Safety of evacuation is of paramount importance in disaster planning for elderly people; however, little effort has been made to investigate evacuation-related mortality risks. After the Fukushima Daiichi Nuclear Plant accident we conducted a retrospective cohort survival survey of elderly evacuees.A total of 715 residents admitted to five nursing homes in Minamisoma city, Fukushima Prefecture in the five years before 11th March 2011 joined this retrospective cohort study. Demographic and clinical characteristics were drawn from facility medical records. Evacuation histories were tracked until the end of 2011. The evacuation's impact on mortality was assessed using mortality incidence density and hazard ratios in Cox proportional hazards regression.Overall relative mortality risk before and after the earthquake was 2.68 (95% CI: 2.04–3.49). There was a substantial variation in mortality risks across the facilities ranging from 0.77 (95% CI: 0.34–1.76) to 2.88 (95% CI: 1.74–4.76). No meaningful influence of evacuation distance on mortality was observed although the first evacuation from the original facility caused significantly higher mortality than subsequent evacuations, with a hazard ratio of 1.94 (95% CI: 1.07–3.49).High mortality, due to initial evacuation, suggests that evacuation of the elderly was not the best life-saving strategy for the Fukushima nuclear disaster. Careful consideration of the relative risks of radiation exposure and the risks and benefits of evacuation is essential. Facility-specific disaster response strategies, including in-site relief and care, may have a strong influence on survival. Where evacuation is necessary, careful planning and coordination with other nursing homes, evacuation sites and government disaster agencies is essential to reduce the risk of mortality. Questions about the safety of evacuation of elderly residents are of paramount importance to residentsResearch on hurricane preparedness in the USA has suggested that evacuation can be associated with an approximately two-fold mortality riskGiven the importance of disaster- and facility-specific factors in determining the success of disaster plans and the possibility of significant increases in mortality due to evacuation, it is important to conduct detailed epidemiological assessments of the efficacy and safety of evacuation procedures in the aftermath of Japan's triple disaster. Almost a year since the nuclear accident, at the request of the local hospital, we conducted a retrospective cohort survival survey of nursing care home residents in Minamisoma city. Minamisoma city was the only town in Japan that was seriously affected by all three elements of the triple disaster, experiencing infrastructure destruction, significant radiation exposure, and a series of evacuation orders simultaneously. The experience of care home operators in Minamisoma thus paralleled many of the challenges faced in the aftermath of Hurricane Katrina. This is the first detailed assessment of mortality risk associated with evacuation of elderly residents after the Fukushima Dai-Ichi nuclear accident, and offers the first opportunity to explore evacuation-related mortality in detail, as well as a chance to generalize from the specific experience of care home operators in Minamisoma to some of the complex policy issues associated with multiple-cause disasters.Ethical approval for the study was granted by the ethics committee of the Institute of Medical Science, the University of Tokyo, authorization number 23-61-3038. For monitoring residents' survival, an information sheet on the research objectives and confidentiality of study participation were sent to the care homes' presidents and verbal consent was obtained. The ethics committees agreed that written consent was not required for each care home resident.th March 2006 and 11th March 2011 were included in this study. Information on demographic and clinical characteristics and entry records was obtained from medical records at the facilities, which were recorded by the care practitioners for all residents at entry to the facility. Evacuation history was also recorded by the facilities at the end of 2011 or the beginning of 2012, and this data was collected along with date of withdrawal.Five of the eight care homes in Minamisoma participated in this study, representing 62% of all individuals resident in a care home at the time of the earthquake; of those that did not participate, one was unable to due to the loss of all records during the tsunami, and one could not provide sufficient quality evacuation or mortality records. Four facilities are intensive care homes for the elderly, which admit those who have difficulty rehabilitating at home. One facility is a rehabilitation facility for the elderly, which make efforts to enable residents to rehabilitate at home. All elderly residents who had been admitted to the five facilities between 11Demographic and clinical characteristics included age at withdrawal or death, sex, and care level, based on the Japanese Category of Condition of Need for Long-Term Care, a number between one and five measuring severity of care needs.To assess the impact of the earthquake on mortality, death incidence density before and after the earthquake was calculated as the number of deaths divided by sum of person-years at risk, which were measured from the date of admission until the end of the study period, death or withdrawal. Person-years of risk were divided into pre-and post-earthquake periods to compare relative mortality and crude relative mortality risk calculated as the ratio of post- and pre-earthquake mortality incidence densities. A seasonal or cohort effect in the data was investigated through visual inspection of a quarterly time-series trend of incidence density. Because Facility 4 lacked data for those who left the nursing home before the earthquake, the total person-years and incidence density before the earthquake was estimated based on the average proportion of person-years for the residents who left the nursing homes before the earthquake in other facilities. Thus the relative incidence density before the earthquake for this facility was an estimate.Survival probability was assessed using the Kaplan-Meier product limit method, comparison of which was on the basis of the Wilcoxon test and Log-rank test, and plotted with survival curves. Effects of the earthquake itself and mortality risk associated with the evacuation procedures implemented by each nursing home were examined using cox proportional hazards multiple regression. Comparison of survival before and after the earthquake was initially conducted without evacuation history data, to measure the effects of the earthquake on mortality. Evacuation history was explored using only post-earthquake data to estimate risks associated with different evacuation patterns. In both analyses, variables were selected using backward-stepwise model-building. Both analyses included a fixed effect to model unobserved, facility-specific confounders. In the analysis comparing pre-and post-earthquake mortality, a facility-earthquake interaction term was included to test for the possibility of facility-specific moderators of evacuation- or earthquake-related mortality. All analyses were conducted using Stata/MP 11.The report is presented in accordance with STROBE guidelines.th March 2006 to 11th March 2011 records were collected for all 596 elderly residents from four of five facilities. Data on residents who had left the facility before the earthquake were missing in one facility. Characteristics of the 715 residents included in this study are shown in From 11The time-series of quarterly death incidence density for the whole study period is shown in Details of the facility-specific relative mortality risk based on the incidence death density are shown in Findings from the multiple regression analysis without any evacuation history data indicated that mortality after the earthquake increased by a factor of three in Facility 1 . The intThis study — the first assessment on the health impact of the evacuation after the Fukushima Dai-ichi nuclear accident — showed that under very different disaster conditions, elderly homes in Minamisoma experienced higher increases in mortality risk than US nursing homes that evacuated in the wake of Hurricane Katrinafuton) and medical supplies ceased for three days. This, rather than evacuation distance itself, might explain the high mortality after the initial evacuation; however, it is difficult to measure the quality and continuity of care quantitatively in evacuation sites because no reliable records exist from that period.There was also a substantial difference in mortality risks across facilities. These differences may be affected by factors such as residents' psychological state or health condition at the time of evacuation, facility-specific evacuation patterns, and the conditions in evacuation sites to which elderly evacuees were admittedBefore the earthquake, the Ministry of Internal Affairs and Communications conducted a national survey to investigate prefectural support for disaster management plans for elderly people. This survey assessed whether facilities had an evacuation plan in accordance with the Evacuation Guidelines for Disaster ManagementThis study had several limitations. There was potential underestimation of relative mortality risk after the earthquake in the Cox proportional hazards analyses, because Facility 4 lacked data on residents who left the facility before the earthquake, which would result in overestimates of incidence density before the earthquake and subsequent underestimation of the relative mortality risk after the earthquake. In addition to this, because there was only one evacuation in Facility 5 it is difficult to compare this facility with the remaining four due to lack of reference to the initial evacuation. However, a sensitivity analysis excluding Facility 5 indicated little influence of this limitation on the regression analysis results. Therefore, the finding that initial evacuation is the most dangerous appears to be robust. Another limitation is that only five of eight nursing homes were involved in this study: one facility lost the residents' records during the tsunami, one had insufficient data for inclusion in the study, and one refused to participate. Because the facilities recorded health information intermittently and/or outsourced health care to external providers, it was not possible to obtain a comprehensive picture of the residents' level of physical health. Unfortunately, the chaotic situation in the prefecture at the time and the rapid reduction in service providers within Minamisoma made obtaining health records from diverse providers within Minamisoma impossibleThe necessity of evacuation of vulnerable residents in a post-disaster setting is a controversial issueIn a post-disaster situation where infrastructure collapse affects the essential conditions for maintaining elderly peoples' healthThis study shows that even under the extreme circumstances experienced in the aftermath of the Great East Japan Earthquake and subsequent radiation accident, some facilities were able to ensure that their residents suffered no significant increase in mortality risk. Balancing the competing risks of radiation exposure and evacuation mortality is of paramount importance when infrastructure collapse and damage do not themselves warrant evacuation. Health planners, disaster coordinators and facility managers in areas that may be subject to similar disasters should consider the lessons of Minamisoma, Fukushima when developing their own plans for disaster response.


Variation in the mechanisms that mediate antigen processing, MHC-loading, and presentation of peptides allows cells to significantly modulate the repertoire of peptides presented by both MHC class I or class II. To more quickly determine how these different modes or modulations of presentation translate into altered immune responses, higher throughput methods for identifying T cell epitopes are needed. Proteomics-based comprehensive cataloging of peptides eluted from MHC is a challenging but ideal way of identifying peptide sequences influenced by variable modes of processing and presentation. Several groups have already been successful with this approach and ongoing technical improvements will broaden its applicability. Subsequently, high content combinatorial peptide-MHC tetramer staining using mass cytometry, as we have recently described, should enable the broad assessment of how these changes are perceived by T cells and translated into an altered immune response. The importance of this analysis is highlighted by evidence that physiologically relevant variation in antigen processing and presentation as well as other factors can give rise to unpredictably different T cell responses. Antigen processing and presentation is centrally important for T cell mediated adaptive immunity. Accordingly, the modes and mechanisms of peptide processing and presentation by MHC class I and class II molecules have been extensively studied and nicely described . ThroughFor a number of pathways alluded to above, including the example highlighting the importance of the thymoproteasome, several studies have demonstrated the consequences of distinct antigen processing and presentation pathways on the T cell response. Another dramatic example of alternate processing and its effects on the T cell mediated immune response was originally described by Unanue and colleagues for alternate processing pathways of the hen egg lysozyme (HEL) model antigen. In this system, some T cells react only with APCs loaded with free exogenous peptide (Type B antigens), while others, Type A antigen-specific T cells, can also react with antigen derived from endogenous processing of intact protein [reviewed in Ref. ]. This tOne of the most profound conundrums of T cell immunology is the unpredictability of epitope use and what factors govern T cell epitope dominance . AlthougIn the past the only available approaches for tracking the antigen-specific T cell response were indirect and relied on response to antigen stimulation. More recently, the use of multimeric peptide-MHC complexes allow for direct detection of antigen-specific cells and accurate quantification regardless of T cell functional capacity . DespiteAside from previously characterized epitopes and predicted MHC-binding peptides, this highly multiplexed approach for identifying and characterizing antigen-specific T cells should also be useful for the screening of both MHC class I and class II ligands identified by proteomics for the purposes of investigating the consequences of variable antigen processing and presentation pathways discussed here. With room for improvement through dedication of additional parameters to multiplexed peptide-MHC tetramer analysis, already the number of candidate epitopes that can be screened is similar in scale to the number of peptides usually identified by mass spectrometry of MHC eluted peptides. Through improved sensitivity of proteomics-based methods of peptide identification and extension of the multiplexing capacity of the T cell analysis approach, it is hopeful that the goal of a truly comprehensive means of assessing epitope usage by antigen-specific T cells will be possible.In line with the theme of this Frontiers in Immunology topic, the relationship between the complexities of antigen processing and presentation and the consequential antigen-specific T cell response were discussed. A great deal of successful research has led to understanding of the mechanisms, pathways, and molecular players of antigen processing and presentation, holding promise for powerful new ways of modulating the T cell mediated immune response. Nonetheless, due to the diversity of pathways and complexity of modulators involved, it is not yet simple to predict how perturbations will influence the repertoire of presented antigens or the resulting T cell response. Thus, the use of proteomics-based identification of peptide-MHC ligands as well as multiplexed methods for analyzing antigen-specific T cell responses should be useful for future studies on this topic.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There is a growing realization that alterations in host-pathogen interactions (HPI) can generate disease phenotypes without pathogen invasion. The gut represents a prime region where such HPI can arise and manifest. Under normal conditions intestinal microbial communities maintain a stable, mutually beneficial ecosystem. However, host stress can lead to changes in environmental conditions that shift the nature of the host-microbe dialogue, resulting in escalation of virulence expression, immune activation and ultimately systemic disease. Effective modulation of these dynamics requires the ability to characterize the complexity of the HPI, and dynamic computational modeling can aid in this task. Agent-based modeling is a computational method that is suited to representing spatially diverse, dynamical systems. We propose that dynamic knowledge representation of gut HPI with agent-based modeling will aid in the investigation of the pathogenesis of gut-derived sepsis.Pseudomonas aeruginosa was developed by translating bacterial and host cell sense-and-response mechanisms into behavioral rules for computational agents and integrated into a virtual environment representing the host-microbe interface in the gut. The resulting gut milieu ABM (GMABM) was used to: 1) investigate a potential clinically relevant laboratory experimental condition not yet developed - i.e. non-lethal transient segmental intestinal ischemia, 2) examine the sufficiency of existing hypotheses to explain experimental data - i.e. lethality in a model of major surgical insult and stress, and 3) produce behavior to potentially guide future experimental design - i.e. suggested sample points for a potential laboratory model of non-lethal transient intestinal ischemia. Furthermore, hypotheses were generated to explain certain discrepancies between the behaviors of the GMABM and biological experiments, and new investigatory avenues proposed to test those hypotheses.An agent-based model (ABM) of virulence regulation in Agent-based modeling can account for the spatio-temporal dynamics of an HPI, and, even when carried out with a relatively high degree of abstraction, can be useful in the investigation of system-level consequences of putative mechanisms operating at the individual agent level. We suggest that an integrated and iterative heuristic relationship between computational modeling and more traditional laboratory and clinical investigations, with a focus on identifying useful and sufficient degrees of abstraction, will enhance the efficiency and translational productivity of biomedical research. The understanding of how microbes cause disease has evolved dramatically since the introduction of Koch's postulates and development of germ theory over a century ago. Humans represent "below the skin" ecosystems, supporting vast and diverse intestinal communities of microbial species that serve important roles in digestion, metabolism and development. There is an increasing recognition of the importance and influence of the gut microbiome in various disease states -3. The hPseudomonas aeruginosa in response to a stressed gut milieu, and the effect of thusly activated P. aeruginosa on that milieu as our model reference system. P. aeruginosa is a gram negative bacillus that is one of the most clinically significant microbes in hospital settings, with a high degree of morbidity and mortality associated with its presence [P. aeruginosa virulence expression has been identified as responding to local environmental cues, many of which are host tissue factors released in response to physiologic stress, such as tissue ischemia [P. aeruginosa virulence activation in the gut of a stressed host as an example of how HPI associated with clinical disease can be investigated through an iterative integration between traditional experimental workflow and dynamic computational modeling. There are four thematic goals in this process:Host-pathogen interactions (HPI) consist of a series of mechanistic molecular-based processes where microbial and host cells sense, respond to and influence their local environments. While mechanisms for this phenomenon have been described for many pathogens, we use the virulence activation in presence . P. aeruischemia , immune ischemia , phosphaischemia -25 and eischemia . Each ofP. aeruginosa virulence activation in the stressed gut. This is primarily reflected in ABM development and initial implementation.1. The integration and dynamic representation of mechanistic knowledge of the complex processes of 2. To use that dynamic representation as a means of knowledge visualization and conceptual model verification: i.e. can the instantiation of the mechanistic hypothesis in achieved in Goal 1 be made to behave in a plausible and recognizable fashion? This is primarily accomplished in the initial model-testing phase of development .in silico adjunct to examine experimental conditions not currently explored using traditional experimental methods. This is primarily manifest in the design and execution of simulation experiments.3. To use the resulting GMABM as an 4. Formulate new hypotheses arising from observed discrepancies between the ABM and real-world observations and suggest how new experiments might be performed to test these new hypotheses. This process takes place during the interpretation and analysis of the simulation experiments.These goals represent a sequential process that mirrors the general scientific method; we aim to demonstrate that the execution of that process within the context of developing and using a computational model can enhance the standard scientific workflow.The spatio-temporal biocomplexity of the host-microbe relationship has come into focus as a key aspect of understanding the pathogenesis of clinical infections ,28. WhilAgent-based modeling represents one technique that offers specific advantages for modeling spatially diverse, dynamic, multi-factorial systems, such as HPI in the gut ,34,35. Amodeling relation between an experimental model  and the biological referent, where the modeling relation is defined as the mapping of the generative processes and generated outputs between the model and its referent [in vitro experimental investigations to the population-level, space-incorporating, tissue and organ level context necessary to represent clinically relevant behavioral dynamics.Being able to capture this type of system-level phenomena is of critical importance in the investigation of biological systems, since there are several levels of organization between the level of mechanism targeted for putative control (often gene/molecule) and the clinical relevance/implications of that intervention (whole organism). Each of these levels of organization, extending from gene to molecule to cell to tissue to organ to organism, represents a potential epistemological boundary where inferred consequences at a higher level of organization cannot be assumed from identified mechanisms at a lower level. These boundaries challenge the fidelity of the referent ,67. Agenexperimental frame [face validity, and thus its plausibility. Face validity is defined as the ability of a particular simulation to behave in a realistic, reasonable and believable manner, and represents the first tier in a validation sequence used for engineering simulations [Related to the issue of explicit representation of the modeling relation in the study of biological systems is the question of what constitutes an appropriate level of model representation and detail? The scope and scale of a modeling project is intimately tied to and informed by its use. This is often termed establishing the al frame ,69. Giveulations -70. OfteP. aeruginosa virulence activation in the stressed gut. We aim to represent virulence-associated signal transduction and gene regulatory processes identified in P. aeruginosa with a relatively high degree of component detail, but abstracted in terms of the actual biochemical kinetics. Rate constants for classes of biochemical events are assumed to operate within qualitative orders of magnitude, and therefore, highly-abstracted representation of biochemical kinetics, as either Boolean, logic-based or algebraic statements, can be of sufficient descriptiveness to produce ABM behavior that pattern-matches those seen in the experimental data [The use of agent-based modeling for this type of knowledge representation has been previously described in the biomedical arena ,71-73, atal data . We notetal data ,67. Our The current ABM represents an initial, relatively abstract example of dynamic knowledge representation of the gut HPI, and in the future the modular nature of ABM will allow graduated addition of agents and variables , as well as more complex rules for individual agents , to produce higher resolution models of the gut HPI. However, we suggest that dynamic knowledge representation using even relatively abstract ABMs can play a useful role in the current scientific process.P. aeruginosa using Netlogo, an agent-based modeling software toolkit [P. aeruginosa were developed using a series of modular submodels, each submodel focusing on a particular set of in vitro experiments examining one particular activation pathway by host-derived stress signals: immune-activation, mediated through the molecule interferon-γ (IFN-γ) [in vivo gut environment of a stressed host. We term this integrated ABM the gut milieu agent based model (GMABM). A text file of the code for the GMABM can be seen in Additional File http://bionetgen.org/SCAI-wiki/index.php/Main_Page.We developed a series of ABMs of virulence regulation in  toolkit . The rul (IFN-γ) , ischemi (IFN-γ) , endogen (IFN-γ) , and pho (IFN-γ) -25. The  (IFN-γ) . Submodein vivo experimental models, is similar to the knowledge transfer associated with "wet lab" progression from in vitro models to more complex animal models, with the added benefit of having explicit transparency in terms of represented mechanisms. Conditions of systemic host stress were then simulated to observe interactions between Pseudomonas agents and the gut barrier manifest as alterations in population characteristics, spatial distribution of effects, and aggregate system-level variables. The results of these simulations were compared with animal models (in vivo referents) to evaluate the plausibility of interactions and to identify knowledge gaps when outcomes were divergent. It should be noted that the agent-rule structures were not changed in the process of submodel integration other than at necessary points of submodel intersection (i.e. shared components).The process of constructing the GMABM, which we treat as an analog to In an effort the move towards standardization of ABM development and analysis, Grimm, et al. have described the Overview, Design Concepts, Details (ODD) protocol to describe the construction and use of an ABM . This prP. aeruginosa. We recognize the potential limitations of this approach, but given the focus of prior investigations we believe that we can provide a novel scientific contribution through our bacteriocentric focus.Existing published ABMs of HPIs during infection have a distinctly immunocentric focus with simplification of the spatial and temporal aspects of phenotypic expression of pathogens ,79-81. AP. aeruginosa bacteria ("Pseudomonas agents") and gut epithelial cells ("GEC agents"). The spatial configuration of the ABM is a 2-dimensional square grid with the 3rd dimension represented as 4 overlying data layers: the intestinal lumen, the gut mucus layer, the gut epithelial layer and the systemic circulation , then bind to and activate Receptor Bif Receptor B is activated, then increase Signal Transduction Enzyme C by 1And so on...For a comprehensive list of entities and state variables included in the GMABM see Table in vitro or in vivo models. The scalar metric output of the GMABM for cross-model validation and simulation experiments are population metrics that represent aggregated output from the individual agents in the GMABM. These scalar metrics correspond to global levels of mediators (measured from the GMABM as a whole) and cell populations, either in total for an agent class or a specific subpopulation. This data can be seen in the outputs of the cross-model validations and simulation experiments. In addition to these scalar metrics, visual patterns of the simulation world observable through Netlogo's graphical user interface. While not quantitative information, the visualized behavior of the GMABM provides a qualitative means of evaluating the plausibility of the dynamics generated.The set of observables for the GMABM is informed and determined by the type of data generate by the biological referents, be they The GBABM uses iterated, discrete time steps, each step corresponding to 5 minutes of real time. As per Netlogo convention, each run step is divided into several sub steps.P. aeruginosa is predominantly controlled by quorum-sensing (QS) regulatory mechanism, a highly conserved "network of networks" regulating hundreds of genes in response to inter-cellular signaling molecules at high population densities [Expression of virulence genes in ensities -84. Whilensities , for theThe GMABM is a spatially explicit model, where interactions between agents and their environment are defined by the parcel of discrete space occupied by the agent ("patch" in Netlogo parlance) as well as the Moore neighborhood of that patch, where the Moore neighborhood on a 2-D square grid consists of the 8 squares immediately adjacent to and surrounding the central square. Biological cell-agent to biological cell-agent interactions are generally mediated through the passage of environmental variables produced and sensed by the various agent types; specific cell-to-cell contact interactions (other than adhesion reflected as cessation of Pseudomonas agent movement) are not included in the current development of the GMABM.The existence of stochasticity in intracellular signaling and gene regulation are well accepted  and thisThere is no dynamic initialization run-period in the GMABM; this means that simulation t = 0 is intended to represent an arbitrary time point in a system that is already at steady state. Baseline simulation conditions represent the reference system in its non-perturbed state, with "normal" levels of bacterial nutrients (including phosphate), fully intact mucus layer, baseline levels of commensal bacteria, GECs with fully intact tight junctions and no active inflammatory mediators. Pseudomonas agents are present, but in the absence of virulence activating cues (see Submodel section below) they do not have their corresponding virulence pathways active. The baseline, non-perturbed state of the GBABM was demonstrated to be stable through as series of non-perturbed simulation runs to 1000 time steps.This section will describe in detail the underlying biology and the implementation of that biology in the two mobile agent classes: Pseudomonas agents and GEC agents. Pseudomonas agent functions are subdivided into response pathways to specific conditions associated with host stress: ischemia, phosphate depletion, inflammation and opioid presence. The GEC agent functions can be classed into two groups: the first represents the representation of gut barrier function, the primary host function affected by microbial virulence, the second group consists of assignment to GEC agents three of the stress conditions discussed above: ischemia, inflammation and opioid production. In addition, while not a specific agent class, a subsection describing the handling of commensal bacteria as a population-based patch variable is described.Entity #1: Pseudomonas Agents:Each virulence activation component was developed with a submodel ABM to allow cross-model validation with their respective experimental referents. Subsequently, the rule sets of these modular submodels were integrated into a single model (the GMABM) intended to be a computational analog to animal models and other more physiologic experimental platforms. Simulated experiments were then performed on the integrated GMABM. Figures Ischemia: Adenosine-mediated virulence activation• in vitro models was in the range of 5-7 hours, and the Pseudomonas agent signal transduction pathway of inosine interaction with the lecA complex was tuned to peak production of i-PAI-lectin at 5 hours. Because expression of PA-I lectin is associated with adhesion to the epithelial cell layer, Pseudomonas agents with positive i-PAI-lectin became fixed to their current patch. Translocation of PA-I lectin to the cell wall was represented as conversion of the agent variable i-PAI-lectin to the patch variable PA-I lectin. The expression and integrity of epithelial tight junctions (occludin) was inversely proportional to patch PA-I lectin concentration, resulting in discrete regions of increased epithelial cell layer permeability around activated microbes. A schematic for this virulence pathway is seen in Figure Intestinal ischemia is a contributing factor in the pathogenesis of gut-derived sepsis ,26. InteP. aeruginosa phosphate sensing and virulence activationPhosphate depletion, • In critical illness and post-surgical stress serum and extra-cellular hypophosphatemia results from phosphatonin-mediated urinary wasting ,88 and sInflammation: interferon-γ (IFN-γ) activation of multiple virulence pathways• P. aeruginosa, cytokine-rich media from activated cultured T-cells induces PA-I lectin expression at transcriptional and translational levels [in vitro studies. The current GMABM does not include inflammatory/immune cells; therefore activation of the inflammatory response and subsequent production of IFN-γ is incorporated as a function of GEC agents controlled at the user interface as a simulation experimental condition. A schematic of this virulence pathway is seen in Figure Early recognition of host immune activation could enhance the efficacy and coordination of microbial defense and virulence strategies against host immunity. In l levels . IFN-γ pEndogenous opioids during host stress and virulence activation• P. aeruginosa through activation of key transcriptional regulator MvfR, expression of its regulated operon pqsABCDE and production of downstream signaling molecules HHQ, HQNO, and PQS [pho box complex and MvfR, which would tie together the pathways for dynorphin and phosphate sensing. This putative interaction is demonstrated in red in the overall schematic for all four virulence pathways seen in Figure Endogenous opioids are diffusely released during host stress and represent a potential early danger signal for microbes in richly innervated tissues such as the intestinal tract -92 and c and PQS . HQNO isNon-adhered Pseudomonas agents move one grid-space per simulation run step in a random fashion; there is no chemotaxis modeled. However, the presence of i-PA-I lectin, produced through pathways for ischemia and inflammation, leads to adhesion of Pseudomonas agents to underlying GEC agents and cessation of movement.Entity #2: Gut epithelial cellsP. aeruginosa virulence activation, we have abstracted and assigned these host functions to the GEC agents as an aggregated proxy for the host component of the gut milieu. The role of gut epithelial cell population behavior as a proxy of host health is represented in their permeability barrier function, reflected as tight junction integrity by the GEC agents.While the epithelial cell layer primarily governs the reactive surface of the host in the gut milieu, there are notable contributions from various epithelial subtypes  and a host of inflammatory cell subtypes. Given our focus on Epithelial permeability and tight junction metabolism• P. aeruginosa [P. aeruginosa produced toxin PQS [The tight junctions are maintained at a steady state though metabolic and localization processes, and these pathways are known to be subject to disruption by inflammatory signals ,71 and, ruginosa . Tight jruginosa . Epitheloxin PQS . GEC ageoxin PQS . WithoutUser controls on the ABM allowed the possibility of independent simulation of specific aspects of host stress as manifest by the epithelial cell agents .Intestinal Ischemia• When "Ischemia" is activated via the User Interface the GEC agents produce HIF-1α, which is added as the initiating factor to the production of adenosine by the GEC agents. The adenosine has an intracellular component, which is a state variable for the GEC agents and represents production, and a secreted version, which is released by the GEC agents and diffuses into the environment. The secreted form of adenosine is the environmental patch variable that activates the ischemia signaling rules of the Pseudomonas agents (see above). Furthermore, during the time when "Ischemia" is active the GEC agents decrement their "life" state variable such that they will die if the "Ischemia" persists for 24 hours of simulated time.Intestinal Inflammation• As noted above, the GMABM does not include inflammatory cells and the inflammatory response of the gut as an organ is abstracted by the production of IFN-γ by GEC agents. When "Inflammation" is activated (via the User Interface) the GEC agents produce IFN-γ as a diffused environmental variable that activates the inflammation signaling rules of the Pseudomonas agents.Endogenous opioid production• When "Stress" or "Ischemia" is activated (via the User Interface) the GEC agents release dynorphin, a representative κ-opioid, as a diffused environmental variable that activates the opioid signaling rules of the Pseudomonas agents.Lactobacillus species, Bacteroides species). While we recognize that a high degree of diversity exists among commensal gut flora, in the GMABM they were represented collectively as a generic microbial species lacking the genetic background or molecular machinery to express significant virulence towards the host. Because populations of commensal flora are typically several magnitudes greater than those of colonizing microbes, commensal flora was abstractly represented as an environmental spatial variable discretized on the model grid space (as opposed to agents) in order to produce a more realistic scale given computational constraints. Populations of both commensal bacteria and Pseudomonas agents were limited by a finite carrying capacity determined by the volume (thickness) of mucous layer at a specific point in the virtual environment [Lactobacillus). At the current time, the dynamics of mucous production, sloughing and turnover were not incorporated into the GMABM.At baseline, both Pseudomonas agents and commensal flora existed within the mucous layer. Commensal flora are represented as patch variables representing aggregate populations of common intestinal bacteria , the discrepancy in HQNO trajectory is accounted for by the inhibitory effect of HQNO on commensal flora growth in the presence of dynorphin based on corresponding in vitro studies [The model output behavior of the effects of dynorphin as implemented in Pseudomonas agents was fitted to the data published in Zaborina et al  see rep and 6. T studies  and seenin vivo" GMABM intended to be a computational analog to animal models and other more physiologic experimental platforms. The behavioral algorithms for sense and response virulence regulation were integrated in Pseudomonas agents maintaining the time-scaled and semi-quantitative properties of signal transduction pathways from the in vitro ABMs. The dynorphin and phosphate sensing submodels connected through the pho box-MvfR interaction; the interferon, dynorphin and adenosine signaling pathways all converge on lecA. Subsequent simulation experiments were performed using the integrated GMABM.The modular submodel rule sets were integrated into a single "in vivo experiments, an initial Pseudomonas agent population = 100 was arbitrarily chosen as the start point for the tuning of virulence gene expression. GMABM simulation runs with initial Pseudomonas agents = 100 produced severe defects in barrier function and marked reduction in commensal flora populations as early as 12 hours after insult, with very high levels of toxin production (HQNO), rapid decrease in commensal flora populations and very rapid Pseudomonas agent colony growth . In our assessment, this appeared to be too severe an effect, not reflective the state space of the clinically relevant situation, and would not allow investigation of the critical dynamics of the "tipping point" of the system. Therefore, the initial Pseudomonas agent population was reduced to produce a more qualitatively realistic progression of injury. This was accomplished with an initial Pseudomonas agent number = 10 . By producing a more realistic injury progression this initial agent level appeared appropriate for the in vivo GMABM's level of resolution, allowing clearer demonstration of the spatial and temporal heterogeneity of the gut environment. Given this tuning, the GMABM demonstrated expression of PA-I lectin and bacterial adhesion appearing at 12 hours post transient ischemic insult, followed by mild disruption of barrier function at 36 hours and then severe regional defect at 48 hours. Similarly, simulated Pseudomonas agent population growth emerged at 36 hours with contiguous colony expansion and barrier disruption at 48 hours  Investigate potential experimental conditions not yet developed.(b) Examine the sufficiency of existing hypotheses to explain the experimental data.(c) Potentially guide future experimental design.In terms of (a); we present a simulated experiment of non-lethal intestinal ischemia/reperfusion, a circumstance very clinically relevant and seen in conditions associated with the later development of gut-derived sepsis, such as hemorrhagic shock, abdominal aortic surgery and initial septic shock of non-gut origin. We use the results of these simulated experiments to fulfill goal (c) and suggest periods of interesting dynamics that might affect the timing of sample acquisition in future laboratory experiments. In terms of (b), we examine a divergence between the simulation output of the GMABM and referent experimental data, and use that insight to effect goal (c) and posit additional factors that might be investigated to afford reconciliation, some of which have been pursued.in vitro experiments to study the effects of endogenous opioids and ischemia byproducts on virulence expression in P. aeruginosa [Pseudomonas virulence expression and host survival using an animal model of non-lethal transient intestinal ischemia. However, the GMABM simulations demonstrated that exposure to select microenvironment changes in the gut subsequent to transient ischemia/reperfusion injury produced significant barrier dysfunction  with which to separate groups.In the traditional laboratory setting, effluent from the lumen of intestinal segments following ischemia reperfusion injury was used in ruginosa ,26. The n Figure , settingP. aeruginosa [P. aeruginosa. Starvation prevents the repletion of phosphate in the mucous layer in addition to the systemic effects of nutritional depletion. The requirement of two stress events to produce morbidity and mortality as a consequence of virulence expression has also been observed in a wet lab model of C. elegans, where the addition of starvation or heat stress was required to potentiate the effect of phosphate depletion [We have developed and published a reproducible model of gut-derived sepsis in mice, where mice were starved preoperatively, underwent laparotomy with 30% hepatectomy, followed by cecal injection of ruginosa . Mice exepletion . The reain vivo results suggesting immune activation alone is insufficient to generate a clinically significant injury , starvation  and the combination of the two (inflammation and phosphate depletion). Simulation of phosphate depletion alone induced a moderate to severe intestinal barrier dysfunction and reduction of commensal gut flora by 48 hours, with the first indications of patchy breakdown between 24 and 36 hours Figure . Alternay Figure . Simulaty Figure  and 19.in vivo studies in the animal model of gut sepsis largely correlate with the behavior observed in the GMABM, with one exception. In the laboratory, starvation induces depletion of phosphate in intestinal mucous, but does not alone lead to morbidity in the animal following cecal injection of P. aeruginosa [The results from ruginosa . The GMABiology is currently a science in its the discovery phase, where incomplete knowledge, ambiguity and uncertainty are inevitably present. We suggest that the detailed, selectively qualitative approach to dynamic knowledge representation lies in the fertile middle ground between high resolution, quantitative predictive models and highly abstracted models of generalize-able functions. This range as previously been well displayed in Hunt, et al . It alsoin silico experimental environments is the ability to implement experimental conditions not yet developed. This can be useful in the future planning of lines of investigation, and can potentially predict behaviors when those experimental preparations are eventually developed [in vivo model of virulence activation in non-lethal intestinal ischemia/reperfusion injury, the GMABM simulations suggest a time frame of interest for future sample collections when those laboratory models are developed. This capability may also be used to refine sampling intervals in existing wet lab models by uncovering unanticipated dynamic shifts within current sampling periods.Expanding on the role of dynamic knowledge representation, a further advantage of eveloped . An examin vivo manifestation of P. aeruginosa virulence effects. We make a series of initial hypotheses concerning the source of discrepancy between the in vivo experiments and the GMABM. These include:The behaviors observed in the GMABM during simulated major surgical stress correlated with existing animal model findings, with only one exception: the GMABM demonstrated substantial barrier defect in the context of mucosal phosphate depletion alone, while in the animal model, phosphate depletion associated with starvation alone did not produce significant morbidity or mortality, but required a second stress signal (hepatectomy). As noted above, we view this discrepancy as an opportunity to investigate additional significant factors involved in the P. aeruginosa virulence effects. The phosphate sensing pathways are explicitly modeled in the Pseudomonas agents, but without potential corresponding control pathways that may result in protective redundancies that provide a damping effect on the low-phosphate sensing triggers of virulence. In order to investigate additional control systems affecting the low phosphate response, we have already initiated a series of experiments concerning the complementary importance of iron sensing and metabolism with respect to low-phosphate induced virulence (manuscript in preparation). Also, as noted above, there is also a suggestion of a potential link between the phosphate sensing pathway and dynorphin response pathway . Negative feedback or concurrent pathway dependencies may result in synergistic damping of the virulence response if this link were established. We are also in the process of investigating this relationship with additional experiments.1. Exacerbation of the importance of phosphate sensing on P. aeruginosa population dynamics. The pathways of virulence expression related to phosphate depletion are dependent on interactions with the quorum sensing system, a highly conserved hierarchical circuitry, but as noted in the Methods section, the GMABM does not incorporate a dynamic switch that links phosphate sensing to quorum sensing. This relationship has been demonstrated [in vitro studies may be incomplete, suggesting that the presence of signaling mediators from other stress signals may be required for complete activation and expression of virulence products. This is further supported by observations in C. elegans models for assessing P. aeruginosa virulence where heat stress or starvation is required to potentiate the effects of phosphate depletion [2. Under-representation of the dynamics of quorum sensing and its influence on nstrated , but wasepletion .P. aeruginosa virulence effects. We note, however, that this discrepancy would have been present in any standard progression of experimental models! The benefit of the use of agent-based modeling as dynamic knowledge representation is that it represents a formal and explicit instantiation of the particular hypothesis, and therefore can provide more directed guidance as to that hypothesis' limitations and means to address them in the next cycle of experiments and modeling.3. Host physiology may be altered in important ways with the addition of a second stressor that are not represented in the current GMABM. These alterations may include accentuating the catabolic state and phosphate sequestration, enhancing immune activation, altering intestinal mucous production and other components of gut barrier defenses. Given the bacteriocentric emphasis of the current GMABM, these host systemic factors are highly abstracted, and therefore may  miss key host dynamics that influence In silico dynamic knowledge representation provides an opportunity for the research to engage in "thought experiments" about plausible lines of investigation, reducing the intellectual and pragmatic overhead in exploring hypothesis-space, thereby enhancing the throughput of hypothesis evaluation. Of course, the direction of the thought experiments would be informed by the researcher's expertise and intuition. For instance, in the case of the GMABM:Resolving discrepancies between ABM and animal model behaviors may require consideration and representation of both the host and microbe in greater detail. However, as evidenced in the recent history of translational biomedical research, the range of possible details to be expanded upon may be too great to be evaluated in an efficient, or even tractable, manner . In silias well as alternative hypotheses in the GMABM. The behavior of the ABM may then suggest the type and design of experiments to test and validate these hypotheses, forming part of an iterative loop of observation, analysis, synthesis and testing.1. Investigation into mucous barrier physiology and immune activation may provide justification and guidance for future modifications of the GMABM to test hypotheses about their role in determining extent of host injury. As noted above, the current GMABM highly abstracts the host functions concerning the dynamics of maintaining the mucus layer and, particularly, the immune response. Researchers with a particular interest and expertise in these areas could speculate as to how their mechanisms of interest would affect the gut HPI. Dynamic knowledge representation would allow them to implement much more detailed instantiations of these mechanisms 2. A more detailed representation of the bacterial ecology in the gut, including metabolism, selection, fitness and succession, could result in the incorporation of more detailed bacterial models into an ABM framework. For instance, there is extensive work in the area of bacterial metabolism, extending to genome scale models of complete bacterial metabolic networks -101; theThe expansibility of ABMs as means of dynamic knowledge representation would allow the consolidation of multiple streams of mechanistic research into aggregated ABMs to simulate increasingly complex biological systems. For instance, in the integrated GMABM the behavior of Pseudomonas agents reflects the combined, simultaneous effects of all four of the central sense and response pathways for virulence regulation in response to host stress. We propose that the GMABM of the HPI in an abstracted model of gut derived sepsis is a starting point to advance the application of dynamic knowledge representation to the translational efforts regarding the role of the gut HPI and its relationship in the pathogenesis of nosocomial infections. Furthermore, we suggest that there is a beneficial role for the use of such relatively abstract and qualitative models in the current research environment. In particular, given the inevitable and unavoidable gaps in knowledge, the use of this type of dynamic knowledge representation as a "virtual sandbox" where researchers can try out new ideas, construct novel hypotheses and instantiate ambiguous or competing mechanisms, will be a necessary capability for dealing with an increasingly data-rich and high-throughput world.The authors declare that they have no competing interests.JS and GA conceived of the project, developed and used the computational model and provided most of the text. OZ and JA provided biological and microbiological background, laboratory experimental results and edited the text. All authors have read and approved the final manuscript.Text File of Netlogo code for the Gut Milieu ABM. The entities, variables and rules are listed in this file; the components of the user interface are seen at the end of the file. Interested parties may download the actual model from http://bionetgen.org/SCAI-wiki/index.php/Main_Page.Click here for fileP. aeruginosa when exposed to media from control, gut epithelial cells over-expressing HIF, and gut epithelial cells exposed to hypoxia Representative data file from original paper concerning the effect of hypoxia and adenosine demonstrates the production of the PA-I lectin by . Reproduced with permission from [ion from .Click here for fileP. aeruginosa production of PA-I lectin (details in the text)Representative data file from original paper concerning the effect of low phosphate is a 48 hr Kaplan-Meier survival curve for mice undergoing major surgery (hepatectomy) , where phosphate is known to be depleted and lethality has been identified to be due to . Reprinted with permission from [ion from .Click here for fileP. aeruginosa exposed to various pro- and anti-inflammatory cytokines Representative data file from original paper on the effect of IFN-γ demonstrates the response of PA-I lectin promoter activity in . Note that the only active curve is that associated with IFN-γ. Reprinted with permission from [ion from .Click here for fileRepresentative data file from original paper on opioid effects demonstrates the production of HQNO in response to the synthetic opioid dynorphin . We note that there is a discrepancy in what appears to be the final trajectory of HQNO production between the ABM (rising) and this figure (plateau or slightly decreasing). However, this discrepancy does not appear to influence the effect of HQNO in inhibiting the growth of commensal bacteria . Additional File Click here for file


Luprops tristis Fabricius (Coleoptera: Tenebrionidae), numbering about 0.5 to 4 million per residential building following summer showers, and their prolonged stay in a state of dormancy, make them an extreme nuisance in rubber tree plantation belts of the Western Ghats in south India. Food preference of post-dormancy adults, larvae and teneral adults stages towards tender, mature and senescent leaves were assessed in three choice and no choice leaf disc tests. All stages have strong preference towards fallen tender leaves and lowest preference towards senescent leaves indicating that leaf age is a major attribute determining food selection and food preference of L. tristis. Ready availability of the preferred, prematurely fallen, tender rubber tree leaves as a food resource is suggested as being responsible for the exceptionally high abundance of L. tristis in rubber tree plantation belts.Massive invasion of the litter dwelling beetle,  Luprops tristis Fabricius (Coleoptera: Tenebrionidae), following summer showers and their prolonged stay in a state of dormancy (oligopause) is a seasonal nightmare for the farming community in the rubber tree plantation tracts of Kerala along the western slopes of the Western Ghats  of L. tristis on different age classes of rubber leaves  available to L. tristis in the litter floor of rubber tree plantations during the period of field presence. The present study provides quantitative evidence in support for the hypotheses that detritivorous L. tristis discriminate between fallen tender, mature and senescent leaves and feed preferably on tender leaves.Massive seasonal invasion of the litter dwelling beetles, rn Ghats . With th tristis  revealed tristis . Similarreported . The sugreported , the chareported , and theviz., an isolated 15 year old rubber tree plantation, RRII 105 clone of Hevea brasiliensis Muell. Arg. (Wild.ex ADR. De Jus) , a building in the vicinity of the plantation with history of beetle invasion, and a laboratory set up.The present investigation was carried out during January-April 2005 on the Devagiri College campus located 6 km east off the Malabar Coast at Calicut , in the Kerala state of India. Three essential components for the study were available, 2) disks of leaves were cut and placed on opposite sides of clay vessel (9 cm diameter × 5 cm height) with thin layer of soil. The vessel was sprinkled with water to moisten the container and soil. Experimental beetles of different developmental stages were introduced into the centre of the vessel and were allowed to feed for 24 h from 8 am to 8 am. Leaf discs were collected, the leaf area consumed by individual beetle and larva from each leaf disc was estimated using a 1 mm2 mesh size reticulated paper glued on a glass slide. The amount of leaf disc consumed during the tests was estimated by subtracting the unconsumed area from the initial area of 400 mm2. All tests were replicated 30 times.Larval and adult food preferences were analyzed with three choice and no choice leaf disk tests. Leaves belonging to three age classes  were collected from the branches of the same height of a randomly selected rubber tree in the middle of the plantation raised from single clone, to avoid the possible influence of intra-plant variation in leaf quality. Based on phenology studies, freshly sprouted leaves of five days of age were categorized as tender, two weeks following sprouting as mature, and the leaves turning yellowish brown prior to the onset of annual leaf shedding as senescent . Collectrd instar larvae in the premoulting quiescence stage were collected from plantation litter and post-dormancy beetles in the refractory phase were collected from their natural aggregation sites in the college hostel premises. Fourth instar larvae were not sufficiently abundant on same day so the tests had to be conducted at two occasions on successive days. Teneral adults and post-dormancy adults that had emerged on a single day were used. All stages were reared in clay vessels placed in an environmental chamber and fed with diced leaves of all three types for four days to reduce the effect of leaf quality variations on growth rate. Each stage was deprived of food for 12 h before the starting of the experiments. Feeding experiments on post-dormancy adults were conducted during the 3rd week of January, while tests on larvae and teneral adults were conducted during the 2nd week of March. Three choice and no choice experiments were conducted on successive days employing frozen leaves.To ensure uniformity of age at the beginning of the experiment, groups of 60 teneral adults and 3Significance levels of variation in food consumption by each life cycle stage  on leaf types  were analyzed with two-way ANOVA in both three choice and no choice conditions. Variations in the quantity of each leaf type consumed by each life cycle stage between three choice and no choice conditions were analyzed with t-test . Megastaviz., larva, pre-dormancy and post-dormancy adults, preferred tender leaves over mature and senescent leaves. Senescent leaf was the least preferred food in three choices as well as in no choice experiment . Among three stages, pre-dormancy adults consumed more food than post-dormancy adults or larva and larva consumed the least.Significant differences in the quantity consumed by three life cycle stages among leaf types as well as in the rate of consumption among three life cycle stages in both tests were distinct  . However, with mature  and senescent leaves , there was significant variation in consumption. Predormancy adults did not show any significant differences in the consumption of tender  and mature  leaves, while they consumed more senescent leaves in no choice tests . Larval stages consumed more of each leaf age class in no choice tests .Post-dormancy adults did not show significant differences in consumption of tender leaf in no choice tests than in three choice tests  for unknown reasons could explain the high preference towards tender leaves (preference hierarchies)  tender leaves. Specialization of L. tristis on highly seasonal tender leaves would have been detrimental to the fitness of all stages, more specifically for post-dormancy adults returning to the plantation litter at a time when only senescent leaves are available. Though leaves of all the age classes are available in plantations when larvae and teneral adults emerge, over specialization on tender leaves would have affected their survival chances if by any reason prematurely fallen tender leaves are not available to feed upon. This is another instance of establishment of preference hierarchies in the feeding behaviour of phytophagous insects in the face of host limitation (All stages of archies) . Howeverarchies)  and non-archies) , suggestmitation . AvoidanL. tristis is oligopause, which does not involve long term advance preparations by accumulation of energy reserves prior to active dormancy  stage is the most aggressive feeder of each leaf type and it may appear that during the preparatory phase of dormancy they are stocking up on energy resources . Howeverdormancy . Therefodormancy  showed tL. tristis beetles, which is uncommon in other natural or monoculture litter stands and it raises the following practical questions. What are the nutritional reasons behind the predisposition towards tender leaves? If tender leaves by way of premature fall of leaves are not available during the period, how will it affect the reproductive capacity of post-dormancy adults, duration of the larval phase, quality and quantity of reserve food accumulated by the teneral adults and the survivability of teneral adults during the dormancy phase? If the high abundance of L. tristis in rubber estates, compared to their very low abundance in natural forests, is related to the advantages gained from feeding on fallen tender leaves, then will not the control of seasonal premature leaf fall in rubber plantations cause by the powdery mildew, Odium hevea, and Corynespora leaf disease, Corynespora cassiicolaPreference of all stages towards tender leaves and the synchronicity of premature leaf fall and the field presence of all three stages indicate that premature leaf fall in rubber plantations contributes to the unprecedented abundance of


We report a case of tumor growth over a period of four decades, presenting with large multicentric lytic lesions of the skull and a profound mass effect, without neurological deficits. Clinical and radiological features of a patient with a giant intradiploic epidermoid and its impact on the choice of treatments are discussed.An 81-year-old Caucasian man, who had first noticed a painless subcutaneous swelling over the left frontal scalp about 40 years ago, presented after a short episode of dizziness, which he experienced after treatment of focal retinal detachment. Computed tomography (CT) and magnetic resonance imaging (MRI) examinations revealed an exceptionally large tumor involving major parts of the skull with extensive destruction of the bone and distinct deformation of the brain. Considering his age and the absence of neurological deficits or pain, the patient refused the option of tumor removal and cranioplasty, yet agreed to a biopsy, which confirmed the suspected diagnosis.The course of the disease demonstrates that even patients with large tumors, inducing distinct pathomorphological changes, do not necessarily experience significant impairment of their quality of life without surgery. This is an impressive example of the chance to lead a long and satisfying life without specific medical treatment, avoiding the inherent risks of these procedures. Yet, there is a clear indication for surgery of intradiploic epidermoids in most cases described in the literature. We report the case of a patient with a progressive swelling of the cranial vault and growing lesions of the skull who refused any further diagnostic procedures over a period of 40 years, although it was urgently recommended by his physicians. When he finally agreed to undergo a magnetic resonance imaging (MRI) investigation, large polycystic calvarial defects and extensive deformation of major parts of the brain were detected.The patient had an exceptionally large intradiploic epidermoid cyst with extensive destruction of large areas of the skull and distinct deformations of the brain without neurological deficits.Cranial epidermoid cysts are rare lesions, representing between 0.2% and 1% of all intracranial tumors. Intradiploic epdermoid cysts account for about 25% of these lesions . Most ofAn 81-year-old Caucasian man presented with a large painless swelling of the cranial vault, covering almost the whole left side and also major parts of the right side of the skull.He had first noticed a small swelling over the left frontal region about 40 years ago. During the following four decades the swelling slowly expanded. For more than 15 years his family physician had recommended an MRI for further examination. The patient refused it over a long time period because he felt no pain or other discomfort. He experienced no significant problems during his customary life activities.He finally agreed to further diagnostic procedures after a short period of dizziness following an ambulatory treatment of a focal retinal detachment. After this episode no further clinical symptoms appeared. Neurological examination was entirely normal. No cognitive deficits were found.Examination of the lesion revealed a subcutaneous mass covering most of the left cranial vault and major parts of the right side. Palpation revealed a soft elastic mass, adherent to the surface, without fluctuation.In contrast to the giant size of the cyst, on first sight no severe deformation of the head had developed. The overlying skin did not show pathologic changes, such as defects or inflammatory signs.A spiral computed tomography (CT) (Philips Brilliance 64 CT-scanner) with surface volume rendering technique revealed a large swelling of the scalp, especially over the left hemisphere Figure . ComputeThe MRI scan T2 weighted images (Philips Achieva 1.5 T) revealed an inhomogeneous, mainly hyperintense mass without penetration of the dura. Significant compression of brain hemispheres and ventricles without cerebral edema suggested a slow growing tumor. In diffusion weighted images there was a restriction of diffusion with low signal in the ADC maps Figure . The conIn summary, radiological features were suggestive of the presence of an extradural intradiploic epidermoid cyst.After thorough discussion of the diagnostic findings with the patient, he favored a biopsy to assure the histological classification of the tumor but refused the option of a complete removal of the tumor and subsequent cranioplasty. A biopsy was taken in the left frontal region. Histological examination revealed laminated keratin material, cholesterol crystals, and cellular debris. Pathological findings were consistent with the diagnosis of an epidermoid cyst. Due to a superficial wound infection surgical revision was necessary. His postoperative course was uneventful.Cushing first described an intradiploic epidermoid cyst in 1922 . A totalClinically intradiploic epidermoid cysts commonly present as slowly growing, painless lumps in any part of the scalp. Differential diagnosis of these tumors presenting with lytic bone defects includes dermoid cyst, hemangioma, eosinophilic granuloma, fibrous dysplasia, especially with widening of diploic spaces, and several others .In most cases a tentative diagnosis can be made based on radiological features and history of clinical symptoms but definitive diagnosis requires tissue histomorphology .Extradural epidermoids account for about 25% of cranial epidermoids and are located on the scalp, cranial vault or skull base . The cauTo the best of our knowledge no intradiploic epidermoid of this large size with extensive bony defects and massive deformation of major parts of the brain has been described in the literature. The time interval between first detection of a subcutaneous swelling by the patient and diagnosis, covering a period of about four decades, also is uncommon, yet corresponds to one other case report, in which a slowly progressive, painless proptosis of an eye has been described .The patient experienced no restrictions of his daily life activities or social contacts. According to the patient the subcutaneous swelling only rarely has been addressed. The patient never felt handicapped by the lesion. Although the tumor did not penetrate the dura, CT and MRI examinations revealed a profound mass effect with an extensive deformation of the brain. In spite of additional large skull defects a jolting of the head or rapid movements did not cause headache or other symptoms. The patient also experienced no problems laying down his head on the left or right side for sleep.Taking into consideration the age of the patient, his obviously good quality of life, and the risks of a total removal of this giant tumor with subsequent cranioplasty, in our opinion there was no indication for surgery in this case, even if the patient would have agreed to it.The origin of these tumors is still debated, yet disontogenetic etiology is widely accepted, assuming that the lesion derives from inclusions of ectodermal remnants during closure of the neural tube . In a feAs evident in our case even giant extradural epidermoids with profound deformation of the brain and extensive lytic skull lesions may allow a normal life without any significant restrictions. The course of the disease, covering a period of more than forty years, also demonstrates that an individual risk-benefit calculation is mandatory in the discussion of a specific treatment, which probably also applies to other diseases. There is no evidence that removal of the tumor within the described time interval would have achieved a positive impact on the life of the patient in spite of the distinct anatomical alterations. Notwithstanding, in most cases surgery is clearly indicated due to clinical or pathomorphological features.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interestsWK examined the patient, interpreted the findings and performed the surgery, and was a major contributor in writing the manuscript. AH examined the patient, interpreted the findings, designed and reviewed the manuscript. HH designed and reviewed the manuscript. JM designed and reviewed the manuscript. DF analyzed and interpreted the radiologic examination findings, and was a major contributor in writing the manuscript. All authors read and approved the final manuscript.


The use of digital imaging and algorithm-assisted identification of regions of interest is revolutionizing the practice of anatomic pathology. Currently automated methods for extracting the tumour regions in basal cell carcinomas are lacking. In this manuscript a colour-deconvolution based tumour extraction algorithm is presented.Haematoxylin and eosin stained basal cell carcinoma histology slides were digitized and analyzed using the open source image analysis program ImageJ. The pixels belonging to tumours were identified by the algorithm, and the performance of the algorithm was evaluated by comparing the pixels identified as malignant with a manually determined dataset.The algorithm achieved superior results with the nodular tumour subtype. Pre-processing using colour deconvolution resulted in a slight decrease in sensitivity, but a significant increase in specificity. The overall sensitivity and specificity of the algorithm was 91.0% and 86.4% respectively, resulting in a positive predictive value of 63.3% and a negative predictive value of 94.2%The proposed image analysis algorithm demonstrates the feasibility of automatically extracting tumour regions from digitized basal cell carcinoma histology slides. The proposed algorithm may be adaptable to other stain combinations and tumour types. The interpretation of digital histology images by pathologists  is revolutionizing the practice of anatomic pathology ,2. A natIn terms of histology, a number of studies have recently looked at image classification algorithms. One recent use of automated image analysis and processing has been as a part of algorithms used to classify breast cancer tissue. Using a supervised learning method, Petushi et al. developed an algorithm capable of classifying breast cancer carcinomas based on histological tissue micro-texture and spatial position . Using tThe aim of our study is to define the operational characteristics (sensitivity and specificity) of a simple colour-based segmentation algorithm for quantifying basal cell carcinoma from photomicrographs. The basis of this algorithm is the observation from anatomic pathology practice that cells with dense chromatin (including many cancer cells) have a different colour spectrum than surrounding normal tissues. Our hypothesis was that the operational characteristics would differ among common basal cell carcinoma subtypes  with the subtypes exhibiting more compact chromatin  demonstrating better operational characteristics than the infiltrative subtypes.Basal cell carcinoma was chosen to examine this question as this cancer presents with a well-defined range of histological subtypes and occurs in association with non-neoplastic chromatin-rich cells present in the epidermis and dermis. Finally, because basal cell carcinomas are the most common malignant neoplasm in humans , access In order to extract and analyse features of a digital image, it is first necessary to identify and separate the ROIs. Image segmentation involves dividing an image into regions of similar characteristics based on features such as brightness or morphology . IdeallyThe slides evaluated with this algorithm were stained with haematoxylin and eosin (H&E). Although H&E stains are easily distinguished visually by colour, digitally separating regions containing stain co-localisation is difficult. Separation via colour deconvolution provides a means of separating stains with overlapping regions. The basis of this method is to separate the component stains by performing an ortho-normal transformation of the image's RGB information . SeveralThe algorithm used to extract the BCC tumours consisted of four steps: pre-processing, segmentation, morphological operations, and feature extraction .The open source image processing and analysis program ImageJ was used in this study. First released in 1997 by software developer Wayne Rastban, ImageJ is an open source program based on the National Institutes of Health's NIH Image. Current features consist of numerous image processing and analysis operations, including image segmentation and extraction, noise reduction, image transformations, and particle analysis. These features are further expanded upon by an active user base. There are currently hundreds of downloadable user plugins and macros . AdditioThe colour deconvolution plugin by Gabriel Landini  was usedThresholding was then used to segment the pixels darker than the threshold value. The ImageJ isodata algorithm  was usedRemove Outliers command. This was followed by a binary closing operation, and median filtering of the dark outliers.Due to the lack of intense haematoxylin staining in the non-basaloid cell regions, the binary images produced during the segmentation process frequently contained holes and disconnected regions in the tumour nests. As a result, morphological operations were performed on the segmented images. Hole filling was achieved using a combination of median filtering and binary closing operations. Initially a median filter was applied to the bright outliers using the ImageJ As other baseloid and chromatin-rich features  could produce false positive results, we attempted to remove these features through a filtering step using the ImageJ particle analyzer feature. A minimum particle size of 750 pixels was used in order to exclude non-tumour nest particles. The extracted tumour was then obtained by removing all particles outside of the ROIs.The evaluation of a given algorithm is inherently subjective and biased towards the author's preferences, as standard methods for evaluating the algorithm do not exist . For theTo accomplish this, one of us (CN) manually evaluated printed photomicrographs of the 30 basal cell carcinoma images: 10 each of nodular, infiltrative and superficial subtypes. For each of these images, all tumour nests present were manually delineated with a black marker, scanned and analyzed with a manual approach. The main challenges in evaluating an extraction algorithm are determining the true dataset (ground truth), and the appropriate performance metrics ,30.A further challenge is the lack of standardized image extraction algorithms, seeing that most existing algorithms are optimized for a specific task. This causes a further problem for evaluating the algorithm, and the colour deconvolution approach in particular. In order to assess the effect of using colour deconvolution, the same set of histology slides were analyzed using grayscale based thresholding in place of the colour deconvolution step. In the comparison algorithm, the image was first converted to an 8-bit grayscale image, and the colour deconvolution step was omitted. The remaining steps were carried out as described by the proposed algorithm.The binary images of the algorithmically extracted tumour nests were subtracted from the binary images obtained by manual evaluation. The resulting image, containing the areas of the image not extracted by the algorithm, was considered to contain only false negative (FN) pixels. Similarly, the binary images of the manually extracted tumours were subtracted from the algorithmically extracted ones. The resulting image quantified the pixels considered to be false positives (FP). In addition, the number of true pixels (TP) was calculated by subtracting the total number of pixels identified by the algorithm from those deemed to be false positives. Finally, the number of true negative (TN) pixels was calculated by subtracting the total number of pixels in the image by the number of pixels identified by the algorithm, and by the number of false negatives.Four different metrics were calculated to assess the performance of the algorithm. The sensitivity of the test evaluates the capability of the algorithm to identify pixels belonging to the tumour nests. The sensitivity was calculated as follows:The specificity of the test evaluates the capability of the algorithm to correctly identify the pixels not belonging to the tumour nests. The specificity was calculated as follows:The proportion of the histology slide occupied by the BCC may vary significantly between different slides. In general, superficial tumours occupy a smaller fraction of the slide compared to the nodular and infiltrative subtypes. For this reason the positive and negative predictive values were calculated. The positive predictive value (PPV) of the test indicates the probability that a positively identified pixel belongs to an actual tumour. As a result, images containing a lower tumour to non-tumour ratio result in lower PPVs. Conversely, the negative predictive value (NPV) is an indication of the probability of a negatively identified pixel actually belonging to non-tumour tissue. The PPV and NPV were calculated as follows:The metrics shown in Table Surprisingly, when compared to grayscale based segmentation Table , the useThis study evaluated a method for digitally extracting the tumour regions from basal cell carcinoma histopathology slides. A combination of colour deconvolution and intensity based thresholding was used with the goal of extracting the tumour nests from the image. The algorithm was evaluated with 3 separate subtypes of basal cell carcinomas: infiltrative, nodular, and superficial. For comparison, the algorithm was repeated using only grayscale based segmentation in place of the colour deconvolution step.The performance of the algorithm varied significantly between the subtypes. The best results were achieved with the nodular subtype, while inferior performance was achieved with the superficial and infiltrative subtypes. One problem encountered with the superficial and infiltrative subtypes was the identification of false positives. This occurred in large part due to the presence of the normal epidermal basal layer in some of the slides which showed similar spectral characteristics to areas of BCC. Because the algorithm uses intensity based segmentation following the colour deconvolution step, regions with similar intensities to the tumour cells were also extracted. Lowering the threshold value would decrease the number of false positives, but would also come at the expense of lower sensitivity. Additional false positives occurred due to the presence of other basaloid elements such as skin adnexae, inflammation, and eccrine glands. A further source of false positives occurred due to blue dye used to mark the deep surgical resection margin of some specimens. Example false positives are displayed in Figure Another challenge for digital feature extraction algorithms is false negatives. In this study, false negatives resulted mainly from two causes: poor contrast between the tumour nest and its surrounding tissue, as well as inadequate hole filling. Although contrast enhancement was performed, some of the images still contained poor contrast between the tumour and its adjacent tissue. This may have been due in part to variation in the intensities of H&E staining of the original sections. One possible approach to this would be to explore the delineation of the tumour based on morphological features, rather than pixel intensities. One possibility would be to use the active contours method in order to evolve a curve representing the boundaries from the ROI . RecentlSimilar to the false positives, the rate of false negatives could be decreased by changing the threshold value. Conversely, lower false negative rates could be achieved at the expense of specificity. Example false negatives are shown in Figure Superior results were achieved by using a colour deconvolution prior to segmentation. Although using colour deconvolution resulted in a slightly lower mean sensitivity, a significant improvement in specificity was gained. This resulted in superior PPV and NPV values. In general, the colour deconvolution decreased the incidence of false positives. This was likely a result of the stain separation achieved using the colour deconvolution plugin.Overall, however, the sensitivities of the colour-based approach were not better than a grayscale-based thresholding approach.This study reports the operational characteristics of a simple colour-based segmentation algorithm using the open-source image analysis program ImageJ. As predicted, the algorithm generally performed best with examples of the nodular basal cell carcinoma subtype. The specificity was unexpectedly low for the superficial basal cell carcinoma examples due to false positive classification of pixels associated with skin adnexae and the normal basal cell carcinoma of the epidermis. However, overall, the finding that the sensitivity of this colour-based approach was not better than a grayscale thresholding approach to the same images suggests that simple colour-based algorithms without the inclusion of more sophisticated texture feature segmentation may have limited utility.The authors declare that they have no competing interests.CN devised the original study design. KL designed the algorithm and evaluated the algorithm performance. Both authors participated in drafting the manuscript. Both authors have read and approved the final manuscript.The ImageJ algorithm we used is available as an Additional file to this manuscript.ImageJ Algorithm.Click here for file