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Aim: To analyze the hypothesis of events or neighborhood interactions that is based upon recognizable structures of systems which possess a surface in a four dimensional space-time constellation {x, y, z, t}. To include the theory of hierarchic order of structures and aspects of thermodynamically open systems, especially entropy, structural entropy and entropy flow., Hypothesis: Any structure is a space-time constellation that occupies a unique space in its environment. The environment can be a system too, and is assumed to be (nearly) constant. Structures can interact in their environment and create a new structure at a higher order level. Interacting structures that create a surface are called a system. Starting from the bottom, such a system is characterized by its inner structures, its surface function, and its neighborhood. Interaction with a neighboring system is called an event. An event can alter a system, create new systems or induce the decay of a system, dependent upon the surrounding lower level system (background)., Results: The hypothesis results in a uniform theory about matter, life, diseases, or behavior. Concrete applications permit the estimation of duration of life in man, for example the effect of solid cancer in man, or appearance of protozoans in sexual or asexual reduplication. In addition, it can successfully describe the development of the universe (small exceed of matter above antimatter at the big bang), or the increase of structures (and systems) with increasing time (development of intelligent systems). The three dimensional space possesses the lowest number of mandatory dimensions to implement such a system.

Pathology is a bridging discipline that involves both basic and clinical biomedical sciences. In this context, it includes both descriptive and mechanistic approaches, with the final goals of further understanding the anatomic and functional changes and underlying molecular events involved in disease-related processes. Pathology studies mainly comprise macroscopic and microscopic examinations, and involve the visual recognition of different patterns in cells and tissues. In time elapsed to this end, it has adopted a dazzling array of methods and techniques from the most varied fields of natural and engineering sciences in order to optimize tissue analysis, which will be summarized in this article.

The technological progress in digitalization of complete histological glass slides has opened a new door in tissue--based diagnosis. The presentation of microscopic images as a whole in a digital matrix is called virtual slide. A virtual slide allows calculation and related presentation of image information that otherwise can only be seen by individual human performance. The digital world permits attachments of several (if not all) fields of view and the contemporary visualization on a screen. The presentation of all microscopic magnifications is possible if the basic pixel resolution is less than 0.25 microns. To introduce digital tissue--based diagnosis into the daily routine work of a surgical pathologist requires a new setup of workflow arrangement and procedures. The quality of digitized images is sufficient for diagnostic purposes; however, the time needed for viewing virtual slides exceeds that of viewing original glass slides by far. The reason lies in a slower and more difficult sampling procedure, which is the selection of information containing fields of view. By application of artificial intelligence, tissue--based diagnosis in routine work can be managed automatically in steps as follows: 1. The individual image quality has to be measured, and corrected, if necessary. 2. A diagnostic algorithm has to be applied. An algorithm has be developed, that includes both object based (object features, structures) and pixel based (texture) measures. 3. These measures serve for diagnosis classification and feedback to order additional information, for example in virtual immunohistochemical slides. 4. The measures can serve for automated image classification and detection of relevant image information by themselves without any labeling. 5. The pathologists' duty will not be released by such a system; to the contrary, it will manage and supervise the system, i.e., just working at a "higher level". Virtual slides are already in use for teaching and continuous education in anatomy and pathology. First attempts to introduce them into routine work have been reported. Application of AI has been established by automated immunohistochemical measurement systems (EAMUS, www.diagnomX.eu). The performance of automated diagnosis has been reported for a broad variety of organs at sensitivity and specificity levels >85%). The implementation of a complete connected AI supported system is in its childhood. Application of AI in digital tissue--based diagnosis will allow the pathologists to work as supervisors and no longer as primary "water carriers". Its accurate use will give them the time needed to concentrating on difficult cases for the benefit of their patients.

Abstract Background Bacterial sepsis induced immunsuppression via antigen hyporesponsibility increases the risk of nosokomial infections and mortality. Pattern recognition receptors (PRR) might have a central role in the pathophysiology of hyporesponsibility. Methods In this study we evaluated in a human E. coli sepsis cohort, the role of PRR including TLR’s and Trem-1. Expression of Trem-1, TLR2, TLR4, CD14 and HLA-DR on blood monozytes and neutrophils were examined using flow cytometry from 22 patients with E. coli sepsis and 6 healthy controls. LPS and LTA stimulated TNF alpha, IL-10, IL-8 and IL-6 production was studied in a 24 h whole blood assay. Free cytokine serum concentration of TNF alpha, PCT and IP-10 were evaluated. Results We found a significant higher expression of Trem-1 and TLR-2 on monocytes and neutrophils in patients compared to healthy volunteers. TLR2 expression (p Conclusions Patients with E. coli sepsis are characterized by an association of Trem-1 expression on blood neutrophils with cytokine inducibility. The TREM-1 pathway on neutrophils might play a role in producing an adequate inflammatory and bactericidal response in bacterial sepsis. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4441869398748313

Abstract Since the existence of pHp was demonstrated, it has been shown that this molecule and its receptor CD163 are regulated by different stimuli. Furthermore, a comparably fast secretion of pHp was described as well as the immuno-stimulatory effects. The intention of this study was to elucidate the role of pHp in the human lungs further. Here we show, by means of confocal microscopy and immune-electron-microscopy, a clear co-localization of pHp with Surfactant protein-B in lamellar bodies of Alveolar Epithelial Cells Type II. These results are underlined by immunohistochemical stainings in differently fixed human lung tissues, which show pHp in vesicular and released form. The images of the released form resemble the intended position of surfactant in the human alveolus. pHp is secreted by Alveolar epithelial cells type II as previously shown. Moreover, pHp is co-localized with Surfactant protein-B. We conclude that the presented data shows that pHp is a native part of the surfactant system in the human lung. Virtual slides http://www.diagnosticpathology.diagnomx.eu/vs/2563584738239912.

Abstract Background Application of virtual slides (VS), the digitalization of complete glass slides, is in its infancy to be implemented in routine diagnostic surgical pathology and to issues that are related to tissue-based diagnosis, such as education and scientific publication. Approach Electronic publication in Pathology offers new features of scientific communication in pathology that cannot be obtained by conventional paper based journals. Most of these features are based upon completely open or partly directed interaction between the reader and the system that distributes the article. One of these interactions can be applied to microscopic images allowing the reader to navigate and magnify the presented images. VS and interactive Virtual Microscopy (VM) are a tool to increase the scientific value of microscopic images. Technology and Performance The open access journal Diagnostic Pathology http://www.diagnosticpathology.org has existed for about five years. It is a peer reviewed journal that publishes all types of scientific contributions, including original scientific work, case reports and review articles. In addition to digitized still images the authors of appropriate articles are requested to submit the underlying glass slides to an institution (DiagnomX.eu, and Leica.com) for digitalization and documentation. The images are stored in a separate image data bank which is adequately linked to the article. The normal review process is not involved. Both processes (peer review and VS acquisition) are performed contemporaneously in order to minimize a potential publication delay. VS are not provided with a DOI index (digital object identifier). The first articles that include VS were published in March 2011. Results and Perspectives Several logistic constraints had to be overcome until the first articles including VS could be published. Step by step an automated acquisition and distribution system had to be implemented to the corresponding article. The acceptance of VS by the reader is high as well as by the authors. Of specific value are the increased confidence to and reputation of authors as well as the presented information to the reader. Additional associated functions such as access to author-owned related image collections, reader-controlled automated image measurements and image transformations are in preparation. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1232133347629819.

Abstract Aim To analyze the hypothesis of events or neighborhood interactions that is based upon recognizable structures of systems which possess a surface in a four dimensional space - time constellation {x, y, z, t}. To include the theory of hierarchic order of structures and aspects of thermodynamically open systems, especially entropy, structural entropy and entropy flow. Hypothesis Any structure is a space - time constellation that occupies a unique space in its environment. The environment can be a system too, and is assumed to be (nearly) constant. Structures can interact in their environment and create a new structure at a higher order level. Interacting structures that create a surface are called a system. Starting from the bottom, such a system is characterized by its inner structures, its surface function, and its neighborhood. Interaction with a neighboring system is called an event. An event can alter a system, create new systems or induce the decay of a system, dependent upon the surrounding lower level system (background). Results The hypothesis results in a uniform theory about matter, life, diseases, or behavior. Concrete applications permit the estimation of duration of life in man, for example the effect of solid cancer in man, or appearance of protozoans in sexual or asexual reduplication. In addition, it can successfully describe the development of the universe (small exceed of matter above antimatter at the big bang), or the increase of structures (and systems) with increasing time (development of intelligent systems). The three dimensional space possesses the lowest number of mandatory dimensions to implement such a system.

Abstract Background As there is no optimal treatment of non small cell lung cancer due to its resistance to common chemotherapeutics, we investigated the effect of human placenta-conditioned medium on tumor tissue. The human placenta constitutes a mixture of maternal and fetal origin and displays a variety of immunomodulatory aspects. Methods Freshly resected non small cell lung cancer tissues were incubated with placenta-conditioned medium in a short-term tissue culture model and A549 cells were challenged, respectively. Term placenta was used for producing conditioned medium and HOPE-fixed stimulated tumor tissue was analyzed for expression of caspase-3 and Ki67 via immunohistochemistry. The effects of conditioned medium on squamous cell carcinoma were further compared to physiological concentrations of Carboplat/Gemzar. Results Conditioned medium caused in 2 of 3 cases elevated expression of caspase-3 and reduced expression of Ki67 in 3 out of 3 cases, while the chemotherapeutic agents caused no comparable expression of caspase-3 or reduction of Ki67. In cell culture up to 50% of karyopyknosis was investigated and even sterile-filtrated medium caused widespread reduction of Ki67 on protein level. Conclusion Human placenta releases substances that mediate apoptosis and reduce proliferation in tumor tissue and cell culture. As even sterile-filtrated medium caused the mentioned effects we hypothesize one or more soluble mediators. The detailed way of promoting apoptosis and nature of these mediators need to be elucidated in further studies.

Abstract Pattern recognition receptors are a key component of the first line host defense against infection, recognizing specific microbial products. We hypothesize that monocyte hyporesponsiveness in human sepsis is associated with a downregulation of the pattern recognition receptors Toll-like receptor (TLR)-2 and TLR4. Protein expression of CD14, TLR2 and TLR4 on blood monocytes was examined using flow cytometry from 29 patients with sepsis and 14 healthy controls. In addition LPS stimulated TNF-α and IL-10 production was studied in a 24 hour whole blood assay. We found an increased expression of CD14, TLR2 and TLR4 in patients with sepsis compared to controls (p < 0.01). In patients with sepsis, death was associated with significant lower CD14 and TLR2 expression at admission (CD14: 25.7 +- 19.1 vs 39.1 +- 17.3 mean fluorescence intensity [MFI], p = 0.02; TLR2: 21.8 +- 9.4 vs. 30.9 +- 9.6, p = 0.01). At 72 hours the TLR2 expression on monocytes was associated with the IL-10 inducibility after LPS stimulation (r = 0.52, p = 0.02) and the CD14 expression with the IL-6, IL-10 and TNF inducibility. We conclude that septic patients are characterized by an increased expression of CD14, TLR2 and TLR4 on monocytes compared to controls. Death is associated with downregulation of TLR2 and CD14 expression on monocytes correlating with reduced cytokine inducibility. We suggest that CD14 and TLR2 are a key factor in monocyte hyporesponsibility during severe sepsis.

Abstract Surfactant Protein-A (SP-A) is the most prominent among four proteins in the pulmonary surfactant-system. SP-A is expressed by alveolar epithelial cells type II as well as by a portion of non small cell lung carcinomas (NSCLC). The expression of SP-A is complexly regulated on the transcriptional and the chromosomal level. SP-A is a major player in the pulmonary cytokine-network and moreover has been described to act in the pulmonary host defense. By the use of cell culture or animal models the functional properties have been repeatedly shown in many aspects, often bearing surprising properties which strongly indicate the physiological importance of SP-A. To date SP-A is recognized as a molecule essential for pulmonary development, structure and function. An upcoming number of reports deals with the role of SP-A for pulmonary pathology. This article gives an overview about the state of knowledge on SP-A focused in applications for human pulmonary disorders and points out the importance for pathology-orientated research approaches using immunohistochemistry or in situ hybridization as promising methods to further elucidate the role of this molecule in adult lung diseases.

Abstract Background A general theory of sampling and its application in tissue based diagnosis is presented. Sampling is defined as extraction of information from certain limited spaces and its transformation into a statement or measure that is valid for the entire (reference) space. The procedure should be reproducible in time and space, i.e. give the same results when applied under similar circumstances. Sampling includes two different aspects, the procedure of sample selection and the efficiency of its performance. The practical performance of sample selection focuses on search for localization of specific compartments within the basic space, and search for presence of specific compartments. Methods When a sampling procedure is applied in diagnostic processes two different procedures can be distinguished: I) the evaluation of a diagnostic significance of a certain object, which is the probability that the object can be grouped into a certain diagnosis, and II) the probability to detect these basic units. Sampling can be performed without or with external knowledge, such as size of searched objects, neighbourhood conditions, spatial distribution of objects, etc. If the sample size is much larger than the object size, the application of a translation invariant transformation results in Kriege's formula, which is widely used in search for ores. Usually, sampling is performed in a series of area (space) selections of identical size. The size can be defined in relation to the reference space or according to interspatial relationship. The first method is called random sampling, the second stratified sampling. Results Random sampling does not require knowledge about the reference space, and is used to estimate the number and size of objects. Estimated features include area (volume) fraction, numerical, boundary and surface densities. Stratified sampling requires the knowledge of objects (and their features) and evaluates spatial features in relation to the detected objects (for example grey value distribution around an object). It serves also for the definition of parameters of the probability function in so – called active segmentation. Conclusion The method is useful in standardization of images derived from immunohistochemically stained slides, and implemented in the EAMUS™ system http://www.diagnomX.de. It can also be applied for the search of "objects possessing an amplification function", i.e. a rare event with "steering function". A formula to calculate the efficiency and potential error rate of the described sampling procedures is given.

Abstract Light emitting diodes (LED), which are available as small monochromatic light sources with characteristic features such as maximum illumination power combined with minimum energy consumption and extremely long lifespan have already proved as a highly potential low-cost alternative for specific diagnostic applications in clinical medicine such as tuberculosis fluorescence microscopy. Likewise, the most reliable evaluation of Her-2/neu (c-erbB2) gene amplification, which has been established in the last few years for routine diagnosis in clinical pathology as determinant towards Herceptin-based treatment of patients with breast cancer, is based on fluorescence in situ hybridization (FISH) and corresponding high priced fluorescence equipment. In order to test the possibility to utilize the advantages of low-cost LED technology on FISH analysis of c-erbB2 gene expression for routine diagnostic purposes, the applicability of a standard bright field Carl Zeiss Axiostar Plus microscope equipped with a Fraen AFTER* LED Fluorescence Microscope Kit for the detection of Her-2/neu gene signals was compared to an advanced Nikon Eclipse 80i fluorescence microscope in combination with a conventional 100W mercury vapor lamp. Both microscopes were fitted with the same Quicam FAST CCD digital camera to unequivocally compare the quality of the captured images. C-erbB2 gene expression was analyzed in 30 different human tissue samples of primary invasive breast cancer, following formalin fixation and subsequent paraffin-embedding. The Her2/neu gene signals (green) were identifiable in the tumor cells in all cases and images of equal quality were captured under almost identical conditions by 480 nm (blue) LED module equipped standard Axiostar microscope as compared to conventional fluorescence microscopy. In this first attempt, these monochromatic LED elements proved in principle to be suitable for the detection of Her-2/neu gene expression by FISH. Thus, our own experiences emphasize the high potential of this technology to provide a serious alternative to conventional fluorescence microscopy in routine pathology; representing a sustainable technological progress, this low-cost technology will clearly give direction also to the growing field of molecular pathology. * AFTER = Amplified Fluorescence by transmitted Excitation of Radiation

Abstract In several tumors the transketolase activity, controlled inter alia by enzymes of the pentose phosphate pathway which is an alternative, energy generating reaction-cascade to glycolysis, has been correlated with proliferation. The increase of thiamine-dependant transketolase enzyme reactions is induced especially through upregulated transketolase-like enzyme 1 (TKTL1)-activity; that shows TKTL1 to be a causative enzyme for tumors enhanced, anaerobic glucose degradation. We investigated TKTL1-expression in 88 human, formalin-fixed non-small cell lung cancer tissues and 24 carcinomas of the breast by immunohistochemical stainings applying a 0 to 3 staining-score system (3 = strongest expression). For means of validation we additionally stained 40 NSCLC fixed and paraffin-embedded utilizing the HOPE-technique; showing comparable results to the formalin-fixed, paraffin-embedded specimens (not shown). Potential correlations with age, sex, TNM-classification parameters and tumor grading as well as tumor transcription factor 1 (TTF1) and surfactant protein A (SPA) expression were investigated. 40.9% of the analyzed lung tumors expressed TKTL1 weakly (Score 1), 38.6% moderately (score 2) and 17.1% strongly (score 3). 3 tumors were diagnosed TKTL1-negative (3.4%; score 0). All Breast cancer specimen stainings were positive and scored 1: 32%; scored 2: 36%; scored 3: 32%. Alveolar macrophages and Alveolar Epithelial Cells Type II were also found to be TKTL1-positive. None of the listed clinical parameters could be found to show a significant correlation to TKTL1 signal appearance. Although we describe the expression of TKTL1 in lung cancers, we need to state that up till now there is no scientific indication for any treatment regimens based upon these findings.

Abstract Background Progress in automated image analysis, virtual microscopy, hospital information systems, and interdisciplinary data exchange require image standards to be applied in tissue-based diagnosis. Aims To describe the theoretical background, practical experiences and comparable solutions in other medical fields to promote image standards applicable for diagnostic pathology. Theory and experiences Images used in tissue-based diagnosis present with pathology – specific characteristics. It seems appropriate to discuss their characteristics and potential standardization in relation to the levels of hierarchy in which they appear. All levels can be divided into legal, medical, and technological properties. Standards applied to the first level include regulations or aims to be fulfilled. In legal properties, they have to regulate features of privacy, image documentation, transmission, and presentation; in medical properties, features of disease – image combination, human – diagnostics, automated information extraction, archive retrieval and access; and in technological properties features of image acquisition, display, formats, transfer speed, safety, and system dynamics. The next lower second level has to implement the prescriptions of the upper one, i.e. describe how they are implemented. Legal aspects should demand secure encryption for privacy of all patient related data, image archives that include all images used for diagnostics for a period of 10 years at minimum, accurate annotations of dates and viewing, and precise hardware and software information. Medical aspects should demand standardized patients' files such as DICOM 3 or HL 7 including history and previous examinations, information of image display hardware and software, of image resolution and fields of view, of relation between sizes of biological objects and image sizes, and of access to archives and retrieval. Technological aspects should deal with image acquisition systems (resolution, colour temperature, focus, brightness, and quality evaluation procedures), display resolution data, implemented image formats, storage, cycle frequency, backup procedures, operation system, and external system accessibility. The lowest third level describes the permitted limits and threshold in detail. At present, an applicable standard including all mentioned features does not exist to our knowledge; some aspects can be taken from radiological standards (PACS, DICOM 3); others require specific solutions or are not covered yet. Conclusion The progress in virtual microscopy and application of artificial intelligence (AI) in tissue-based diagnosis demands fast preparation and implementation of an internationally acceptable standard. The described hierarchic order as well as analytic investigation in all potentially necessary aspects and details offers an appropriate tool to specifically determine standardized requirements.

Abstract As part of an investigation aimed at illuminating the possibilities and limits of the HOPE-fixation and paraffin-embedding technique we here describe a novel procedure which was developed in order to combine the benefits of the HOPE-technique with the capabilities of laser microdissection. The presented procedure avoids the need for amplification of template-RNA thus facilitating reliable and reproducible results. The excellent preservation of nucleic acids, proteins, and morphology in HOPE-fixed, paraffin-embedded tissues enhances the molecular applications available to date with materials acquired by laser microdissection when compared to formalin fixed, paraffin-embedded tissues, thus substantially extending the methodological panel in tissue based research.