drugs significantly increased urinary excretion while heart rate and arterial pressure remained largely unaffected. The urine flows during non-surgery were 43, 147, and 176 ml in the control, esmolol, and phenylephrine groups, respectively (medians, Esmolol doubled and phenylephrine almost tripled urinary excretion during anesthesia-induced depression of renal fluid clearance. In contrast, α1-receptor stimulation by phenylephrine increased urinary excretion [During general anesthesia and surgery, renal clearance of crystalloid fluid is only 15% to 20% of that found in conscious volunteers . If not xcretion ,5.1-receptor blocker) or phenylephrine in patients undergoing laparoscopic gynecological surgery during intravenous anesthesia. Based on the previous animal studies [The aim of the present study was to examine to what degree the slow turnover of lactated Ringer's solution during anesthesia and surgery can be rectified by infusing esmolol  I patients between 21 and 49 (mean of 32) years of age were studied during elective laparoscopic removal of ovarian cysts or laparoscopic supracervical hysterectomy (uterus less than 1 kg) performed under intravenous general anesthesia. The protocol (reference number 080186), including the sampled blood volume of approximately 75 mL, was approved by the ethics committee of Zhejiang University . Each patient gave her informed consent to participate.Patients fasted overnight. No premedication was given. A radial artery catheter was inserted for sampling and for monitoring arterial blood pressure. Anesthesia was induced with midazolam 50 μg/kg, propofol 1.5 mg/kg, cis-atracurium 0.15 mg/kg, and fentanyl 3 μg/kg and this was followed by endotracheal intubation and mechanical ventilation. The anesthesia was maintained with propofol 6 mg/kg per hour and cis-atracurium 0.15 mg/kg per hour; fentanyl was infused at a rate of 2 μg/kg per hour during the first 30 minutes and then at 1.5 μg/kg per minute; these rates could be slightly adjusted depending on the depth of anesthesia.1-receptor blocker esmolol ; or 0.01 μg/kg per minute of the alpha1 adrenergic (can we use α1 here instead?) receptor agonist phenylephrine . The line was primed to provide the drug from the first minute. The anesthesia and the research were managed by two anesthesiologists blinded to the study drug.An indwelling catheter was placed into the bladder. The patients were then randomly assigned by the Excel random number generator  to receive a 3-hour continuous infusion of 10 mL/hour lactated Ringer's solution that provided no drug (controls); 50 μg/kg per minute of the βAfter 10 minutes for adjustment to the effects of the drug, plasma volume expansion was induced by infusing 20 mL/kg of lactated Ringer's solution  over the course of 30 minutes via an infusion pump. No other fluid (except drug vehicles) was given. No study data were collected after termination of the anesthesia.Monitoring included pulse oximetry, electrocardiography, heart rate, and invasive mean arterial pressure (MAP). Data were displayed on a multifunction monitor  and saved digitally. The depth of anesthesia was monitored by a bispectral index (BIS) sensor applied to the forehead. The signal was recorded on a BIS monitor Model A-2000TM . Urine output was measured every 20 minutes during the study. Arterial blood samples (2 mL each) were collected every 5 minutes during the first 60 minutes and every 10 minutes during the following 120 minutes. The blood hemoglobin (Hb) concentration was measured on a GEM Premier 3000 . Duplicate samples collected at baseline ensured a coefficient of variation of 1.5%. Blood loss was assessed from the content of blood in suction bottles and on sponges.Ro to increase the volume of central body fluid space Vc to vc. The rate of elimination was given as the product of the volume expansion of Vc and the elimination rate constant, k10 (unit: per minute). Perspiratio insensibilis was accounted for by constant ko, which was preset to 0.4 mL/minute. Distribution of fluid to the peripheral body fluid space Vt was governed by k12 and its return from vt to vc by rate constant k21. The differential equations are the following:The distribution and elimination of fluid were analyzed by a two-volume kinetic model -7 with aVc resulting from the infusion:The Hb-derived fractional plasma dilution was used to indicate the volume expansion of where Hct stands for hematocrit. Symbols in capital letters denote baseline values. A correction for the effect of blood sampling and blood loss was made on the plasma dilution .Vc, k12, and k21) were estimated by applying a non-linear least-squares regression routine (fminsearch) by using Matlab R2010a software , whereas k10 was calculated as a secondary parameter as follows:The primary parameters in the model  to detect a doubling of urinary excretion in any of the study groups as compared with the controls on the basis of the ratio between the excreted urine and infused fluid previously found during surgery ,3. An inU test for post hoc analysis. Changes were assessed by the Wilcoxon matched-pair test. Correlations between variables were studied by simple and multiple regression analysis by using square root transformation if distribution was skewed. A P value of less than 0.05 was considered statistically significant.Data are presented as the median and 25th to 75th percentile range. Comparisons between the three groups were made by the Kruskal-Wallis test, which, if significant, this is so. was followed by the Mann-Whitney P < 0.01), whereas no change versus baseline occurred in the other two groups . Phenylephrine was also associated with a slightly higher overall MAP than the other groups    Figure . Studyin) Figure .Vc, k12, and k21 - could be estimated in all experiments  than in the control group. Moreover, k21 was lower in the phenylephrine group than in the controls (P < 0.02).Figure ts Table . The elivc during the infusion was similar among the groups  than in the control and esmolol groups (P < 0.03).Simulations of the fluid distribution showed that the volume expansion of s Figure , top rowvt in the two treatment groups. At 60 minutes, 59% of the fluid resided in vt in the control group, 48% in the esmolol group, and 43% in the phenylephrine group  . However, multiple regression of the effects of MAP and the two treatments in turn showed that only the treatment drugs served as statistically significant predictors of the urinary excretion and the ratio of excreted/infused fluid.Simple regression showed that MAP explained 11% of the variability in the urinary excretion  was similar to that found for acetated Ringer's during laparoscopic cholecystectomy  and thyr% was simWhy the diuretic response to plasma volume expansion is blunted during anesthesia and surgery is poorly understood but probably can be attributed to vasodilatation, reduced arterial pressure, and activation of the renin-aldosterone hormonal axis . Beta-revc. Similarly high percentages have been reported during isoflurane anesthesia [Vc is low, and this is typical of anesthesia-induced vasodilatation [Vc can also be explained by the relatively low body weight (50 to 55 kg) of the Chinese women studied. The study drugs might have altered Vc, although the average volume in the esmolol and phenylephrine groups differed very little from that of the controls.The physiological effects of intravenous anesthesia also promote a pronounced plasma volume expansion in response to crystalloid fluid. When the infusions ended, 60% to 70% of the infused fluid remained in the functional plasma volume, esthesia  and duriesthesia ,3,10. Prvc to vt and this probably reflects minimally decreased filtration due to vasoconstriction. This counteracted any hypovolemic effect of the increased diuresis during the first half of the experiments. The fluid volumes residing in vc became reduced at the end of the study, but, at that time, differences were hardly worth noting. The fluid volumes in vt did not differ significantly at 180 minutes (Figure A key finding of the kinetic analysis is that both study drugs slowed down the distribution of fluid from s Figure .1-receptor blockade in conscious humans lowers the resting plasma levels of renin, angiotensin, and aldosterone but without affecting the diuretic response to volume loading with saline [Variable results for the diuretic effect of esmolol in previous work suggest that the present study may be relevant only to anesthetized subjects, possibly because the renin-aldosterone axis is then stimulated. Complete βh saline . In awakh saline . Moreoveh saline  and thisPhenylephrine increases the urinary excretion by pressure diuresis if the increase in MAP is profound, but also by increasing the release of atrial natriuretic peptides . The smaAnother limitation was that the operations were performed on ASA I patients only. Therefore, the effects of the study drugs on patients with cardiovascular disease are unclear. Operations were also performed in a teaching hospital and this explains why more than 1 hour had passed before the surgery started. The restoration of baseline MAP between 70 and 90 minutes in all groups can be understood on the basis of the late start of the surgery Figure , middle.The main conclusion still appears to be valid when the urinary excretion is calculated for periods of apparently different MAPs and for periods of surgery and non-surgery Tables  and 4. TVt is not estimated, although it can be inferred in retrospect. The output is given in the form of Vc and three rate constants. The latter the three rate constants are implied here should be multiplied by Vc to be converted into clearances, which is used in virtually all previous publications of volume kinetics. Yes, it should be "are". I originally meant clearance as a concept, but I guess we should aim at them being three different types of clearance.The novel kinetic analysis used here has the advantage of being able to estimate all parameters without having to discriminate between two models . Drobin'Low doses of esmolol doubled and phenylephrine almost tripled urinary excretion. These drugs may potentially be used as diuretics when renal fluid clearance is reduced by anesthesia and surgery-associated stress. Further work is needed to elucidate how the diuretic effects of these drugs are best employed. Phenylephrine might be an alternative to a conventional diuretic such as furosemide if the hemodynamics is unstable. Esmolol could be used as a mild diuretic in patients with supraventricular arrhythmias.• The clearance of crystalloid fluid is very low during intravenous anesthesia.• Small doses of esmolol and phenylephrine increase the clearance but not up to the normal values for conscious volunteers.• These drugs at least doubled the urinary excretion in comparison with controls.• The increased diuresis did not cause hypovolemia as both esmolol and phenylephrine slowed down the rate of distribution of lactated Ringer's from plasma to interstitium.ko: fluid loss by evaporation through skin and airways; k10: rate constant for fluid leaving the system; k12: rate constant for fluid passing from vc to vt; k21: rate constant for fluid passing from vt to vc; Ro: rate of infusion; vc: size of central body fluid space during fluid therapy; Vc: size of central body fluid space at baseline; vt: size of peripheral body fluid space during fluid therapy; Vt: size of peripheral body fluid space at baseline.Hb: hemoglobin; MAP: mean arterial pressure; The authors declare that they have no competing interests.YHL wrote the ethics application, organized the study, and collected data. HBZ, XZ, HJC, and LS collected data and performed the operations. RGH designed the trial, performed the calculations, and wrote the manuscript. All authors read and approved the final manuscript.


Single fiber electromyography (SFEMG) was established by Stalberg and Eskedt in the 1960s, and is of proven value in the diagnosis of neuromuscular disorders, especially myasthenia gravis.3Single fiber electromyography typically requires the use of a specially contracted single fiber EMG needle electrode or faciaThe validity of the technique has been proven by examining a large number of myasthenia patients with a sensitivity of up to 99% in detecting a neuromuscular transmission defect. in generalized myasthenia gravis has been reported.4 When a Most of the modern nerve conduction and EMG machines have a software to perform and analyze the SFEMG examination. There are two methods to perform this. One is stimulated and the other is under a volitional effort. However, most physicians, including the author, prefer to perform SFEMG under the volitional effort. The goal of SFEMG is to study the adjacent action potentials from the same motor unit, known as ‘pairs’. This is achieved by using a specially constructed single fiber needle electrode or facial concentric needle electrode. This identifies and selectively records the action potentials from individual muscle fibers. The selectivity of the recording is further strengthened by adjusting the filter settings. (Low frequencies filter 500 HZ – high frequencies filter 10 KHZ). This filter setting selectively abolishes low frequency components from distant muscle fibers. Action potentials should be greater than 200 mictovolts in amplitude and the rise time should be less than 300 microseconds. Around 20 potential pairs are collected from the same muscle by three-to-four insertions. The subject is asked to maintain a steady contraction if volitional SFEMG is undertaken, until 100 consecutive discharges are recorded from each pair.Stimulation SFEMG is particularly useful in children, uncooperative, comatose patients, and those who have tremors. A branch of the motor nerve is stimulated by using a mono polar needle electrode and recording is made by SFEMG or a concentric needle electrode. Stimulation is delivered at 2 - 10 HZ and the stimulus intensity is adjusted accordingly.Jitter is the measurement of variation of the inter-potential interval. This is calculated between the triggered potential and the time-locked, second single muscle fiber action potential Figures –7. This When neuromuscular transmission is sufficiently impaired, nerve impulses fail to elicit an action potential and this is called ‘blocking’ . This usThe normal jitter values have been determined for many muscles in a multicenter collaborative study7Figure .The study is considered abnormal if one of the following criteria is met,Mean jitter value exceeds the upper limit of the normal valueMore than 10% of the pairs have increased jitter (two out of twenty pairs)Normal values apply only if the inter-spike interval is up to four microseconds. Errors may be encountered if this is higher and may produce a false jitter.In patients with myasthenia gravis, jitters are greater in the weak muscles, but they are also increased in muscles with normal strength. Jitter is abnormal in the orbicular oculi > 95%, followed by frontalis and extensor digitorum communis in 85% of the patients. Jitter is abnormal even when patients take anticholinesterase inhibitors.. The SFEMG was highly sensitive (99%), followed by the acetyl choline receptor antibody, and the least sensitive was repetitive stimulation (76%), if the proximal muscles were tested.0. The SF12 The reSingle fiber electromyography is highly sensitive, but not specific to the diagnosis of myasthenia and myasthenic syndromes. It must be emphasised that increased jitter values are not pathogonomic for myasthenia, but indicate disturbed neuromuscular transmission. Increased jitter values are seen during the early stages of reinnervation, when motor unit remodeling occurs. Such changes can be seen in motor neurone disease, polyneuropathies, polymyositis, and Facioscapulohumeral dystrophy. However, it is also true that if SFEMG is normal in a weak muscle, it almost completely excludes the diagnosis of myasthenia.Single fiber electromyography is the most sensitive test to demonstrate an impaired neuromuscular transmission like myasthenia gravis. However,


The authors would like to add the following grant to their funding information: "Faculty of Engineering research grant (2010), Burapha University Thailand (Grant #33/2553)."


Varicella-zoster virus (VZV) vaccines induce immunity against childhood chickenpox and against shingles in older adults. The safety, efficacy, and widespread use of VZV vaccines suggest that they may also be effective as recombinant vaccines against other infectious diseases that affect the young and the elderly. The generation of recombinant VZV vaccines and their evaluation in animal models are reviewed. The potential advantages and limitations of recombinant VZV vaccines are addressed. Varicella-zoster virus (VZV) vaccines provide immune protection against diseases that affect both the young and the elderly. The live, attenuated varicella vaccine (VARIVAX) immunizes children against chickenpox, a childhood disease characterized by fever and vesicular skin rash. The VZV Zostavax vaccine protects older adults against herpes zoster (shingles), a vesicular skin disease caused by VZV reactivation from latently infected neural ganglia.  The proven safety and effectiveness of the varicella and shingles vaccines provide support for recombinant VZV (rVZV) vaccines to induce immunity against not only VZV but also against other pathogens. The ability of VZV vaccines to safely induce long-lasting humoral and cellular immune responses provides advantages over other live, attenuated vaccine vectors and over killed and subunit vaccines. This review summarizes research to develop and evaluate VZV vectors as recombinant vaccines against other diseases.  Varicella is a highly contagious disease of children and adolescents . The inf Following resolution of the primary disease, VZV establishes life-long latent infection within neural ganglia . Later i Acyclovir and related nucleoside analogs may be effective against chickenpox and shingles, particularly if administered early in the course of infection. However, prevention against VZV disease outbreaks has been facilitated by development of VZV vaccines. The VZV VARIVAX vaccine induces immunity and protection against chickenpox in children . The vac The shingles (Zostavax) vaccine is also composed of live, attenuated VZV Oka but is administered at a >10-fold higher viral dose than the varicella vaccine . The vacThe size of the VZV genome (125 kb) allows stable insertion of multiple heterologous genes into specific loci without effecting viral replication .VZV replicates in the cell nucleus, so foreign genes may be properly spliced, and viral antigens are expressed, processed, and presented in infected cells as they are in natural infection .The replication competent virus amplifies foreign antigen expression—a potential advantage over replication defective vectors. A protective Th1 type T-cell immune response against VZV and the foreign antigen is induced .VZV DNA does not contain genetic elements associated with oncogenesis.The VZV host range is limited to man, limiting uncontrolled environmental spread.Periodic subclinical reactivation of VZV from latency may provide restimulation of immune responses to VZV and foreign antigens as discussed below . A recombinant varicella or shingles vaccine could be ideally suited for prevention of other diseases that affect children or the elderly, respectively. An rVZV vaccine offers greater safety as a general population vaccine compared to other live viral vectors, such as vaccinia virus, and the varicella vaccine is the only live viral vaccine approved for certain groups of cancer and otherwise immunosuppressed patients, including children with HIV infection .–20.E. coin vitro replication and are targets for insertion of foreign genes. For example, several rVZVs have incorporated the foreign gene within the viral thymidine kinase (TK) gene [− viruses replicate as efficiently as wild-type VZV in cell culture. Generation of rVZV-TK− is facilitated by selection with nucleoside analogs such as bromodeoxyuridine. However, rVZV vaccines generated by insertion of foreign genes within the viral TK gene may not be clinically useful as effective antiviral treatment for VZV infections with acyclovir and other nucleoside analogs depends on a functional TK. In other cases, the exogenous genes are inserted within the intergenic regions of VZV ORFs 12 and 13 or between ORFs 65 and 66. The large size (125 kb) of VZV DNA facilitates insertion of one or multiple foreign genes within the VZV genome. VZV encodes several genes which are nonessential for TK) gene , 21, 22.RecA+ recombination and Red recombination may be used to insert genes of other pathogens into specific sites within VZV cosmids or BACs taking advantage of homologous flanking VZV sequences [ A variety of recombination methods including equences , 23. Tn7equences .  Foreign genes within rVZV have been expressed from an endogenous VZV promoter, such as the TK or gE promoters , 21. In  Several studies provide support for the VZV vaccine virus as a vector for the expression of foreign genes. Initially, an rVZV expressing the Epstein-Barr virus (EBV) membrane glycoprotein (gp350/220) was generated and plaque purified . The VZV An rVZV Oka expressing the hepatitis B surface antigen (HBs) was generated by inserting the HBs gene into the viral TK ORF , 25. The The herpes simplex type 2 (HSV-2) glycoprotein D (gD) was inserted into the unique short region of the VZV Oka genome within the intergenic regions between ORFs 65 and 66 using the VZV cosmid genetic system . HSV-2 g An rVZV vaccine expressing the HIV-1 env gene was constructed by insertion of the HIV-1 env gene encoding amino acids 296–463 fused to HBs antigen within the VZV TK gene . The rVZ The mumps virus (MV) hemagglutinin-neuraminidase (HN) gene was inserted within the VZV Oka ORF 13 using the VZV-BAC system . The HN  The ability of an rVZV vaccine to induce immunity against simian immunodeficiency virus (SIV) was investigated in a nonhuman primate model . The SIVLuc-infected melanoma cells by confocal microscopy and the virus replicated in vitro as efficiently as wild-type VZV. The rVZV-63/70-Luc retained pathogenicity in the SCIDhu mouse model of VZV pathogenesis. In vivo IE63 expression over the course of at least 21 days was measured in rVZV-63/70-Luc-infected human skin and dorsal root ganglia xenografts by whole animal imaging.  rVZV expressing foreign genes can also be employed to investigate the molecular basis of viral pathogenesis. The VZV cosmid system was used to generate rVZV Oka expressing a fusion protein consisting of the immediate early gene ORF 63 (IE63) and its duplicate gene ORF 70 fused in-frame to click beetle luciferase genes . IE63 ex Studies on VZV pathogenesis, antiviral therapy, and vaccines are hampered because VZV inoculation of laboratory animals, ranging from mice to monkeys, fails to induce a varicella-like disease. However, a natural disease closely resembling human varicella occurs in nonhuman primates with symptoms characterized by fever and vesicular skin rash . Simian  The causative agent, simian varicella virus (SVV), is a primate herpesvirus with properties resembling human VZV. The SVV and VZV genomes are similar in size and structure, share 70–75% DNA homology, and are colinear in gene organization –35. SVV  Based upon the genetic and antigenic relatedness of SVV and VZV and the clinical similarities between simian and human varicella, SVV infection of primates is a useful animal model for studying viral pathogenesis and for evaluating the effectiveness of antiviral agents and vaccines , 40, 41. The SVV model offers an approach to evaluate recombinant varicella vaccines. The initial recombinant SVV (rSVV) was generated by insertion of the green fluorescent protein (GFP) gene expressed from the Rous sarcoma virus (RSV) promoter into the intergenic region between SVV ORFs 65 and 66 by homologous recombination . Inserti An SVV cosmid genetic system was employed to generate an rSVV expressing SIV antigens. The SIV gag and env genes were inserted within the SVV glycoprotein C (ORF 14) gene, and SIV antigen expression in infected Vero cells was confirmed by immunofluorescence and immunoblot analysis using SIV monoclonal antibodies . The rSV A subsequent study evaluated the ability of the rSVV-SIVenv and rSVV-SIVgag vaccines to immunize rhesus macaque monkeys against SIV infection . The rSV An rVZV vaccine expressing respiratory syncytial virus (RSV) antigens could be effective for immunization of children or older adults who are most susceptible to RSV-induced respiratory disease. To evaluate a recombinant varicella vaccine, rSVV expressing the RSV glycoprotein G and the M2 matrix protein were constructed employing the SVV cosmid system . ImmunizE. coli [Red-mediated recombination. The SVV BAC genetic system will facilitate the generation of rSVV expressing foreign genes and evaluation of candidate recombinant vaccines. The 125 kb SVV genome has recently been cloned into a BAC and stably maintained in E. coli . The SVV A recombinant varicella vaccine may be effective for VZV seronegative children. However, older children and adults are generally VZV seropositive either by natural VZV infection or by prior VZV vaccination. Preexisting antibodies to VZV could potentially hamper the effectiveness of an rVZV vaccine, possibly by neutralization of the vaccine virus. Indeed, humoral and cellular immune responses to a model antigen expressed by a recombinant HSV-1 were reduced in HSV-1 seropositive mice . However The VZV vaccine virus, like wild-type VZV, can establish latent infection in neural ganglia and has the potential to reactivate to cause herpes zoster , 57. How Viral vectors offer novel opportunities as vaccine platforms, particularly against diseases for which traditional vaccine approaches have not been effective. Recombinant viral vaccines against several human diseases are currently in clinical trials . Most of


The correct grant number for NCI P20 is CA103736.


FS anti-VZV) in patients with MS and VZV reactivation has hitherto not been compared.Primary infection with or reactivation of varicella zoster virus (VZV) can cause neurologic complications, which typically result in an intrathecal production of VZV-specific antibodies. Intrathecal antibodies to VZV are detectable by an elevated antibody index (AI). However, elevated VZV AIs are also found in more than half of patients with multiple sclerosis (MS), where they are thought to be part of a polyspecific intrathecal immune response. Determination of the fraction of intrathecally-produced virus-specific antibodies among all intrathecally produced antibodies may discriminate between virus-specific and polyspecific intrathecal immune responses, but the fraction of intrathecally-produced VZV-specific immunoglobulin (Ig)G of the total intrathecally produced IgG  or MS (n = 20) and in patients with VZV reactivation (7 samples from 5 patients), which all had elevated VZV AIs. The median FS anti-VZV was 35-fold higher in patients with VZV reactivation  than in patients with CIS/MS . While there was thus no overlap of FS anti-VZV values between groups, VZV AIs completely overlapped in patients with CIS/MS (1.6-14.8) and VZV reactivation (2.1-8.1).The fraction of intrathecally-produced VZV-specific IgG of the total intrathecally produced IgG discriminates between patients with VZV reactivation and MS. Our results provide further evidence that intrathecally-produced VZV antibodies are part of the polyspecific immune response in patients with MS. Thus, the median amount of intrathecally-synthesized anti-VZV antibodies was 35-fold higher in patients with VZV reactivation compared to patients with CIS/MS (p = 0.0001). There was no overlap of FS anti-VZV values between patients with CIS/MS and VZV reactivation. Conversely, VZV AI values overlapped between both groups, re-confirming that AIs do not discriminate between a virus-driven and a polyspecific intrathecal immune response. A comparison of FS anti-VZV between CIS/MS patients in relapse and remission revealed no significant difference (p = 0.34). To confirm the specificity of elevated FS anti-VZV values for VZV reactivation we additionally determined FS anti-VZV in 2 patients with HSV encephalitis with a concomitant intrathecal VZV antibody synthesis. FS anti-VZV values of these 2 patients (0.3% and 4.96%) were in the same range as those of patients with CIS/MS.The median FS anti-VZV is about 35-fold higher in patients with VZV reactivation than in patients with CIS/MS. FS anti-VZV values did not overlap between both groups, suggesting that determination of FS anti-VZV can accurately discriminate between a polyspecific immune response in CIS/MS and a virus-specific immune response in VZV reactivation. This is in accordance with previous reports demonstrating that patients with HSV encephalitis, subacute sclerosing panencephalitis (SSPE), or Epstein-Barr virus (EBV) replication in the CNS have a fraction of the intrathecally-produced virus-specific IgG that is 20- to 60-fold higher than in patients with MS [FS anti-VZV observed in this work was even higher (45.1%), indicating that VZV reactivation with CNS complications results in a highly-focussed VZV-specific intrathecal antibody response. Consistent with previous observations in 4 patients with HSV encephalitis [FS anti-VZV values determined in 2 patients with HSV encephalitis in this work were in the same range as those of CIS/MS patients, suggesting that intrathecal production of VZV antibodies represents a formerly-recognized non-specific anti-VZV antibody synthesis in patients with HSV encephalitis [FS anti-VZV elevation in patients with VZV reactivation observed in the present study might be specific for this condition. Nevertheless, further studies involving larger patient numbers will be required to establish more definitively the specificity of elevated FS anti-VZV values for VZV reactivation and to determine a cut-off above which elevated FS anti-VZV values can be considered specific for VZV reactivation.This study shows that  with MS . In HSV  with MS . The medFS anti-VZV values in patients with CIS/MS were in the same range as previously reported FS values for VZV, HSV, measles virus, rubella virus, and EBV in patients with CIS/MS [FS values for VZV, HSV, measles virus, rubella virus, and EBV in patients with CIS/MS as well as the marked difference in FS anti-VZV between patients with CIS/MS and VZV reactivation, strongly suggest that the intrathecal production of VZV antibodies is part of the polyspecific intrathecal humoral immune response in MS. Our finding that FS anti-VZV values did not differ between CIS/MS patients in relapse and remission does not support the idea that FS anti-VZV could be influenced by clinical disease activity.The h CIS/MS ,7. The sA previous study reported the presence of VZV DNA in CSF of patients with MS during relapses, leading to the proposal of a participation of VZV in the pathogenesis of MS ,11. HoweAn intrathecal IgG synthesis is found in ~95% of patients with MS ,7, our rFS anti-VZV (QIgG > Qmean and VZV AI ≥1.5). Nevertheless, the difference between both groups was clear-cut, highly significant, and corresponded well to previous findings on the intrathecal production of antiviral antibodies in MS.One limitation of this study is its relatively small patient numbers. Especially, the number of patients with VZV reactivation was limited as less than half of these patients met the requirements for determination of FS anti-VZV allows discrimination between patients with VZV reactivation and CIS/MS. It also provides further evidence that intrathecally produced VZV antibodies are part of the polyspecific immune response in MS. Determination of FS can be applied to any intrathecally produced microorganism- or antigen-specific antibody and may therefore prove a useful tool to differentiate between microorganism/antigen-triggered and polyspecific intrathecal humoral immune responses in different contexts and diseases.Altogether, our study demonstrates that determination of CO, JH, CF, and MZ report no disclosures. KR has received speaker’s honoraria and travel grants from Merck Serono, Teva, Biogen Idec, Novartis Pharma GmbH, and Bayer Schering as well as scientific grant support from Novartis Pharma GmbH. KR is supported by the German ministry of education and research .CO participated in the design of the study, carried out the experiments, and collected and analyzed data, JH participated in the design of the study and analyzed data, CF and MZ collected and analyzed data, KR conceived of the study, participated in the design of the study, analyzed data, and wrote the manuscript. All authors read and approved the manuscript.


A second grant from the National Institutes of Health to the twelfth author was incorrectly omitted from the funding statement. The number of this grant is: R21 AI073230-01.


Ongoing quality control of the gluconate assay has uncovered problems with assay performance and data processing. Significant, and unpredictable, matrix-interference discovered in an independent re-validation have led the authors to conclude that gluconate is not a reliable analyte for measuring cervicovaginal fluid dilution. As a result, gluconate adjusted cervicovaginal fluid volumes reported in the manuscript are incorrect and are not correctible. Other findings and conclusions of the study remain unaffected.