In response to activation by WASP-family proteins, the Arp2/3 complex nucleates new actin filaments from the sides of preexisting filaments. The Arp2/3-activating (VCA) region of WASP-family proteins binds both the Arp2/3 complex and an actin monomer and the Arp2 and Arp3 subunits of the Arp2/3 complex bind ATP. We show that Arp2 hydrolyzes ATP rapidly—with no detectable lag—upon nucleation of a new actin filament. Filamentous actin and VCA together do not stimulate ATP hydrolysis on the Arp2/3 complex, nor do monomeric and filamentous actin in the absence of VCA. Actin monomers bound to the marine macrolide Latrunculin B do not polymerize, but in the presence of phalloidin-stabilized actin filaments and VCA, they stimulate rapid ATP hydrolysis on Arp2. These data suggest that ATP hydrolysis on the Arp2/3 complex is stimulated by interaction with a single actin monomer and that the interaction is coordinated by VCA. We show that capping of filament pointed ends by the Arp2/3 complex (which occurs even in the absence of VCA) also stimulates rapid ATP hydrolysis on Arp2, identifying the actin monomer that stimulates ATP hydrolysis as the first monomer at the pointed end of the daughter filament. We conclude that WASP-family VCA domains activate the Arp2/3 complex by driving its interaction with a single conventional actin monomer to form an Arp2–Arp3–actin nucleus. This actin monomer becomes the first monomer of the new daughter filament. This paper provides the biochemical and biophysical basis for actin filament formation, necessary for cell shape and motility The actin cytoskeleton determines the shape, mechanical properties, and motility of most eukaryotic cells. To change shape and to move, cells precisely control the location and timing of actin filament assembly by regulating the number of fast-growing (barbed) filament ends . The actThe Arp2/3 complex must be activated by both a member of the Wiskott–Aldrich syndrome protein (WASP) family and a preexisting actin filament before it will nucleate a new actin filament . The strBoth the Arp2 and Arp3 subunits of the complex bind ATP . HydrolyWe show here that the Arp2/3 complex rapidly hydrolyzes ATP on the Arp2 subunit upon filament nucleation. There are several events in the Arp2/3 nucleation reaction that might trigger ATP hydrolysis on Arp2: (1) binding of VCA to the Arp2/3 complex, (2) binding of VCA-Arp2/3 to the side of a preformed filament, (3) binding of a VCA-tethered actin monomer to the Arp2/3 complex, or (4) binding of a second or third actin monomer to form a stable daughter filament. We find that ATPase activity requires the combination of a preformed actin filament, a VCA domain, and an actin monomer, but does not require actin polymerization. This indicates that hydrolysis is triggered relatively early in the nucleation reaction—before completion of a stable daughter filament. Capping the pointed ends of actin filaments also stimulates Arp2 to rapidly hydrolyze ATP in the absence of monomeric actin and VCA and without branch formation. Thus, ATP hydrolysis on Arp2 is stimulated directly by interaction with conventional actin, presented to the complex either as a monomer attached to the VC domain of the WASP-family protein or as one of the subunits making up the pointed end of a preformed filament. To our knowledge this is the first direct evidence that the monomer supplied by the VCA domain is the first monomer of the new daughter filament. From these observations we propose a model for the mechanism of Arp2/3 complex activation by WASP-family proteins.32P-8-AzidoATP to the Arp2 and Arp3 subunits of the Arp2/3 complex  exposure to UV light. In the presence of γ-32P-AzidoATP at concentrations above the KD for ATP  to show that UV irradiation covalently crosslinks α- complex . Here we for ATP , γ-32P-Aficiency A. Additiaffected A. The ef32P-AzidoATP-Arp2/3 with 2 μM monomeric actin in polymerization buffer and initiated polymerization by adding 750 nM VCA, which activates rapid actin filament nucleation by the Arp2/3 complex . (Unless otherwise stated, VCA refers to 6-histidine [6His]-N-WASP-VCA [398-502]. Cleavage of the 6His tag did not affect the kinetics of Arp2/3-mediated actin polymerization [unpublished data].) We assayed timepoints both by SDS-PAGE and thin-layer chromatagraphy (TLC) during the same reaction to monitor remaining and cleaved 32P, respectively (32P cleavage) and cleavage has ceased by 90 s (32P is cleaved rapidly from Arp2 upon addition of VCA, but not significantly from Arp3 (hyd > 0.1 s–1, noting that the true rate constant is probably much higher. Isolated Arp2/3 complex in polymerization buffer shows very slow spontaneous cleavage of γ-32P from both Arp2 and Arp3 (<1 × 10–4 s–1) (unpublished data). As a control, 32P-ATP hydrolysis is only seen when the Azido-ATP is covalently crosslinked to the Arp2/3 complex -bound Arp2 is inaccessible to the enzyme and remains unconjugated orthophosphate. We quantified hydrolyzed 32P-ATP and released phosphate by TLC  to calibrate the stoichiometry of ATP hydrolyzed by Arp2 , coincident with filament debranching , and nucleation is rapid (using N-WASP VCA), initiation of the polymerization reaction causes striking and near-complete ATP hydrolysis on Arp2  the Arp2/3 complex binds the sides of preformed actin filaments; (2) the Arp2/3 complex binds to the pointed ends of filaments, either by remaining associated with the daughter filament following nucleation or by capping preformed pointed ends; and (3) the Arp2/3 complex may interact with an actin monomer bound to the VCA domain of a WASP-family protein. There is abundant experimental evidence for filament side- and pointed-end binding by the complex , 2001b. Based on sequence conservation and biochemical similarities, ATP hydrolysis on Arp2 is probably driven by a mechanism similar to that which stimulates ATP hydrolysis on actin. The molecular details of how polymerization activates ATP hydrolysis on conventional actin, however, are not well understood. A leading hypothesis is that a “hydrophobic plug”—a loop between subdomains 3 and 4 of actin  and Arc4 (p20) subunits . The ArcATP release = 330 s; 32P signal is unchanged by the addition of free ATP. The 32P signal is only equivalent to hydrolyzed ATP in the absence of free ATP. The addition of free ATP should cause the excess of uncrosslinked Arp2/3 complex to compete with the small fraction of crosslinked 32P-ATP-Arp2/3 complex and thereby significantly reduce the 32P signal. The observation that the 32P signal is not reduced, rather than confirming that removal of free ATP has no effect, instead confirms that contaminating ATP is present for the latter part of the “ATP-free” condition, presumably released slowly from monomeric actin. The lag in the polymerization created by the initial absence of ATP would be present in the experimental ATP hydrolysis measurement, but may not have been present in the nucleation data presented because this was generated by a model-dependent computer simulation  are more than an order of magnitude faster than debranching of Arp2/3-generated dendritic networks (approximately 1000 s) . The kin2). This is consistent with the fact that in vitro the binding energy of this interface is sufficient to drive the interaction and promote the active conformation of the complex directly, even in the absence of VCA or a mother filament  and gel-filtered before use. Rat N-WASP VCA (398–502) and Human Scar1-VCA (489–559) with N-terminal 6His tags and TEV cleavage sites were bacterially expressed and purified by nickel affinity chromatography.We purified Arp2/3 from tography . We flas2, 1 mM EGTA, 10 mM Imidazole [pH 7.0]). We took care not to unintentionally shear the phalloidin-stabilized actin filaments by using wide-bore pipette tips.We prepared phalloidin-stabilized actin filaments by adding 1/10 volume of 10× KMEI to monomeric actin at room temperature for 20 min to initiate polymerization, then added twice the concentration of phalloidin and incubated for a further hour at room temperature (1× KMEI buffer: 50 mM KCl, 1 mM MgCl2, 50 mM KCl, 10 mM Imidazole (pH 7.0) and added 6 μM γ-32P-labeled 8-AzidoATP (Affinity Labeling Technologies, Lexington, Kentucky, United States). After a 2-min incubation to allow nucleotide exchange, we crosslinked for 9 s using a UV hand lamp , added 1 mM ATP and 1 mM DTT to quench the reaction and buffer exchanged into 1× KMEI plus 100 μM ATP, 1 mM DTT using a NAP5 column . We used the Arp2/3 for assays within 10 min of crosslinking. The same actin (including 7% pyrene–actin) was used for both ATP hydrolysis assays and correlative pyrene–fluorescence polymerization assays. We took ATPase time points by mixing 400 μl of the reaction mixture with premixed 400 μl of methanol and 100 μl of chloroform. We ran the precipitated protein on SDS-PAGE gel to separate the subunits and quantified 32P-labeling using a phosphoimager . For phosphate cleavage assays, we quenched timepoints into 1/10 volume 26 M formic acid, spotted on cellulose TLC plates, and separated the components in 0.4 M KH2PO4 (pH 3.4). We separately ran 32P-ATP and 32P-ATP treated with apyrase as standards to confirm the separation of 32P-ATP and cleaved 32P, respectively (unpublished data). As an alternative method of quantifying cleaved 32P, phosphomolybdate was extracted as in We diluted freshly thawed aliquots of Arp2/3 to 2.0 μM in 1 mM MgClAcanthamoeba actin with 7% pyrene–actin to monitor actin polymerization by fluorescence (λex = 365 nm, λem = 407 nm, 25°C) (–1) (cf. 2+ cation on monomeric actin was preexchanged with Mg2+ 30 s before use.We doped m, 25°C) . We calc(–1) cf. . PolymerWe prepared filamentous actin as above and stabilized filaments with stoichiometric Alexa-488 phalloidin (Molecular Probes, Eugene, Oregon, United States). We mixed 2 μM Alexa-488 phalloidin–F-actin with 20 nM Arp2/3, passed twice through a 30-gauge needle to shear the filaments, and incubated at room temperature. Timepoints were taken by diluting 500-fold and rapidly applying to poly-L-lysine–coated coverslips for visualization. Filament images were quantified for length distribution and branch frequency by a custom MATLAB (MathWorks Inc., Natick, Massachusetts, United States) routine.


With the rapid expansion of scientific research, the ability to effectively find or integrate new domain knowledge in the sciences is proving increasingly difficult. Efforts to improve and speed up scientific discovery are being explored on a number of fronts. However, much of this work is based on traditional search and retrieval approaches and the bibliographic citation presentation format remains unchanged.Case study.The Telemakus KnowledgeBase System provides flexible new tools for creating knowledgebases to facilitate retrieval and review of scientific research reports. In formalizing the representation of the research methods and results of scientific reports, Telemakus offers a potential strategy to enhance the scientific discovery process. While other research has demonstrated that aggregating and analyzing research findings across domains augments knowledge discovery, the Telemakus system is unique in combining document surrogates with interactive concept maps of linked relationships across groups of research reports.research report schema, a document surrogate of extracted research methods and findings presented in a consistent and structured schema format which mimics the research process itself and provides a high-level surrogate to facilitate searching and rapid review of retrieved documents; (2) research findings, used to index the documents, allowing searchers to request, for example, research studies which have studied the relationship between neoplasms and vitamin E; and (3) visual exploration interface of linked relationships for interactive querying of research findings across the knowledgebase and graphical displays of what is known as well as, through gaps in the map, what is yet to be tested. The rationale and system architecture are described and plans for the future are discussed.Based on how scientists conduct research and read the literature, the Telemakus KnowledgeBase System brings together three innovations in analyzing, displaying and summarizing research reports across a domain: (1)  An unfortunate consequence of specialization in the sciences is poor communication across research domains – which can hamper the knowledge discovery process. Research findings in one area may be pertinent to another, researchers may be unaware of relevant work by others that could be integrated into their work and important findings just outside a researcher's focus can be overlooked. Compounding this problem is the difficulty of keeping current with new research findings that continue to grow at an exponential rate.Reliance on keywords and/or subject indexing to find relevant literature limits the researcher's ability to precisely search for and locate specific research findings. For example, a typical database query to locate all research articles reporting a statistically significant relationship between caloric restriction and cancer would retrieve articles reporting both concepts as represented by the indexing and keyword search – but not necessarily linked together as a research finding, with information regarding reported statistical significance of the finding, nor, perhaps most importantly, lacking representation of the linkages among the retrieved document sets.This lack of "interactivity" among retrieved citations is a critical limitation of traditional search and retrieval systems. As stated by Swanson (1986) in his examination of "mutually isolated literatures:""Knowledge can be public, yet undiscovered, if independently created fragments are logically related but never retrieved, brought together, and interpreted [..] This essential incompleteness of search and retrieval therefore makes possible, and plausible, the existence of undiscovered public knowledge ."In addition to this limitation of search and retrieval, there are questions about representing a set of documents: What format or display of the retrieval set most enhances users' ability to identify which documents need to be examined in more detail? How can users navigate across document sets (i.e., to explore linkages) to enhance the discovery process? The bibliographic citation format is used by virtually all bibliographic databases today to report the results of database searches. However, it does not provide a way for the user to quickly review retrieved results for research methods and findings or to quickly view the relationships among the documents in the document set. Abstracts, even structured abstracts, simply do not provide a format conducive to rapid review of retrieved citations. In fact, the bibliographic citation format itself has changed little for the past two hundred years – even though it does not present an accurate representation of either the research methods or the research findings in a document .In spite of great improvements in document retrieval over the past twenty years, most information systems developed to promote scientific discovery e.g., -6), are , are 6])A comprehensive approach to these challenges is the goal of the Telemakus research program. Telemakus was named for the son of Odysseus who went searching for his father, the legendary Greek hero of Troy. Similarly, the Telemakus research program is developing approaches and tools for searching, knowledge discovery and mapping domain knowledge. The overall vision is to enhance the knowledge discovery process through retrieval, visual and interactive interfaces and tools. In close collaboration with researchers in the biology of aging, a working knowledgebase system has been designed to present aggregated citation information and research methods and findings for display in a conceptual schema.The Telemakus KnowledgeBase System provides the user with both a macro- and micro-view of domain knowledge. The macro-view facilitates identification of patterns – both expected and unexpected occurrences of relationships among research concepts – and permits visualization and dynamic navigation of scientific domains. The resulting maps are analogous to citation mapping work done by Small  but, insThis article describes the theories underlying the Telemakus KnowledgeBase System, provides an overview of its implementation, reports initial user feedback and explores future directions. Telemakus system builds on prior research in the areas of: (1) schema theory, (2) concept representation and (3) information visualization.Schemas are generalized mental models that provide a guide for structuring the process of production and comprehension of texts: "...at the simplest level, a schema is a description of a complex object, situation, process or structure. It is a collection of knowledge related to the concept ." Accord"The crucial fact is that the cognitive constraints on information processing which require the formation of semantic macro-structures and which organize acts and speech acts in global units, at the same time have social implications: they determine how individuals wish, decide, intend and plan, execute and control, "see" and understand their own and others' speaking and acting in the social context. Without them the individual would be lost among a myriad of detailed incoherent pieces of visual, actional and prepositional information ."Research on the application of schema theory to scientific research includes the schematic representation of psychological reports , clinicaUnderstanding a written text is a process of fitting it into a larger schema known to reader as part of their previous knowledge about the world. It is reasonable to expect that presenting written texts in familiar formats can enhance and potentially speed up an individual's ability to review and analyze large document sets rapidly.Fuller  investigThe predictability provided by schemas also applies to a document's metastructure. For example, Dillon  has propA second core component of the Telemakus system is based on concept representation.Concept representation is an important component in accurately representing facts from the document. Characterizing the location of concepts in a scientific document can greatly facilitate accurate document representation.Indexing a document – using a vocabulary or thesaurus of terms to represent the document – is a standard method employed to improve retrieval of relevant documents. Yet traditional approaches to indexing fall short of true document representation: reducing the words found in the abstract, title or full-text of the document may be suggestive of the content but are not truly representative of the methods and research findings. The indexing literature is replete with studies documenting interindexer inconsistency, even among experienced professionals using familiar well-documented systems . Studies"The information retrieval (IR) problem can be described as a quest to find the set of relevant information objects (i.e., documents D) corresponding to a given information need, represented by a query Q. The assumption is that the query Q is a good description of the information need N. An often used premise in IR is the following: if a given document D is about the request Q, then there is a high likelihood that D will be relevant with respect to the associated information need. Thus the information retrieval problem is reduced to deciding the aboutness relation between documents and queries ."Another problem with current indexing practice lies in the way the unique structure of the information elements in the document is obscured. Scientific research reports have a highly predictable structure, with an introduction, methods, results and conclusion. Concepts mentioned in the introduction or conclusion section of a scientific article may not be the primary focus of the research described within the document. For example, the Introduction may include discussion of research among several animal models whereas the target of the research study itself is a specific breed of mouse. However, current indexing processes (whether human or automated) rarely discriminate between locations of concepts in the document for indexing purposes. In addition, index terms do not represent the connections between the various elements in the document; thus, a significant amount of critical information for the scientist is lost.® or any other bibliographic database today that will answer the question: "Has anyone ever published data that supports a connection between cancer and caloric restriction? If so, what was the intervention, what type of experiments were done and what were the findings?" A successful response to a query of this type is extremely difficult or impossible in traditional information retrieval systems because: " [..] conventional IR systems that employ isolated term assignments seem inadequate for queries which are specific and empirical in nature. If, on the other hand, retrieval systems provide a link to represent the relationships between the variables of interest as reported in the documents, queries [..] would be better answered. That is, precision might be enhanced for specific and empirical queries when the relationships between the index terms were specified in retrieval systems [For example, it is not possible to unambiguously retrieve citations from PubMed systems ."In other words, the researcher asking the questions above can retrieve a set of citations that contain both topics but still must go through the full-text of each document to determine if the research specifically answers the question.Several research studies have explored the utility of relationships captured from data tables and figures in scientific research studies. Fuller, et. al.  describeThe importance of data tables for expert decision-making was underscored by Malogolowkin, et. al. who found that cancer researchers rely on ideas presented in numerical displays in published research studies for much of their design of new research protocols . MalogolOh ,29,30 inIdentifying semantic relationships in text involves looking for certain linguistic patterns in the text that indicate the presence of a particular relationship (or research finding) using pattern-matching to identify the segments of the text or the parts of the sentence that match with each pattern: "If semantic relationships can be identified accurately in the text, retrieval results can be improved by eliminating documents containing the required concepts but not the desired relationships between the concepts ."The third component of the Telemakus system is based on visual mapping of reported research findings.As previously mentioned, indexing strategies rely on "isolated term assignments." This approach leads to the loss of two important sources of information: (1) intra-document information, i.e., the research relationships studied and tested and (2) cross-document information, which captures and links research relationships across groups of documents or domains. This loss is the result of breaking apart the context of clearly linked in research findings in the data tables and figures, concepts typically linked together (the x-y axes of the tables and graphs).Once the research relationships have been extracted, concept mapping, a means of spatially representing knowledge in a visual format, provides a potential solution to the challenge of maintaining the inter-relationships between documents and reported research findings. Spatial representations can assist in understanding conceptual relationships across a domain. They can also assist in identifying previously overlooked potential research connections.Numerous approaches to visualizing an information retrieval space have been explored e.g., ,32,33), ,33, 32,3While a review of information visualization strategies is outside the scope of this paper, there is a growing body of work related to mapping metaphors and visualizing large document sets and database search results to provide the user with the ability to visualize relationships among documents and their contents ,39. In aConcept mapping represents knowledge graphically through networks of ideas. Such networks consist of nodes (points) and links (arcs/edges). Nodes represent concepts and links represent connections between concepts. Concept mapping has been used for a variety of purposes, including to communicate complicated ideas and, as in the Telemakus system, to demonstrate connections among research findings.How might one apply the theories previously described in developing a comprehensive "real world" information retrieval and knowledge discovery system? As reviewed in the previous section, the Telemakus system is built on and extends prior research in the areas of concept representation, schema theory and information visualization. Work on components of what has become the Telemakus system has been underway for many years with a particular emphasis on the importance and utility of relationships extracted from data tables and figures ,42-44. FBased on how scientists use and want to use the research literature, Telemakus brings together three innovations in analyzing, displaying and summarizing research reports across a domain:1. Research Report Schema: Research methods and findings are extracted and presented in a consistent, coherent and structured schema format which mimics the research process itself and provides a high-level research report surrogate to facilitate searching as well as rapid review of retrieved documents.2. Research Findings extracted from data tables and figures are used to index the documents, allowing searchers to request research studies which report a relationship between two concepts of interest.3. Visual Exploration Interface provides a dynamic map of extracted research findings to graphically display what is known as well as, through gaps in the map, what is yet to be tested.The Telemakus system consists of a database, research report schema and tools to create relationship maps among concepts across documents. The research report schema serves as a surrogate for the study, methods and research findings for each document as well as providing an interactive search interface. The schematic representations include standard bibliographic information , information about the research design and methods (age, sex, number of subjects, pre-treatment and treatment regimen, organism and source of organism) and, most importantly, research findings derived from data tables and figures.® (UMLS®) Metathesaurus® as the basis for creating a controlled vocabulary.The elements extracted by the Telemakus system from full-text documents are listed in Table The UMLS Metathesaurus is a rich database of information on concepts that appear in one or more of a number of different controlled vocabularies and classifications used in the field of biomedicine. It provides a uniform, integrated distribution format of over 95 biomedical vocabularies and classifications and contains syntactic information. All Metathesaurus concepts are assigned to specific types or categories – e.g., "Disease or Syndrome," "Virus" – and the Semantic Network contains information about the permissible relationships among these types – e.g., "Virus" causes "Disease or Syndrome" . The 200The thesauri are reviewed (curated by expert indexers) in order to create a consistent controlled vocabulary structure. As indicated in Table At present, data extraction utilizes systems with both manual and automated processes. An evolving thesauri-building and revising approach are important components of the Telemakus system to ensure that vocabulary identification and management reflect the specialized needs of the knowledge domain as new research concepts are identified and reported.®, etc.). Database elements are extracted and verified against the relevant thesaurus. As new concepts are identified the UMLS is checked for the preferred term and it is added to the appropriate Telemakus thesaurus – along with synonyms, narrower and broader terms.The knowledgebase construction process begins with an Internet search of a bibliographic database (e.g., PubMed, Web of ScienceOne of the key innovations in the Telemakus system is the use of the data tables and figures for locating the concepts studied (and tested) by the researchers. Concentrating on the legends from data tables and figures focuses the extraction process and reduces the background noise of the full-text document, making the process tractable. In general, the information content of data tables and figures can be broken into two types: "facts" and "findings." Facts include reporting experimental design and comparative characteristics of animals in the study group (e.g., weight, age, pre-existing conditions, etc.). Findings are the results of the study (the research findings). Research findings are extracted from each of the "findings" data tables in a process described in Figure Table ®, (NLM®) is being tested as a means of automatically parsing the legends from the data tables and figures to identify preferred UMLS concepts for addition to the Telemakus thesauri. MetaMap maps arbitrary text to concepts in the UMLS Metathesaurus; or, equivalently, it discovers Metathesaurus concepts in text. With this software, text is processed through a series of modules. First it is parsed into components including sentences, paragraphs, phrases, lexical elements and tokens. Variants are generated from the resulting phrases. Candidate concepts from the UMLS Metathesaurus are retrieved and evaluated against the phrases. The best of the candidates are organized into a final mapping in such a way as to best cover the text [A current focus is the application of natural language processing (NLP) techniques to assist in the automation of concept extraction process. MetaMap, a program developed by the National Library of Medicinethe text .The Telemakus system architecture centers on: a relational database; a set of tools used to populate the knowledgebase with data extracted from bibliographic databases and full-text research reports; and several server side tools and programs responsible for delivering the content of the database to the public via the WWW. The entire system is built from open-source components, leveraging standard protocols and tools whenever possible.The document processing system is initiated by an analyst who runs, reviews and edits as necessary extractions from the document being processed. It currently consists of a number of discrete phases to download, extract and analyze each document. These services are built primarily in Java running behind Tomcat and Apache and accessed by the analyst through the browser.For the public Telemakus website interface, a number of open-source solutions have been selected and configured. An Apache web server intercepts all requests and delegates them to surrogate processes dedicated to each respective task. For requests to display the data from the database, the request is delegated to Zope, a content management service, for responding to the user's request. This typically includes running SQL queries against a PostgreSQL database and rendering the results in the conceptual schema that serves as a surrogate for each document. For tasks beyond simple queries and HTML requests, a Java Servlet™ is employed. As plain HTML is insufficient to effectively display and interact with the relationship map, a Java™ applet, TouchGraph, is used.TouchGraph is an open-source concept-mapping tool for creating and navigating links between information sources. The tool was chosen for Telemakus because of its flexibility, customizing capabilities, high quality source code and compatibility with most browsers and operating systems (OS). The TouchGraph visualization package serializes maps to and from XML. By using Java, HTTP and XML, TouchGraph makes it easy to dynamically feed content to generate interactive nodes-and-edges maps.Figure Figures The research report schema also serves as a convenient interface for searching for related research concepts, offering a rapid way of following research connections through the database. For instance, clicking on "killer cells, natural – ad libitum" would retrieve additional articles that present data tables linking those two concepts.The "map it" function, at the bottom of the retrieval set Figure  provides. Caloric restriction was an ideal starting point for Telemakus because it is an important and rapidly expanding specialized area of the biology of aging that is also highly interdisciplinary. Telemakus is a component of the Science of Aging (SAGE) project funded by the Ellison Medical Foundation. Other SAGE partners include the American Association for the Advancement of Science and Highwire Press, Stanford University (SAGE Knowledge Environment web site: ).The first completed Telemakus knowledgebase focuses on caloric restriction in aging and is freely available at Formal usability testing of Telemakus is underway and will be the subject of a future article. Because a major goal of the Telemakus research program is to study scientists' approaches and preferences for accessing and using the scientific literature in order to create models and approaches for user-centered knowledgebases, researchers have been involved in the iterative design and testing of the system from its inception. The primary goals of this evaluation are to:1. Determine scientists' preferences for working with the research literature.2. Model preferred features based on those preferences.3. Test the completeness of schema elements and structure as a document surrogate.4. Experiment with and identify optimal visual representations to meet user needs.5. Iteratively review/evaluate/test for improved performance in response to user feedback.6. Identify domain(s) for future knowledgebase creation.In general, response to each successive iteration of Telemakus has been positive and included constructive feedback for system enhancements and expansions. User feedback affirms that retrieval based on research findings is a unique and highly desirable core function. Further, the Telemakus schematic document surrogate has been enthusiastically received as a major improvement over the traditional citation format with abstract. As one researcher stated , "The strengths of Telemakus are doing what PubMed does not do, which is to give an outline of the main points and to allow searching off the figure/table legends, organisms/sources and outcome fields."Additional feedback relates to the labeling of concept relationships as "statistically significant." Some researchers are interested in knowing the level of reported significance  and asked for a detailed labeling to document this. In addition, there have been requests to consider labeling the relationships . Early testing of the mapping function resulted in the observation that color-blind individuals would not be able to see lines that were labeled with red or green, which led to a change in the mapping color scheme.There have been many additional suggestions for improving the visualization, including addition of three-dimensional representations and allowing more user control of the presentation itself. Some researchers have expressed interest in being able to build maps based on the date a particular research finding was reported. This functionality would create time sequence maps that show the progression of research over time and, perhaps, will demonstrate paths of research that have been discarded prematurely and may be worth re-visiting. A number of researchers have indicated the utility of this approach for teaching purposes – for a student to quickly get a sense of the research "facts" in a domain. There have also been requests for tools to support downloading subsets of the knowledgebases, as well as tools to allow individuals to manipulate maps and add their own research findings and ideas to the concept maps.While initial Telemakus development has focused on the research literature related to caloric restriction and the biology of aging, the goal is to expand into additional domains. For example, tables of genetic sequence information, which display reported relationships between gene sequences and diseases, are a natural area of expansion for Telemakus. There is great potential for building linkages between Telemakus knowledgebases and other factual databases, e.g., NCBI entrez resources. In addition, scientists from other domains beyond biomedicine  have indicated that a customized schematic representation of research findings could be very useful in their domains.Speeding up document processing so Telemakus can easily and efficiently scale for comprehensive treatment of domains is a key priority. As discussed previously, the UMLS Metathesaurus resources (in particular, MetaMap) are proving extremely useful. In addition, the Semantic Network will be tested for enhancing searching and visualization of research findings.We will continue to utilize an iterative development method so that results of usability evaluation can immediately inform development of additional features. In particular, we want to test our hypothesis that the mapping feature will promote knowledge discovery by showing graphically what is known as well as, through lack of links, what research linkages have not yet been tested.Since basic sciences researchers tend to initially focus on the data found within a report's tables and figures , extracting the headings and providing linked research concepts mimics a researcher's traditional approach to reading the research literature ,48. WhenOne of the long-term goals of the Telemakus system is not to build knowledgebases "ad infinitum" but rather to create flexible tools for users to quickly and efficiently locate and visualize aggregate research findings from any domain which reports research findings as data. As more and more full-text research reports are available on the Internet, we believe the tools we are developing will provide an important approach for focusing on research findings and providing visual cues for quick review and assimilation.The Telemakus KnowledgeBase System builds on a good deal of prior research in a variety of domains. It provides a flexible new approach for creating knowledgebases to facilitate retrieval and review of scientific research reports. In formalizing the representation of the research methods and results of scientific reports, Telemakus offers a potential strategy to enhance the scientific discovery process. While other research has demonstrated that aggregating and analyzing research findings across domains augments knowledge discovery, the Telemakus system is unique in combining informative document representations with interactive concept maps of linked relationships across groups of research reports. Telemakus presents a novel approach to creating useful and precise document surrogates and may re-conceptualize the way we currently represent, retrieve and assimilate research findings from the published literature.None declared.SF conceived the study and contributed to its design, coordination and evaluation. DR and PB participated in the design of the study. DR led the overall coordination and drafted the manuscript. PB led the technical implementation. GMM contributed to the design, coordination and evaluation. All authors read and approved the final manuscript.


Symptomatic hypogammaglobulinemia in infancy and childhood (SHIC), may be an early manifestation of a primary immunodeficiency or a maturational delay in the normal production of immunoglobulins (Ig). We aimed to evaluate the natural course of SHIC and correlate in vitro lymphoproliferative and secretory responses with recovery of immunoglobulin values and clinical resolution.Children, older than 1 year of age, referred to our specialist clinic because of recurrent infections and serum immunoglobulin (Ig) levels 2 SD below the mean for age, were followed for a period of 8 years. Patient with any known familial, clinical or laboratory evidence of cellular immunodeficiency or other immunodeficiency syndromes were excluded from this cohort. Evaluation at 6- to 12-months intervals continued up to 1 year after resolution of symptoms. In a subgroup of patients, in vitro lymphocyte proliferation and Ig secretion in response to mitogens was performed.32 children, 24 (75%) males, 8 (25%) females, mean age 3.4 years fulfilled the inclusion criteria. Clinical presentation: ENT infections 69%, respiratory 81%, diarrhea 12.5%. During follow-up, 17 (53%) normalized serum Ig levels and were diagnosed as transient hypogammaglobulinemia of infancy (THGI). THGI patients did not differ clinically or demographically from non-transient patients, both having a benign clinical outcome. In vitro Ig secretory responses, were lower in hypogammaglobulinemic, compared to normal children and did not normalize concomitantly with serum Ig's in THGI patients.The majority of children with SHIC in the first decade of life have THGI. Resolution of symptoms as well as normalization of Ig values may be delayed, but overall the clinical outcome is good and the clinical course benign. Streptococcus pneumonia, Haemophilus influenza and Staphilococcus aureus, but the infections may be of unusual severity, persistence, or frequency.Pediatric patients with "recurrent infections" within our area are referred to the pediatric immunology clinic in the Kaplan Medical Center. Few fulfill the clinical criteria of the immune deficiency "red flags", Table In the last 10 years, tremendous advances in the fields of molecular medicine and genetics, have made possible the definitive diagnosis of most combined immunodeficiency patients, agammaglobulinemia patients and clinical syndromes associated immunodeficiency patients, on the basis of a recognized genetic aberration leading to a protein product dysfunction ,4. NeverIn this study we aimed to evaluate the natural course of disease in symptomatic hypogammaglobulinemia of infancy and correlate in vitro lymphoproliferative and secretory responses to mitogens in this population with recovery of immunoglobulin values and clinical resolution.Children more than 1 year of age, with recurrent infections, defined as more than three episodes of acute otitis media and/or more than one episode of acute sinusitis and/or more than one episode of pneumonia or the presence of a severe deep seated infection (meningitis, septicemia, etc.) within the last 6 months, or fulfillment of one of the "red flags" of immunodeficiency, see Table All procedures were performed according to accepted ethical standards of the Institutional Review Board of Kaplan Medical Center. The parents of all the children were informed accordingly and gave their permission for participating in the study and blood sampling.Total serum Ig levels were measured by nephelometry  and serum IgG subclasses have been assayed with an immunodiffusion commercial kit (Human IgG Subclasses single dilution BINARID, Birgminham, UK). Specific antibody production was not evaluated.5 cells were stimulated for four days with 0.01% w/v SAC , 2.5 μg/ml PWM, 20 μg/ml E. coli: O55:B5 LPS or with 20 μg/ml PHA (Sigma, St. Louis, MO, USA). The cells were then pulsed with 1 μCi/well of [3H]-Thymidine (Nuclear Research Center, Negev, Israel) and incorporated radioactivity was measured by a β scintillation counter. Proliferation was expressed as stimulation index (SI).Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood of patients and age-matched donors  on ficoll isopaque gradients (Sigma, St. Louis, MO, USA). Patient and normal donor cells were cultured in microtiter plates with culture medium: RPMI medium supplemented with 10% FCS , 10 mM Hepes buffer, 100 U/ml penicilin 100 μg/ml streptomycin, 2 mM L-glutamine, and 100 μg/ml kanamycine (Sigma Israel), and were grown at 37°C with 7.5% CO2 in air. 5 × 10In parallel, cell culture supernatant aliquots were harvested and Ig isotype concentrations were measured in the culture supernatants, by a solid-phase immunoassay, in Nunc- Immunoplate Maxisorp 96 wells (Nunc, Denmark). The plates were coated with goat anti-human IgM, IgG or IgA antibodies (Jackson Immunoresearch Laboratories, West Grove PA, USA). Biotinilated goat anti-human IgM, IgG or IgA (Jackson, West Grove, PA, USA) and streptavidin-alkaline phosphatase (Amersham, Buckinghamshire, England) were used for measurement of IgM, IgG and IgA respectively. Resulting yellow dye intensity was read by an ELISA reader (Microplate Auto-Reader Bio-Tek Instruments, VT, USA). Dye units were converted to immunoglobulin concentrations by extrapolation from standard curves determined by using purified myeloma proteins of known concentration in every assay.We used non-parametric tests to compare the means  and a standard analysis of variance to compare between groups. Logistic regression analysis was used for comparison of distribution of dichotomous values between the groups. Analysis was performed using SPSS for windows ver 9.0.32 patients were included in the study, 24 males (75%) and 8 females (25%) with a mean age at diagnosis of 3.4 years (range 1.2 – 7.0). Clinical presentations included severe and recurrent Ear-Nose-Throat (ENT) infections – 22 patients (69%), pneumonia, bronchopneumonia or severe, recurrent upper respiratory infections – 26 patients (81%), diarrhea – 4 (12.5%) and atopy related complaints – 20 patients (63%). A positive family history of recurrent, unusual or severe infections was obtained in 7 patients (22%). Demographical and clinical data of patient cohort is summarized in Table Out of the initial 32 patients, 17 (53%) spontaneously corrected their Ig abnormalities. This group included 15 boys (88%) and 2 girls, mostly All defects were partial, no patient in this group, showing a complete absence of a given Ig isotype. The clinical course was benign, only 9.4% (3/32) patients requiring IVIg (2 in the THGI group, 1 in the non corrected group). 38% (12/32) of patients received prolonged antibiotic prophylaxis (4/17 in the THGI and 8/15 in the non corrected group, p = 0.09) and resolution of clinical symptoms occurred in 84% of patients (14/17 in the THGI and 13/15 in the non corrected group). All calculated p values, non significant for comparison between the 2 groups.Comparative analysis of serum Ig isotype levels at diagnosis showed no significant difference, between the transient and non-transient group.In vitro lymphocyte proliferation and Ig secretion were measured in 9 patients (5 patients with THGI and 4 with non-corrected IGD) on one or more occasions. Lymphocytes proliferative responses to SAC, PWM and LPS showed no differences between the groups and no significant differences from childhood norms. The proliferative response to PHA was significantly increased (p < 0.005) after the correction of Ig abnormalities, overshooting normal controls Fig .Quantification of the in-vitro immunoglobulin secretion in response to the various mitogens showed significant isotype and mitogen dependent variation. The IgM secretory response to PWM and LPS, was low in hypogammaglobulinemic patients. After normalization of serum Ig values, the IgM response to LPS stimulation increased, see table For both IgG and IgA, the response in normal children was significantly better than in either group of patients. The IgA secretion index to all 3 stimulatory mitogens was minimal in hypogammaglobulinemic patients, even after serum Ig correction and differed significantly from age matched controls (p < 0.005 for SAC, p < 0.05 for PWM, p < 0.05 for LPS), table Young patients with recurrent infections represent a sizeable portion of the daily practice of all primary care pediatricians and family physicians, with parents clamoring for a solution with the accumulation of lost daycare or school days. The physician is faced with the dilemma when parental assurance will suffice, rather than initiation of a costly immunological investigation. Often, the presenting clinical signs and symptoms are insufficient for an educated diagnosis, as well as Ig levels in infants below the age of one year. The present investigation was initiated in order to try to contribute additional understanding how to differentiate between cases of primary immune deficiency and those who are not.We prospectively studied the outcome of SHIC in 32 patients during a period of 8 years, mean follow up of 3.2 years. During this time more than half corrected their Ig abnormalities. The mean follow up of the non-corrected IGD group is slightly shorter  than that of the THGI group (2.5 years and 3.5 years respectively) which leads us to speculate that some patients in the "non-transient" group may eventually correct. This is consistent with previously published reports from Dalal et al. who, after a follow up of 10 years, found that 70% of patients had complete resolution of their Ig abnormalities .Interestingly, the majority of our patients were males (75%), and a higher proportion of males corrected their serum Ig . This finding of male preponderance is not uniquely ours. In the reports of Dalal et al. , 24/35  resolved their tendency for recurrent infections irrespective of their Ig values. Only a minority of patients required any medical intervention, 38% received antibiotic prophylaxis and only 9% intravenous Ig replacement therapy. We observed no difference in the clinical presentation or follow up of the transient group as compared to the non corrected IGD patients and resolution of serum Ig abnormality did not cause a complete clinical remission in all patients. This may be due to a residual inability to mount an adequate antibody response to specific antigen challenge, data that has not been evaluated in our series. The impaired IgG and IgA in vitro secretory responses, seen in hypogammaglobulinemic patients, even after serum Ig normalization, may be an expression of such an inability, which warrants further investigation.Atopy was a prominent associated complaint in 21/37 (57%) of our patients. Especially so in comparison with the prevalence of asthma in this age group in Israel – 7% ,23 and tThough no clinical evidence of T-cell functional impairment was observed in our patients , previous reports -28 suggeTHGI is a relatively common cause of symptomatic hypogammaglubulinemia in infancy in our area. Most children will spontaneously correct their Ig abnormalities during the first decade of life. Though tests of cellular or humoral stimulation index, are not as yet capable of differentiating the transient from the non-transient patients upon their presentation, significant isotype and mitogen specific variability is evident. The relative preservation of the in vitro IgM secretory response and the lack of IgA/IgG response in patients with hypogammaglobulinemia, argues for a delay in isotype switching as the molecular basis underlying the clinical entity of transient hypogammaglobulinemia of infancy.The author(s) declare that they have no competing interests.SHIC – Symptomatic Hypogammaglobulinemia in Infancy and ChildhoodTHGI – Transient Hypogammaglobulinemia of ChildhoodIg – ImmunoglobulinIGD – Imunoglobulin deficiencySAC – Staphylococcus aureus cowan IPWM – Pokeweed mitogenLPS – LipopolysaccharidePHA – PhytohemagglutininIVIg – Intravenous ImmunoglobulinENT – Ear, nose & throatMIK – carried out the patient care and follow-up, was responsible for the database organization, data analysis and for manuscript coordination and writingZT – is one of the research initiators, carried out the patient care and follow-up and contributed to the manuscript writingIA – carried out the in vitro cell proliferation and Ig secretion studiesRS – carried out the in vitro cell proliferation and Ig secretion studiesMS – carried out the patient care and follow-up, was responsible for the database initiationIZ – is one of the research initiators, carried out of the laboratory evaluation and contributed to the manuscript writingAll authors read and approved the final manuscriptThe pre-publication history for this paper can be accessed here:


This review article combines four disparate observations about Neural Tube Defects (NTDs). They are the worldwide decline in the birth incidence that began prior to prenatal diagnosis; family recurrence risks; the effect of prenatal diagnosis and termination of affected pregnancies; and the effect of folic acid. NTDs are due to many different causes . The incet al [Family studies suggest the recurrence risk for first-degree relatives of affected individuals is approximately 1 in 30. For second-degree relatives (the children of the mother's sisters and brothers) the risk is approximately 1 in 220 . Howeveret al  report tet al [et al [The third aspect, prenatal diagnosis and termination of affected pregnancies, is one that should be discussed with all women in the reproductive age range and, more importantly, with patients who have a family history of an NTD. Folic acid taken orally on a daily basis is shown to lower the occurrence and recurrence of NTDs in their own offspring and in their relatives. The Medical Research Council  was the et al  have recl [et al  demonstrl [et al  and dietl [et al ,18.When recent trends in the birth incidence of NTDs are reported, they focus additionally on the effect that folic acid has on the early second trimester prevalence of affected fetuses. As for the epidemiological studies noted above, these reports include varying types of cases; some report only "spina bifida", others "spina bifida" and anencephaly, and still others mention these two types and encephalocele with or without hydrocephalus. Some studies report only deaths due to complications in these groups of patients as stillborns, or deaths in the neonatal time period; other reports study all affected newborns, and still others cover selective or spontaneously aborted fetuses. Those that include time intervals after the introduction of intrauterine diagnosis and selective termination do not take into consideration the variations in incidence at different gestational ages and at birth, whether stillborn or live . Creasy Our data and this review clearly demonstrate the effects of intrauterine diagnosis and selective termination prior to the recommendation for supplementation and fortification of foodstuffs with folic acid. Because the reason for termination of a pregnancy is not reportable in our state and the USA, we cannot determine the effect of folic acid on the prevalence of myelomeningocele and anencephaly in first and early second trimester fetuses. Studies of the effect of folic acid in reducing the birth incidence in communities with a low incidence, and active prenatal diagnosis associated with termination of affected fetuses, require longer-term studies than published to date. The differences in data discussed above need to be considered if one is to evaluate the effect of prenatal diagnosis and elective termination as well as the effects of fortification or supplementation with folic acid. We recommend that these variables be discussed with women of reproductive age, particularly if they are relatives of a patient with an NTD. Regardless of the uncertainties, we recommend supplementation of the diet of women, beginning three months prior to an anticipated pregnancy. We recommend all women of childbearing age take at least 400 mcg of folic acid daily when they begin sexual activity. Relatives of a patient with an NTD should take 4.0 mg daily, beginning three months prior to conception.The authors declares that he has no competing interests.


Absenteeism due to communicable illness is a major problem encountered by North American elementary school children. Although handwashing is a proven infection control measure, barriers exist in the school environment, which hinder compliance to this routine. Currently, alternative hand hygiene techniques are being considered, and one such technique is the use of antimicrobial rinse-free hand sanitizers.A systematic review was conducted to examine the effectiveness of antimicrobial rinse-free hand sanitizer interventions in the elementary school setting. MEDLINE, EMBASE, Biological Abstract, CINAHL, HealthSTAR and Cochrane Controlled Trials Register were searched for both randomized and non-randomized controlled trials. Absenteeism due to communicable illness was the primary outcome variable.Six eligible studies, two of which were randomized, were identified (5 published studies, 1 published abstract). The quality of reporting was low. Due to a large amount of heterogeneity and low quality of reporting, no pooled estimates were calculated. There was a significant difference reported in favor of the intervention in all 5 published studies.The available evidence for the effectiveness of antimicrobial rinse-free hand sanitizer in the school environment is of low quality. The results suggest that the strength of the benefit should be interpreted with caution. Given the potential to reduce student absenteeism, teacher absenteeism, school operating costs, healthcare costs and parental absenteeism, a well-designed and analyzed trial is needed to optimize this hand hygiene technique. Routifection" . This stfection" -5. The efection" ,7. Ignazfection" ,7. This fection" ,7. Thesefection" ,9.The elementary school environment is also negatively impacted by outbreaks of disease causing microorganisms ,11. Theset al. (1997) reported that proper handwashing compliance, with soap and water, in school-aged children ranged from 8 to 28 percent. Reported reasons for the observed inadequacy in compliance included insufficient time during the day, and the use of substandard washing facilities in hard to access locations of the school environment [Despite the scientifically proven evidence of the effectiveness of handwashing, and the increasing promotion of proper hand hygiene techniques, observational studies in school settings have indicated that handwashing practices are often lacking ,14. Guinironment ,14.In attempts to overcome the obstacles of routine handwashing in school environments, antimicrobial rinse-free hand sanitizers are being used as an alternative hand hygiene technique. The concern is that such programs may be carried out in the absence of evidence of effectiveness in the school environment. Thus, it is timely to review the evidence currently available for the effectiveness of antimicrobial rinse-free hand sanitizer programs in reducing absenteeism due to communicable illness. The aim of this systematic review was to determine whether antimicrobial rinse-free hand sanitizer interventions are effective in preventing illness-related absenteeism in elementary school children.A detailed written protocol was prepared and reviewed in advance (complete protocol can be obtained from the corresponding author).A comprehensive search was conducted to identify all relevant studies regardless of publication status. Six electronic databases were searched for studies published in any language. The databases included: Biological Abstracts (1990-May 2003), CINAHL (1982–2003), the Cochrane Controlled Trials Register (1981–2003), EMBASE (1980-May 2003), HealthSTAR (1975-May 2003), and MEDLINE (1966-May 2003). A detailed search strategy was developed for use in MEDLINE and an iterative process was completed to refine the MEDLINE search for each database. Descriptions of the database search strategies are presented in Appendix 1 . The interventions of interest were those that administered antimicrobial rinse-free hand hygiene programs compared with no intervention or placebo treatment arm in a school setting. The outcome of interest was the comparison of the number of absences due to communicable illnesses in children who received the antimicrobial rinse-free hand hygiene intervention with the number of such absences in those who received a placebo or no intervention. We included cluster randomized controlled trials (RCTs) and cluster non-randomized controlled trials regardless of publication status.et al. 2002 and Moher et al. 2003 indicate that the exclusion of trials in languages other than English (LOE) does not bias measures of effectiveness- however, both are cautionary, advocating language inclusive search strategies [All relevant citations, titles and abstracts, were imported into a reference database where duplicates were manually removed. Priority in downloading was given to MEDLINE. Reviews were excluded, but the bibliographies from such articles were examined for relevant studies. The screening was completed in an unblinded manner; there is inconclusive evidence that blinding introduced bias into the process . One indrategies ,19. Due Two reviewers (EM, NLS) independently abstracted data from all studies meeting the eligibility criteria, excluding one abstract where EM independently abstracted pertinent information , using pTwo reviewers (EM, NLS) independently assessed the quality of each of the included studies, excluding the abstract by Thompson 2004) previously mentioned , using t04 previoData synthesis and analysis was performed in accordance with the Cochrane Reviewers' Handbook . FirstlyA flow diagram of the search results is illustrated in Figure After review of the full text of these studies, 21 were excluded for the following reasons: no outcomes of interest (n = 1), inappropriate population (n = 1), inappropriate interventions (n = 9), inappropriate study design (n = 3), irrelevant subject matter (n = 5), and review (n = 2). Thus, a total of 4 trials fulfilled the inclusion criteria ,27,31,41Of the 6 remaining studies, 2 were crossover studies ,27, 1 waAll six trials were conducted in the United States, 4 with reported industrial sponsorship, and were published between 2000 to 2004. The trials varied in size , and geographic locations  and Dyer et al. (2000), provided each student with alcohol-free instant hand sanitizer [et al. (2000), Guinan et al. (2002), Morton et al. (2004), and Thompson et al. (2004) provided each class with alcohol-based instant hand-sanitizer dispensers [The trials also varied with respect to the intervention administered. White anitizer ,41, whilspensers ,15,16,31spensers ,41, educspensers , one stuet al. (2001) reported a significant number of dropouts (857 of 1626 students did not complete the study) however no explanations were offered [The quality of reporting of the 5 trials that were examined in detail was low. Only one study was described as being randomized and double-blinded, however, it failed to describe a detailed and appropriate method of randomization and allocation concealment was unclear . Four of offered . Four st offered . Other cet al. (2000), the experimental group had a 20% (95% CI = 19–21%) reduction in absences due to communicable illness, the experimental group in the trial completed by Guinan et al. (2002) had a 49% (95% CI = 42–56%) reduction, White et al. (2001) demonstrated a 33% (95% CI = 17–45%) reduction in the experimental group, Dyer et al. (2000) had a 34 % (95% CI = 10–50%) reduction in absences due to communicable illness in the experimental group in the first phase and a 56 % (95% CI = 31–72%) in the second phase, and Morton et al. (2004) reported a significant odds ratio for McNemar's test .All six studies varied in their definition of communicable illness-related absenteeism, refer to Table et al. 1999, indicated this item to be of great concern for the parent's of school-aged children [Many studies have examined the importance of preventing the transmission of infectious diseases in the school environment, one such studied completed by Cramer children . The moschildren ,8,10,28.children -50. EffeCan the evidence from these six trials reported here be used to promote this type program in elementary schools at the present time? This systematic review of antimicrobial rinse-free hand sanitizers for prevention of illness-related absenteeism in elementary school children is the first review, of the author's awareness, to assess this issue. Although randomized controlled trials are the study design least likely to provide biased estimates of effect, due to the nature of school-based interventions, the inclusion of both randomized and non-randomized cluster controlled trials was required . Of the Four of the six studies used an alcohol-based product, the other two using a benzalkonium chloride based disinfectant. The FDA in the United States has indicated that insufficient data exits to classify the latter compounds as safe and effective to use as antiseptic handwashes. They are also adversely affected in the presence of organic material such as food residues, which may be an issue in schools . Four stSeveral limitations were encountered when completing this review, the major one being the scarcity of high quality studies. Additionally, although content experts, primary authors and industrial companies were contacted, no grey literature was found. The possible existence of unpublished non-significant trials should not be discounted. The validity of performing a quantitative synthesis was considered, however based on a qualitative inspection of heterogeneity and estimates of intervention effectiveness this was not deemed appropriate. Sources of heterogeneity included study designs, population characteristics, intervention characteristics, case definition and primary outcome measure. Thus, sensitivity and subgroup analyses were not performed, and publication bias was not assessed quantitatively. Another limitation was the fact that one reviewer was used to do the broad screen of articles and review the two citations identified between September 2003 and the present time. This may have biased the results; however, it is believed that this reviewer would overestimate the citations to be included.In wake of the recent worldwide emergence of Severe Acute Respiratory Syndrome (SARS), the importance of proper hand hygiene has been brought to the spotlight. Comprehensive hand hygiene programs with occasional reinforcement are an inexpensive intervention, which potentially can work for a broad population, with minimal adverse effects. Future research should concentrate on developing study protocols that are scientifically sound with regards to randomization generation, blinding, allocation concealment and other factors that will minimize or avoid bias. Hand hygiene programs are the most important infection control measure in the school environment and have potentially large public health and economic implications therefore their design, implementation, and analysis should be carried out with the rigour.The author(s) declare that they have no competing interests.EM conceived and designed the study as part of a graduate course in systematic reviews, reviewed trials for inclusions, abstracted data, participated in data analysis, and drafted and revised the manuscript.NLS participated in initial study design, reviewed trials for inclusion, abstracted data, participated in data analysis, and revised the manuscript.Both EM and NLS agreed upon the final revision.Appendix 1 – Syntax for searchesAppendix 2 – List of corresponding authors, content experts and industrial companies contactedAppendix 3 – Data collection formThe pre-publication history for this paper can be accessed here:Additional file 1 - Syntax for searchesClick here for fileAdditional file 2 - List of corresponding authors, content experts and industrialcompanies contactedClick here for fileAdditional file 3 - Data collection formClick here for file


Peromyscus maniculatus) are the principal natural hosts of SNV, in which the virus establishes life-long persistence without conspicuous pathology. Little is known about the mechanisms SNV employs to evade the immune response of deer mice, and experimental examination of this question has been difficult because of a lack of methodologies for examining such responses during infection. One such deficiency is our inability to characterize T cell responses because susceptible syngeneic deer mice are not available.Human infections with Sin Nombre virus (SNV) and related New World hantaviruses often lead to hantavirus cardiopulmonary syndrome (HCPS), a sometimes fatal illness. Lungs of patients who die from HCPS exhibit cytokine-producing mononuclear infiltrates and pronounced pulmonary inflammation. Deer mice (Mus musculus) granulocyte-macrophage colony stimulating factor. These cells are capable of processing and presenting soluble protein to antigen-specific autologous helper T cells in vitro. Inclusion of antigen-specific deer mouse antibody augments T cell stimulation, presumably through Fc receptor-mediated endocytosis.To solve this problem, we have developed an in vitro method of expanding and generating competent antigen presenting cells (APC) from deer mouse bone marrow using commercially-available house mouse (The use of these APC has allowed us to dramatically expand deer mouse helper T cells in culture and should permit extensive characterization of T cell epitopes. Considering the evolutionary divergence between deer mice and house mice, it is probable that this method will be useful to other investigators using unconventional models of rodent-borne diseases. Bunyaviridae) are rodent-borne and can cause hemorrhagic fever with renal syndrome (HFRS4) or hantavirus cardiopulmonary syndrome (HCPS) [Hantaviruses (family e (HCPS) . While He (HCPS) -4. In NoPatients afflicted with HCPS exhibit pronounced pulmonary inflammation due to capillary leak syndrome, with the consequent hypotension often leading to rapid decline and death . Virus iPeromyscus maniculatus) are the principal reservoir host of SNV [Deer mice  [Recent advances in hematopoietic stem cell research have identified an important role for granulocyte-macrophage colony stimulating factor (GM-CSF) in the expansion and maturation of bone marrow cells into competent APC -40. We pusculus) . This leusculus) , it is pWe used RACE to obtain the complete 5' end of GM-CSF. This sequence was translated using the default translation table within MacVector. The polypeptide is predicted to have a 25 residue signal peptide based upon orthologous sequences from other species -45 Figu. The recDeer mouse bone marrow cultures contained mostly cells that appeared dead or dying after 24 hours in culture with GM-CSF. However, at 48 hours clusters of cells were apparent, while control wells without GM-CSF had fewer live cells than at 24 hours. By day 3, adherent stromal cell foci were conspicuous, while semiadherent and nonadherent cells were more evident and these became the prominent cells for the duration of culture. Day 12 bone marrow cells incubated for an additional 48 hours were large, ranging from 12 to 18 μm in diameter, and possessed macropinocytic vesicles and processes Figure . AlthougDay 8 deer mouse bone marrow cells were cultured with various concentrations of house mouse GM-CSF. Two days later, proliferation was assessed Figure  and maxiBM-APC and T cells were examined for the expression of orthologous I-Eβ and TCRβC, respectively, by RT-PCR Figure . For I-EDeer mice were immunized with keyhole limpet hemacyanin (KLH), and 10 days later the lymph nodes, spleens and bone marrow were processed for in vitro expansion of polyclonal T cells (lymph node cells), while the bone marrow cells and splenocytes were frozen. Sera were tested for antibodies to KLH by ELISA and in each deer mouse tested the titer was greater than or equal to 8,000 (data not shown). For recall proliferation, KLH, in vitro-propagated T cells (14 days with IL-2) and BM-APC (14 days with GM-CSF) or freshly thawed splenocytes were cultured together for 72 hours, and proliferation was assessed by MTS assay Figure . For eacDeer mouse antiserum raised against KLH and incubated with antigen for one hour prior to addition of cells increased the sensitivity of T cell proliferation Figure , suggestTo our knowledge, no previous efforts have been made to develop long-term cultures of T cells from unconventional laboratory rodents. The principal reason for this is that highly inbred strains, required for conventional long-term T cell work, are not available from rodents not routinely used in laboratory work. At least for deer mice, we have developed a method of fulfilling this need by using commercially-available house mouse GM-CSF. This cytokine apparently binds to the GM-CSF receptor on deer mouse cells such that it generates competent APC from the bone marrow. These cells are capable of processing and presenting soluble antigen to autologous antigen-specific helper T cells.Our initial suspicions that house mouse GM-CSF might bind to deer mouse GM-CSF receptor was the result of previous work  in whichWe used a method that has been shown to generate dendritic cells in house mice; however, the cells obtained from deer mouse bone marrow more closely resembled macrophages rather than DC. These cells contained many large macropinocytic vesicles, but conspicuous dendrites typical of DC were not observed. Microscopically, these cells also appeared sensitive to TNF, which decreased macropinocytosis, but it had no effect on the capacity of these cells to present antigen to T cells as has been reported for human DC derived from blood mononuclear cells . TNF can7 bone marrow cells from a deer mouse, which is sufficient for freezing 5 vials at 2 × 106 cells each. Each vial is used to seed a 100 mm bacterial Petri dish, which produces about 107 BM-APC at 14 days of culture. For deer mice, the most significant limitation for cells is from the spleen. Although deer mice are slightly smaller than BALB/c mice, their spleens are disproportionately small (unpublished observations). We routinely recover 7 × 106 splenocytes from a deer mouse, while BALB/c house mice usually provide 10-fold more. Because of this limitation, we have begun to use BM-APC to propagate T cells. This method involves culturing of bone marrow cells with GM-CSF for 10 days, then freezing aliquots of 106 cells. Three days before T cell restimulation, the 10-day BM-APC are thawed and cultured with GM-CSF, then used for restimulation with fresh antigen in one well of a 24-well plate. The T cells are fed fresh IL-2 DMM-5 at two-day intervals for expansion.Since the deer mice are outbred, this method requires the immunization and collection of cells from individual animals Figure . These cWe have used this method to establish nine T cell lines, six specific for KLH and three specific for SNV nucleocapsid antigen (data not shown). Based upon typical cell yields, it should be possible to assay several thousand wells on 96-well plates, which we estimate to be sufficient for many T cell activities, including cloning, peptide epitope mapping, TCR variable gene segment usage, and cytokine profiling.We believe the methods described in this work will allow the characterization of antigen presentation and T cell responses in infected deer mice. Many viruses impair pathways involved in APC and T cell functions so that they can evade a sterilizing immune response. With hantaviruses and their rodent hosts, millions of years of evolution have presumably allowed a coadaptation of the viruses and host immune responses such that pathology does not occur and the virus is not eliminated. It is possible that hantaviruses possess some as yet unidentified mechanism for suppressing an aggressive inflammatory immune response in rodent hosts, which is ineffective in human infections and often leads to inflammatory immunopathology.Because of the substantial evolutionary divergence of deer mice and house mice (about 25–50 million years) , it is lA limitation of this approach is that BM-APC lose their ability to proliferate in the presence of GM-CSF after four to six weeks, similar to what has been observed with house mouse bone marrow-derived cells ,54. SincWe have developed a method for generating large numbers of competent antigen presenting cells from deer mouse bone marrow using house mouse GM-CSF. This method resulted in the production of antigen-specific T cell lines from outbred deer mice. Inclusion of antigen-specific antibody in cultures augments T cell proliferation, suggesting the APC express Fc receptors. This method will allow characterization of APC and T cells in deer mice and may be extended to other rodent species that are important in infectious disease research.The deer mice used in these experiments were from a colony of animals established with deer mice trapped in western Colorado . All proSigmodon hispidus, AAL55394) and human (NP_000749) were aligned using MacVector's (Accelrys, San Diego, CA) clustal algorithm.The 5' end of the deer mouse GM-CSF cDNA was obtained by using RACE. Briefly, a primer Table  was desiTotal RNA from activated splenocytes was reverse-transcribed using an oligo-dT primer and Superscript II (Invitrogen, Carlsbad, CA). TCRβC cDNA sequences from house mouse, rat and human were aligned with MacVector. PCR primers Table  were desDeer mice were bilaterally immunized subcutaneously at the base of the tail with 20 μg of KLH  emulsified in CFA (Sigma). Ten days later, draining lymph nodes, spleens and bone marrow were recovered for in vitro experiments. For production of high-titer KLH antiserum, deer mice were immunized i.p. with 20 μg of KLH emulsified in CFA and boosted with 20 μg in IFA one month later. Sera were collected 7 days after boosting.6 cells per vial in 1 ml of 10% DMSO/DMM-5 and stored at -70°C.Immunized deer mice were euthanized by cervical dislocation and the draining lymph nodes, spleens and bone marrow from individual animals were separately collected in Hank's balanced salt solution for processing. The lymph nodes served as a source of antigen-specific T cells, while the splenocytes were treated with ammonium chloride  to lyse RBCs, then frozen in 10% DMSO/5% FBS deer mouse medium (DMM-5: 5% FBS, RPMI-1640 supplemented to 315 mOsm [with 2.5 ml of 5 M NaCl/L], 2.5 μg/ml Fungizone, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamicin, 50 μM β-ME, 10 mM HEPES, 2 mM L-glutamine) in aliquots for use as autologous APC for additional rounds of in vitro T cell stimulation. The bone marrow cells were collected from tibiae and femurs, washed twice in DMM-5, and aliquotted at 2 × 106) was quick-thawed in a 37°C water bath and cultured without washing in 100 mm bacterial Petri dishes in DMM-10 containing 20 ng/ml recombinant house mouse GM-CSF (R&D Systems, Minneapolis, MN) at 37°C under 7% CO2. Fresh GM-CSF/DMM-10 was provided on days 3, 6, 8, 10, and 12 for the generation of APC. Cells were collected with a scraper for use in experiments. Cells were processed by cytospin and stained with Wright's stain for morphological examination.Bone marrow-derived APC (BM-APC) were generated with modification of a previously described method for dendritic cells (DC) . One viaDeer mouse splenocytes depleted of RBC by ammonium chloride treatment were incubated with a suboptimal dose of PHA (2 μg/ml) (Sigma) in DMM-5 and recombinant human IL-2 (R&D Systems) for 48 hours. Proliferation was determined by MTS assay (Cell Titer-96 AQ, Promega). The means and standard deviations of duplicate samples were calculated, with the mean of cells without IL-2 subtracted from sample means.Total RNA was extracted from 14 day T cell and BM-APC cultures (Versagene RNA, Gentra Systems, Minneapolis, MN) and converted into cDNA. Class II expression of the bone marrow cells was assessed by PCR using a forward primer from exon 2 and a reverse primer that overlaps the boundaries of exons 2 and 3 of deer mouse I-Eβ (Table P. leucopus IgG (H&L) (KPL, Gaithersburg, MD) for 1 hour, then horse anti-goat IgG-HRP conjugate for 1 hour (Vector, Burlingame, CA). ABTS substrate (Sigma) was incubated for 15 min, and plates were read at 414 nm. Means were calculated with the background  subtracted.Sera were collected at euthanasia by cardiac puncture. Plates were coated with 5 μg/ml KLH in PBS overnight and washed 5× with wash buffer (PBS-0.1% TWEEN-20). Plates were then blocked with blocking buffer (5% nonfat powdered milk in wash buffer) for 1 hour at room temperature. The sera and remaining reagents were diluted in blocking buffer. Sera were incubated in duplicate for 2 hours at room temperature, followed by goat anti-6 cells per well (24 well plate) with 20 μg/ml KLH in DMM-5. After a 4 day incubation, the lymph node cells were collected and washed twice in DMM-5. The number of recovered lymph node cells varied between animals, but between 2 × 105 and 5 × 105 cells were recovered and plated with fresh antigen and 3 × 106 thawed autologous splenocytes in DMM-5 in 24 well plates in 1 ml of DMM-5. At 2 day intervals, cultures were fed by removing 750 μl of media and replacing it with DMM-5 containing 20 U/ml of recombinant human IL-2. When cultures were greater than 80% confluent, cells were passaged 1:2 into additional wells or into T25 tissue culture flasks. This process was continued for 14 days to expand T cells. Cultures were also restimulated at two week intervals with fresh mitomycin-C treated autologous splenocytes as above, or 2 × 106 BM-APC to continue propagation of T cell lines.In vitro stimulation of helper T cells was performed essentially as described elsewhere ,57. Lymp4) or splenocytes (2 × 105) were used to stimulate 105 T cells in the presence of KLH in 96-well plates. In other experiments, the effects of house mouse tumor necrosis factor (20 ng/ml) were evaluated on APC morphology and capacity to stimulate T cell proliferation. Lastly, antisera to KLH were produced in deer mice and used to assess the capacity of the BM-APC to capture and process antigen for presentation to T cells. In these experiments, antiserum or normal deer mouse serum were diluted to 1:2,000, a saturating dilution in ELISA as described above, with KLH and incubated for 1 hour at room temperature. BM-APC and T cells were then added and the cultures incubated for 72 hours prior to determining proliferative responses by MTS. Means and standard deviations were calculated from duplicate samples.BM-APC were examined for functional capacity to process and present KLH to sensitized autologous deer mouse T cells expanded in culture. For these experiments, mitomycin-C treated BM-APC -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumBD conducted bone marrow cell culture work. DGW and TAC performed RT-PCR experiments. JP cloned and sequenced the TCRβ cDNA. RMF cloned and sequenced the MHC class II cDNA. TS immunized deer mice and generated T cell lines.


Esophagectomy is considered the gold standard for the treatment of high-grade dysplasia in Barrett's esophagus (BE) and for noninvasive adenocarcinoma (ACA) of the distal esophagus. If all of the metaplastic epithelium is removed, the patient is considered "cured". Despite this, BE has been reported in patients who have previously undergone esophagectomy. It is often debated whether this is "new" BE or the result of an esophagectomy that did not include a sufficiently proximal margin. Our aim was to determine if BE recurred in esophagectomy patients where the entire segment of BE had been removed.Records were searched for patients who had undergone esophagectomy for cure at our institution. Records were reviewed for surgical, endoscopic, and histopathologic findings. The patients in whom we have endoscopic follow-up are the subjects of this report.Since 1995, 45 patients have undergone esophagectomy for cure for Barrett's dysplasia or localized ACA. Thirty-six of these 45 patients underwent endoscopy after surgery including 8/45 patients (18%) with recurrent Barrett's metaplasia or neoplasia after curative resection.Recurrent Barrett's esophagus or adenocarcinoma after esophagectomy was common in our patients who underwent at least one endoscopy after surgery. This appears to represent the development of metachronous disease after complete resection of esophageal disease. Half of these patients have required subsequent treatment thus far, either repeat surgery or photodynamic therapy. These results support the use of endoscopic surveillance in patients who have undergone "curative" esophagectomy for Barrett's dysplasia or localized cancer. The incidence of esophageal adenocarcinoma has increased more rapidly than any other form of cancer since the 1970s and now represents the majority of esophageal neoplasms in the West . Other rDetection of dysplastic Barrett's esophagus or mucosal adenocarcinoma is important because it allows the opportunity to intervene prior to the development of invasive neoplasia. Unfortunately, no medical or surgical GERD treatment has been consistently and convincingly demonstrated to prevent the development of adenocarcinoma . TraditiAfter approval by the Mayo Foundation's Institutional Review Board for Research, the electronic medical records of Mayo Clinic patients in Jacksonville, Florida, were searched to find all patients who had undergone esophagectomy for cure at the Mayo Clinic surgical facility, St. Luke's Hospital, Jacksonville, Florida, since 1995. This time period was chosen to coincide with the routine availability and clinical use of pre-operative staging with endosonography in our institution. The records of these patients were reviewed for pre-operative and post-operative staging results including computed tomography and endosonography studies. In addition, endoscopic, surgical studies and histopathological studies were studied. Specifically, the surgical specimens were reviewed to ensure that the esophagectomy specimen, including lymph node sampling, was adequate and the proximal margin was completely free of Barrett's metaplasia, dysplasia or carcinoma. The patients, in whom we have at least one follow-up endoscopy study, with biopsies obtained for histologic confirmation of mucosal disease, are the subjects of this report. Esophageal disease was staged according to the Tumor-Lymph node-Metastasis (TNM) criteria .Since 1995, 45 patients have undergone esophagectomy for Barrett's dysplasia or localized adenocarcinoma with curative intent in our institution. At operation, none of these patients were found to have extension of malignant disease to paraesophageal lymph nodes and all esophageal glandular mucosa was resected with only normal squamous mucosa remaining at the proximal surgical margin. Subsequently, 36 of these patients (80%) have undergone endoscopy after surgery including 8/45 patients (18%) who were found to have recurrent Barrett's glandular mucosa after curative resection and are described in the table.Open transthoracic esophagectomy (Ivor Lewis procedure) with pyloroplasty was performed in most patients (39/45) including the patients diagnosed with recurrent Barrett's disease. Five different surgeons performed these operations. Most patients had evidence of gastric stasis (retained food) at endoscopy . Anastomotic dilation was performed at endoscopy in 16/36 patients . It is possible that patients with anastomotic strictures may be at increased risk of recurrent Barrett's esophagus because of worse reflux although their swallowing symptoms may, alternatively, be related to other factors such as anastomotic ischemia or surgical sutures. Patients frequently used aspirin (42%) or COX-2 specific non-steroidal anti-inflammatory agents (25%). Twice-daily proton pump inhibitors were routinely prescribed for these patients although patient compliance is difficult to assess because of high drug costs and limited symptomatic improvement. While the small number of patients limits our analysis, these factors were found to occur in a proportional number of patients with Barrett's disease and no clear trends could be identified.2N0M0 in a 78-year-old man. This metaplastic glandular epithelium was detected at a follow up of 90 months and 17 months, respectively. In the other 3 patients, short segment Barrett's low-grade dysplasia has been found in lengths between 10–25 mm after complete resection of the esophagus for Barrett's high-grade dysplasia (1 patient) and Barrett's T3N0M0 carcinoma (2 patients). This recurrent Barrett's glandular dysplasia was detected at a follow up of 42–47 months. Erosive esophagitis was also noted in 4 of 5 patients indicating uncontrolled reflux disease. Subsequently all patients have been treated with high doses of proton pump inhibitors (such as esomeprazole 80 mg twice a day or 40 mg three or four times per day) in an attempt to maximally control reflux of acid and digestive juices from the stomach into the cervical esophagus.Five of these patients have been diagnosed with Barrett's metaplasia or low-grade dysplasia have been followed for more than 12 months in surveillance endoscopy programs monitoring the stability of the glandular epithelium. Two of these 5 patients have been found to have short segment Barrett's metaplasia with lengths of 10 mm and 15 mm, after complete esophageal resection for Barrett's high-grade dysplasia in a 72-year-old man and Barrett's adenocarcinoma T2 or T3N0M0 adenocarcinoma. These patients varied in age from 58–80 years of age. These patients were found to have more severe erosive esophagitis suggesting worse acid reflux and mucosal injury compared to the non-carcinoma recurrent Barrett's patients. Recurrent Barrett's multi-focal high-grade dysplasia, over a 10 mm segment length was detected 88 months after esophagectomy in one patient and was successfully ablated with porfimer sodium photodynamic therapy using the methods described elsewhere [1N0M0 was confirmed at repeat esophagectomy. Finally, a diminutive polypoid mass proximal to the surgical anastomosis was found in a 58-year-old woman who had 7 months previously undergone esophagectomy for Barrett's mucosal adenocarcinoma. Computed tomography with contrast enhancement noted esophageal wall thickening and suspicious lymphadenopathy. Repeat resection confirmed the tumor histologic grade of T2N1M0 adenocarcinoma.Three other patients developed recurrent Barrett's disease after curative resection of esophageal Tlsewhere . High-grOver the past four decades, the incidence of esophageal adenocarcinoma has risen dramatically, particularly in older white men . PreviouAfter undergoing esophageal resection, the native squamous mucosa of the cervical esophagus will be brought into contact with the acid-secreting mucosa of the gastric body. This reconstruction allows acid and duodenal juice to reflux from the gastric conduit to the remaining cervical esophagus. Reflux of gastric and duodenal content is an important factor in the pathogenesis of Barrett's metaplasia, dysplasia and esophageal adenocarcinoma -20. It iIn the study of Oberg et al, despite the use of potent acid-suppressing medications, severe esophageal acid exposure was noted in most patients. Patients with recurrent Barrett's epithelium were found to have significantly more severe acid exposure that occurred predominantly in the supine position . There wis or T1) were detected at surveillance endoscopy and were treated with endoscopic laser ablation, mucosal resection or surgical resection. While squamous cell carcinomas are not related to gastroesophageal reflux, this paper also suggests that esophageal cancer patients (squamous or adenocarcinoma) are predisposed to the development of metachronous carcinomas in the remnant cervical esophagus. This is consistent with DeMeester's experimental model of Barrett's dysplasia and adenocarcinoma occurring after complete gastrectomy with esophago-jejunostomy and reflux of bile and digestive enzymes into the cervical esophagus [Murata and colleagues recently reported the diagnosis of metachronous squamous cell carcinomas in five of 253 patients (2%) who had undergone esophagectomy for thoracic esophageal squamous cell carcinoma more than two years previously. These superficial carcinomas (Tsophagus . Also, iKonishi et al reported finding an adenocarcinoma in Barrett's esophagus following a total resection of the gastric remnant in a 52-year-old man who had undergone distal gastrectomy for gastric cancer nearly twenty years previously . The BarThese studies have demonstrated that the cervical esophagus is exposed to high amounts of acid and refluxate despite the use of proton inhibitor medications and often in the absence of severe reflux symptoms. Although our group of patients has been observed for only a median of 2 years after esophagectomy, our study confirms that the development of metaplastic columnar mucosa in the cervical esophagus is a common complication related to reflux associated injury to the squamous epithelium. Further, our findings suggest that this recurrent glandular mucosa is unstable and predisposed to the development of dysplasia and invasive carcinoma, as has already developed in most of patients.The early detection of this recurrent disease remains vitally important to preserve all possible treatment options including surveillance endoscopy follow-up, endoscopic ablation with porfimer sodium photodynamic therapy, and if necessary repeat esophagus resection surgery. Our specific recommendations include surveillance endoscopy every 6–12 months for patients who have undergone "curative" esophagectomy for Barrett's dysplasia or adenocarcinoma. In addition, we also routinely recommend indefinite use of proton pump inhibitors, regardless of symptom status, starting at twice daily dosing and increasing as necessary to control reflux symptoms and mucosal damage due to acid, bile and digestive enzymes. Whether these drug doses should be titrated based on ambulatory pH and impedance test results remains to be determined. We have generally been disappointed by prokinetic agents such as metoclopromide in improving reflux symptoms in these patients. For esophagectomy patients who develop recurrent Barrett's metaplasia we recommend the use of COX-2 inhibitors or aspirin chemoprevention to protect against the development of metachronous Barrett's carcinoma. ,30.None declared.All authors participated in the study design and coordination as well as case collection and review of histopathologic and endoscopic results. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: