In addition to the traditional differentiation between implicit and explicit knowledge we outline the concepts of general and individual knowledge. We connect general knowledge with the "frame problem," a fundamental issue of artificial intelligence, and individual knowledge with another important paradigm of artificial intelligence, case-based reasoning, a method of individual knowledge processing that aims at solving new problems based on the solutions to similar past problems.The "applied" nature distinguishes applied sciences from theoretical sciences. To emphasize this distinction, we begin with a general, meta-level overview of the scientific endeavor. We introduce the We outline the fundamental differences between Medical Informatics and theoretical sciences and propose that Medical Informatics research should advance individual knowledge processing (case-based reasoning) and that natural language processing research is an important step towards this goal that may have ethical implications for patient-centered health medicine.We focus on fundamental aspects of decision-making, which connect human expertise with individual knowledge processing. We continue with a knowledge spectrum perspective on biomedical knowledge and conclude that case-based reasoning is the paradigm that can advance towards personalized healthcare and that can enable the education of patients and providers.We center the discussion on formal methods of knowledge representation around the frame problem. We propose a context-dependent view on the notion of "meaning" and advocate the need for case-based reasoning research and natural language processing. In the context of memory based knowledge processing, pattern recognition, comparison and analogy-making, we conclude that while humans seem to naturally support the case-based reasoning paradigm (memory of past experiences of problem-solving and powerful case matching mechanisms), technical solutions are challenging.Finally, we discuss the major challenges for a technical solution: case record comprehensiveness, organization of information on similarity principles, development of pattern recognition and solving ethical issues.Medical Informatics is an applied science that should be committed to advancing patient-centered medicine through individual knowledge processing. Case-based reasoning is the technical solution that enables a continuous individual knowledge processing and could be applied providing that challenges and ethical issues arising are addressed appropriately. Solomonoff's ideas [Our aim is to place Medical Informatics in the context of other sciences and to bring coherence in its formal education . This wi's ideas , summari's ideas , contrib's ideas , a relathuman knowledge as a collection of individual theories elicited from scientific observations. The immense number of theories that comprise the collective human knowledge about every possible subject, as well as its extraordinary dynamics, have forced us to divide it into what we commonly refer to as knowledge domains, thereby reducing the contexts of our observations to smaller space-time continuums. The attempts to process with computers the knowledge in a domain have taught us that we need to recognize the reality of the "knowledge acquisition bottleneck" [knowledge spectrum.The four-dimensional space-time continuum we live in (i.e., our universe) forms the reality (i.e., the context) of all scientific observations. The compression of the immense complexity and dynamicity of this reality in concise "theories of everything" was already demonstrated by Zuse ,6 and retleneck"  and to ntleneck"  and [10-tleneck" ). The pacomplex reality  to high-level abstractions (e.g., theories, hypotheses, beliefs, concepts, formulae etc). Therefore, it comprises increasingly lean modalities of knowledge and knowledge representations media and the relative boundaries and relationships between them. Two forces manifest on the knowledge spectrum: that of creating abstractions and that of instantiating abstractions for practical applications. The former is the theory elicitation and is synonymous to processes of context reduction and knowledge decomposition. The latter, theory application, equates to context increase and knowledge composition processes. The engines behind the two knowledge spectrum forces are the knowledge processors, natural or artificial entities able to create abstractions from data and to instantiate abstractions in order to fit reality.The knowledge spectrum Figure  spans frimplicit and explicit  is the rich, experiential, sensorial kind of knowledge that a knowledge processor acquires when immersed into an environment (i.e., grounded in an environment), or presented with detailed representations of that environment (e.g., images, models, recordings, simulations). It is very well applicable to specific instances of problems and relies on processing mechanisms such as feature selection, pattern recognition and associative memory.Explicit knowledge (E) is the abstract, symbolic type of knowledge present explicitly in documentations of knowledge such as textbooks or guidelines. It requires a representation language and the capability of a knowledge processor to construe the meaning of concepts of that language. It is applicable to both specific and generic problems and relies on explicit reasoning mechanisms.explicit knowledge in order to describe, with various degrees of precision, particular instances of problem solving and theory application. This represents the rationale for further dividing the knowledge spectrum into general and individual knowledge  is the explicit, abstract, propositional type of knowledge (e.g., guidelines), well applicable to context-independent, generic problems. However, it is more difficult to use in specific contexts because of the gap between the general knowledge itself and a particular application context. This knowledge gap translates into uncertainty when a general knowledge fact is instantiated to a specific situation. For example, knowing generally that a certain drug may give allergic reactions but being uncertain whether a particular patient may or may not develop any, is an example of what we consider the uncertainty associated with general knowledge. The creation of general knowledge  is a relevance-driven process done by "stripping away irrelevancies" [vancies" . This cavancies" .Formal representations of explicit knowledge have been common in early artificial intelligence (AI) applications in the context of expert system development. They operated under the "closed world assumption" and were meant to make the representation of knowledge manageable, reproducible and clear. However this assumption also rendered the expert systems "brittle" or completely unusable when applied to real world problems . The com   1. first, that the brick is on something that is not a pyramid;   2. second, that there is nothing that the brick is on and that is on the brick as well; and   3. third, that there is nothing that is not a brick and the same thing as the brick."This definition could have the predicate calculus representation:This representation shows that an intelligent agent who has no implicit knowledge of the hypothetical physical world and no capacity of generalization or analogy making, must be explicitly provided with all knowledge necessary to reason about "bricks" in that limited reality. Such approaches are known to suffer from a fundamental shortcoming, the "frame problem."Daniel Dennett was the first philosopher of science who clearly articulated the "frame problem" and promoted it as one of the central problems of artificial intelligence   ignoring some thousands of implications it has determined to be irrelevant" and its batteries were therefore lost in the inevitable explosion.• The third robot failed because it was "The frame problem can therefore be recast as a problem of relevance [only because in the real world we do have time constraints(see preface), which is compounded by time constraints. It demonstrates that relevance judgment mechanisms based on general knowledge are time consuming and cause the failure to solve time-constrained decision problems. It is a problem .elevance  or instance specific knowledge, on the other hand, is a knowledge modality very well applicable to real problems, because it identifies uniquely and matches precisely an application context. The knowledge gap and uncertainty are reduced but still exist because of our changing reality (time dimension) which may render individual knowledge about a patient collected in the past , less applicable in the present or future. Because it preserves context (i.e., it is more grounded), individual knowledge has a higher complexity than general knowledge and hence is more difficult to manage (i.e., has high memory requirements). For example, knowing the drugs and the precise description  of the allergic reactions that they caused in a certain person, as well as many other particular knowledge facts about individual, is what we call individual knowledge. The uncertainty and knowledge gap related to the application of such knowledge to future instances of decision making involving that individual are reduced: individual knowledge is supposed to fit very well the application context where it was originally captured.Individual knowledge captured from a very specific context (e.g., diagnosing a particular patient with a particular disease) can be extrapolated to similar contexts. The higher the similarity between contexts, the smaller the knowledge gap and instantiation uncertainty and the higher the chances for a successful solution to a new problem. For this reason, individual knowledge processing has become increasingly important for artificial intelligence applications and is defined as the approach to solving new problems based on the solutions of similar past problems ,19-21. IThere are four steps (the four "RE") that a case-based reasoner must perform ,20,21:1. RETRIEVE: the retrieval from memory of the cases which are appropriate for the problem at hand; this task involves processes of analogy-making or case pattern matching;2. REUSE: the decomposition of the retrieved cases in order to make them applicable to the problem at hand;3. REVISE: the compositional adaptation and application of the knowledge encoded in the retrieved cases to the new problem; and4. RETAIN: the addition of the current problem together with its resolution to the case base, for future use.CBR entails that an expert system has a rich collection of past problem-solving cases stored together with their resolutions. CBR also hinges on a proper management of the case base and on appropriate mechanisms for the matching, retrieval and adaptation of the knowledge stored in the cases relevant to a new problem. Ideally, the individual knowledge in a case-base will progress asymptotically towards an exhaustive knowledge base, which represents the "holy grail" of knowledge engineers. From a learning systems point of view, similarly to artificial neural networks ,23 and iThe absolute positions and shapes of boundaries between the four knowledge modalities, although admittedly not as precise as drawn on the knowledge spectrum in Figure • Individual knowledge has a higher complexity than the explicit knowledge elicited from the same context. This is equivalent to stating that, for example, the picture of a person encodes more knowledge than the textual description of that person's appearance.• Implicit knowledge is a subset of the individual knowledge.• General knowledge is a subset of the explicit knowledge.• The set of individual knowledge represented explicitly formed by the intersection of individual knowledge with explicit knowledge is a nonempty set. This is equivalent to stating that it is possible, for example, for an explicit textual description to identify a context uniquely (e.g., the complete name and address of a person at a specified moment in time).The meta-level overview of sciences and the definitions and properties of the knowledge spectrum and knowledge modalities enable us to draw some fundamental differences between theoretical sciences and applied sciences such as Medicine  and Medi• Make use of observations which are highly abstract symbolisms and create far more limited contexts of application of their theories, when compared to the complexity of the human body or of any social or biological system,general knowledge comprising valid, powerful theories which explain precisely and completely the observations, and therefore,• Have as a primary purpose the creation of • Include a relatively limited number of precise theories which are evaluated primarily by their power of explaining experimental observations, elegance, generality, andindividual knowledge required by the practical implementation and by the application of results to real world problems.• Are less concerned with the acquisition of the Applied sciences such as Medicine and Medical Informatics, on the other hand:individual knowledge) from very complex systems [• Gather extensively data and observations ,• Have as a primary purpose not only distilling data and observations into • May lack the incentive to refine existing theories which are objectively wrong as long as practical success is achieved ,general knowledge) that can solve individual problems completely or explain and predict accurately [• Contain very few simple, "elegant" theories  ,25,• Abound in general theories (e.g., guidelines) which are "lossy"  and which are evaluated statistically by their practical success relative to existing ones (e.g., cancer therapy),knowledge gap between general knowledge and the reality where knowledge is applied, by employing experienced clinicians who require extensive training and information technology (e.g., decision support), and, in addition,• Attempt to make up for the • Are compounded by time-constrained circumstances and largely unsolved ethical issues .individual knowledge. By doing so, Medical Informatics will provide a solution to the problems that arise during the use of general knowledge and, in the same time, will enable clinical research as well as advanced decision support and education of both healthcare providers and patientsMedical Informatics should complement the traditional quest for general biomedical knowledge with the advance of acquisition, storage, communication and use of .Given the special circumstances of our applied science in the context of other sciences and the increasing recognition of the importance of knowledge processing to Medical Informatics , we propIf EHR are to be used in a CBR context, the issues pertinent to the design of case-bases automatically become pertinent to the EHR design, and the CBR paradigm becomes important to Medical Informatics.Individual knowledge processing equates to a case-based reasoning (CBR) approach that employs collections of patient cases. Currently, such collections are the focus of research on Electronic Health Records (EHR). Envisioned as "womb to tomb" collections of patient-specific data, EHR contain a wealth of data that could be used to support case-based decisions. knowledge processing capacity of healthcare systems can be thought to be distributed between two sources: human resources  and information technology . An ideal CBR approach would increase this knowledge processing capacity by allowing for the automatic processing  of individual knowledge present in increasingly rich knowledge media such as natural language artifacts, images, videos and computer simulations of reality  research is a very important component of Medical Informatics, required to advance the organization and processing of individual knowledge in reusable case-bases. Further, the goal to advance processing of increasingly complex knowledge representations  and create intelligent machines that can hear, see, think, adapt and make decisions, brings Informatics even closer to what traditionally was the concern of Artificial Intelligence (AI).The overall y Figure . The stomoving towards the ideal of individual knowledge processing, no matter how slowly, may also have ethical implications because it proves that medical informaticians are trying to do everything they can in order to serve the interest of the individual.Finally, because the knowledge processing capacity of human resources tends to remain relatively constant, In order to support our thesis, the following discussion will focus initially on fundamental aspects of medical decision-making and biomedical knowledge creation from the standpoint of the knowledge spectrum. This will lead to a discussion of fundamental knowledge representation and processing principles and the proposal of a CBR perspective on EHR, including challenges and potential solutions.Medicine is a knowledge intensive domain where time-constrained decisions based on uncertain observations are commonplace. In order to successfully cope with such situations, health professionals go through a tedious learning process in which they gain the necessary domain knowledge to evolve from novices to experts. As experts, health professionals have attained, among other things, two important, highly interrelated abilities:determining efficiently what is relevant for solving a problem in a particular situation, and,• To be able to reduce knowledge complexity by reduce the knowledge gap between knowledge facts and reality which translates into being able to reduce the uncertainty of knowledge instantiation to a particular context.• To be able to For example, both the presence and the absence of a past appendectomy are relevant and contribute  to reducing the uncertainty of instantiation of the biomedical knowledge of an expert to a particular context of a patient with right lower abdominal pain. Fundamental to decision making, relevance judgments and uncertainty reduction seem both closely connected with the quality and quantity of knowledge available for solving a problem as well as with the nature of knowledge processing mechanisms. Studies of expert-novice differences in medicine  have shoThe discussion around the amount of problem solving experience of a decision maker becomes critical in time-constrained decision circumstances. The exhaustiveness of the knowledge base and the efficiency of retrieval mechanisms now become paramount to the decision speed. Empirical evidence that shows the existence of "systematic changes of cognitive processes" related to time stress, comes from the studies on the psychology of decision-making under time constraints . AlthougSince humans are able to make sound relevance judgments and reduce instantiation uncertainty of knowledge most of the times, the following questions arise: What is their strategy for increasing the exhaustiveness of their knowledge base while managing its exponential complexity? How do they represent and organize their knowledge and how do they manage time-constrained situations? At least some of these questions have been under intense scrutiny that has resulted in important empirical work on naturalistic decision-making ,33,36,37From the perspective of the knowledge spectrum, it seems reasonable to associate expert decision makers with individual knowledge and novices with the more abstract general knowledge about a subject, available in explicit knowledge artifacts (e.g., textbooks, guidelines). It is also conceivable that mental models of experts span a great length of the knowledge spectrum, causing them to efficiently perform implicit processing  and also just-in-time explicit reasoning Figure . The abiNovices, on the other hand, have limited mental models of reality situated towards the abstract region of the spectrum. This causes them to have difficulties with construing appropriate meanings of concepts due to the increased knowledge gaps between their mental models and reality. Novices are therefore unable to make sound relevance judgments and limited in their ability of interpreting data and creating abstractions. They are also usually overwhelmed by the explicit, general knowledge present in textbooks and guidelines and unable to fully construe the meanings of concepts present in such knowledge artifacts.In conclusion, in information and knowledge intensive domains such as medicine, explicit reasoning is important but individual knowledge acquisition (i.e., experience) and processing (i.e., CBR) are crucial for decision-making. Because the nature of expertise seems largely connected with individual knowledge processing, it follows that the evolution of novices into experts is unattainable only by the provision of extensive general knowledge. In addition, not only the individual learning but also the collective sharing of experiences  between individuals and between generations, contribute to the way humans deal with decision problems.The major driving force of science is universally applicable knowledge . While creating and communicating new knowledge, scientists move across the knowledge spectrum from the data that captures the reality of their experiments and observations towards abstract representations that allow them to communicate their theories. In biomedical research, such an example is the randomized controlled trial (RCT), currently regarded as the gold standard for knowledge creation. The correct design of an RCT is crucial for the validity of the medical evidence obtained. A correct randomization process in RCTs will limit the bias and increase the chance for applicability of the evidence obtained, to a specifically selected group of patients (e.g., "women aged 40–49 without family history of breast cancer"). However, at the same time, the randomization process removes the circumstances of individual cases and creates a knowledge gap between the RCT evidence and future application instances. As with any statistical approach, the RCT-based evidence is best applicable at the population level rather than at the individual level.This depersonalization of medical knowledge and evidence was also noted by others ,40 and cOthers  have alsindividual knowledge and personalized health care, the general knowledge gained through populational studies (e.g., RCTs) targets the ideal of universal applicability , providing the decision makers have access to it and are able to apply it to specific situations. However, general knowledge is unlikely to be used as such in many naturalistic decision-making processes, because it does not support the way expert decision makers think. The knowledge gap and inherent instantiation uncertainty manifested in the application of general knowledge does not fully enable the education of providers and patients which would require additional knowledge about individual contexts of successful or unsuccessful application instances. Informatics, on the other hand, by advancing individual knowledge processing, provides an alternative solution to the problems that arise from the use of general knowledge that targets universal applicability. An integral part of individual knowledge, genomic data is already recognized [as being of extreme importance for a solution to the problems of general knowledge.cognized ,40as beiTherefore, intelligent agent must be endowed with efficient mechanisms for determining the relevance of particular facts for a decision.The application of formal knowledge representations to real problems suffers from a fundamental shortcoming: the frame problem. As explained above (see "The frame problem"), the frame problem can be recast as a problem of relevance. Given the capability of relatively effortless human relevance judgments, the frame problem seems a rather "artificial" creation, difficult to grasp and which usually goes unnoticed. In order to circumvent its abstract nature, Dennett uses a story-telling approach. However, the frame problem also applies to and could be illustrated from the perspective of humans, who in their first years of life, learn and can easily and efficiently reason about the side effects and the implicit changes of the complex four-dimensional spatio-temporal physical world in which they live. As this learning gradually becomes common sense knowledge, it causes us to efficiently determine the relevant implicit changes while ignoring the non-relevant ones for a given situation. For example, such facts as that the clothes we are wearing are moving with us while walking or traveling are most of the times irrelevant given the context of a planned trip. However, if the trip involves some rapid movement through the air such as riding a motorbike, suddenly wearing a sombrero becomes a relevant fact. As experts at managing our physical world, we are able, through an effortless but powerful mental simulation, to determine the relevance of such a particular fact. The recall of our personal experiences of moving fast through the air and of the dragging force of the air becomes paramount. We suggest that what made the robots vulnerable was their creators' choice for knowledge representation and reasoning: the robots did not have quick access to implicit knowledge about the relevance of particular facts (i.e., records of problem solving instances) but only to explicit facts in frames which had to be employed in time-consuming, immense number of explicit relevance judgments about the effects of particular actions. Although they were supposed to be experts at their task, the robots were behaving like novices. The frame problem is not a problem of the knowledge representation per se, but a problem of the choice for representation of knowledge needed to solve time-constrained decisions. In other words, formal representations and logic reasoning work, but not in time constrained, complex situations.intended meaning of concepts: a retrieved document is considered to be relevant to a query if the intended meanings of the authors of a document are relevant to the intended meaning of that query. We propose that "meaning," a property that characterizes all concepts present in explicit knowledge, is intimately connected  with the notion of context. According to this rather paradoxical view, meaning, a property which characterizes the abstract side of the knowledge spectrum, is strongly coupled with context which, by definition, is a feature of the reality side of the knowledge spectrum. Therefore, in order to construe meaning appropriately one needs to be able to efficiently move from abstractions towards richer representations of reality. This movement on the knowledge spectrum is necessary in order to fill the knowledge gap between abstract concepts and the richer mental representations required for construing their meaning.From the perspective of our knowledge spectrum, explicit, formal representations sit on the abstract side of the spectrum Figure . The retExplicit, formal representations attempt to capture general truth and generally applicable problem solving strategies, but become too abstract in nature. Through the abstraction process, which is essentially a reduction driven by the relevance judgments of knowledge creators, the context of a problem is lost. Losing context creates difficulties with construing meaning (which is context-dependent by definition) and widens the knowledge gap between the representation itself and the reality of a future problem-solving instance. The knowledge gap translates into the instantiation uncertainty that characterizes the application of general knowledge to specific problems. Making up for the knowledge gap through explicit relevance reasoning becomes time consuming and consequently takes its toll on the applicability of the representation. In sensitive applications such as medical decision-making and health research, general knowledge may potentially be harmful . In addition, abstractions and general methods and theories of problem solving and decision making (e.g., guidelines) do not fully enable the education of individuals and the learning from successes and mistakes.Knowledge representation approaches must therefore preserve to the extent possible, the context of a problem-solving instance. By efficiently recalling similar past instances of problem solving and their contexts, intelligent agents are immediately provided with implicit knowledge about relevance, encoded in the retrieved contexts and, in the same time, with more possibilities to reduce the instantiation uncertainty of general knowledge when applied to specific problems. To enable this, informatics research must advance the processing of rich representations of the knowledge encoded in past problem solving cases: this is the definition of CBR research.Similar to formal specifications  natural language uses abstractions, i.e., concepts. Its richness and power of expression place it in the knowledge spectrum to the left side of formal specifications but to the right side of rich descriptions consisting of images, sounds, video-clips and simulations of reality Figure . NaturalIn order to illustrate our point about ambiguity we direct the reader to the previous, natural language definition of the concept of "a brick." Although the definition may look unequivocal, there are subtle ambiguities that make a difference in the predicate calculus representation. The first condition of an object to be "a brick" (i.e., "the brick is on something that is not a pyramid," highlighted in the equations 2 and 3) is an ambiguous natural language construction and could have slightly different formal representations:In (2) this condition has been interpreted as: "the brick being on something IMPLIES that that something is not a pyramid" and was therefore represented as "for all Y, if X is on Y, this implies that Y is not a pyramid." In (3), which is identical to (1) but is repeated to the benefit of the reader, this condition was interpreted as "the brick MUST BE  on something that is not a pyramid" and that was represented as "there exists Y such as X is on Y and Y is not a pyramid."The first definition is therefore more "relaxed" as it allows the possibility that a brick sits on nothing. The second definition is more restrictive, because it requires the brick to be on something that is "not a pyramid" or otherwise X is not a brick anymore. Therefore, the first definition is more general and defines the concept of "a brick" in such a way that the definition would be true even in a world with no gravity (i.e., the brick is on nothing). In addition, definition (3) does not reject the possibility that an object sits on both another brick and a pyramid, at the same time Figure .The point is that, most often, humans receive and transmit knowledge without the deep understanding and completeness required by an exact mathematical representation of the knowledge to be transmitted. This shallowness has also been recognized by others  who are If possessing the necessary knowledge, humans are able to effortlessly fill the knowledge gaps between natural language representations and their richer representations of reality , and to easily construe the appropriate meaning of potentially ambiguous concepts. Although current technology allows for its storage, knowledge present in richer media (e.g., images, videos, simulations) is currently very difficult to process  using today's technology.Because natural languages are used by people universally and allow rich representations that no other language specification can attain, natural language processing (NLP) research is a first step that Informatics should take in order to advance the organization and processing of individual knowledge in case-bases that can be reused. The insights gained will advance knowledge processing towards richer knowledge representation media, will reduce the knowledge processing gap and consequently increase the knowledge processing capacity currently supported largely by human knowledge processors.One of the main features of information processing systems is their memory. It is accepted that storage and manipulation of information are necessary for complex cognitive activities in humans . Memory From a computational point of view, one could easily argue that without a random access memory structure there can be no effective processing. In the context of "the computational architecture of creativity," this argument is clearly outlined in . It is bRecent natural language processing (NLP) research stresses the importance of memorization of individual natural language examples . The impThe tradeoff between learning effort and communication efficiency seems to be biased naturally towards memorization rather than towards logical reasoning. The processing complexity of natural language might therefore not be an intrinsic quality of the algorithms, but rather a function of the memorization capabilities of the language processor, given the sparseness of natural language pattern space. By analogy, the advanced knowledge processing in humans might not be the result of very sophisticated reasoning strategies, but rather the utilization of a limited reasoning apparatus on a huge knowledge base, consisting of rich representations of one's experience. The limitations in reasoning are balanced by complex spatio-temporal pattern recognition capabilities operating on a case base built from years of experience. This case base includes common-sense knowledge.Furthermore, people and computers memorize information differently. Both have a short term, working memory and long-term memory for storing data and information. However, the memory access is carried out in different ways. Computers can reliably store large streams of data, which most of the times have a very well defined spatial and temporal structure (e.g., a movie clip). In contrast, people can only store information and knowledge rather than data and their storage is unreliable, temporally fragmented and spatially incomplete. Computers have very reliable memories capable of error checking at the bit level while the human memory supports only a high-level semantic consistency check. Finally, computers access their memory in a random seek fashion, being able to position their "reading heads" at any position in the data streams in order to extract a certain block of data. People can access their memory by content, by being provided with an incomplete description of a potentially complex, spatio-temporal pattern serving as a retrieval key. Therefore, one of the main differences between computers and humans is that computers have address-based random access memories, while humans possess content-addressable memories.In conclusion, from a case-based reasoning perspective, humans seem to be naturally endowed with the necessary structures for efficient case base acquisition, organization and retrieval while computers do not directly support this way of processing information and knowledge.Pattern recognition is an undisputed feature of human cognitive abilities and a research area in its own right. However, it does not seem to be as pervasive as it should, in the information processing systems in current use. Natural language, as a product of human cognition, offers compelling evidence that people are naturally inclined toward processing information using pattern recognition and similarity principles. This evidence is supported by the widespread use of language devices such as the simile and the metaphor. These are examples of comparison and analogy making that humans perform without effort, in contrast to the difficulty of implementing them in the artificial information processing systems . AnalogyThe patterns and analogies that humans are able to handle are often represented by complex spatio-temporal events with a potentially multi-sensorial impact. For example, while humans have no difficulty in understanding a metaphor like "the computer swallowed the disk," an artificial information processing system that has no visual input sensors and which lacks the capability of image understanding, would probably never be able to perceive this particular analogy with the same speed, because of the extensive reasoning and amount of explicit knowledge needed to bring the swallowing process, as it occurs in living things, close to the action of inserting a disk into a computer's disk drive.In addition to operating on high dimensional, spatio-temporal complex patterns, analogy making in humans may also possess a dynamic component that could yield different relevance judgment outcomes, depending of context. A very illustrative example is given by French and Labiouse in , using tPolyhierarchy and multiple inheritance are indisputable desiderata of terminology systems . HoweverThe dynamicity of concept meanings and relevance judgments may offer at least one of the reasons why fixed classification schemes, controlled terminology systems or open domain ontologies have not turned out satisfactory. It may also explain why existing lexical databases based on carefully handcrafted knowledge such as WordNet  often coFrom a case-based reasoning perspective, humans are naturally equipped with powerful pattern matching and classification capabilities which allow them to cope with complex, time-constrained relevance judgments, to easily construe meaning of concepts and to tolerate the ambiguity of natural language. Only relatively recently have computers come close to this functionality with the introduction of data mining and machine learning techniques such as self organizing maps and clustering algorithms based on similarity metrics [feature selection equates to a problems of relevance. metrics . In suchIatrogenic causes are said to be important causes of death in the US . The repOne very effective form of medical education is the retrospective analysis of case records where health professionals, both experienced or novices, learn from their own and from others' successes and failures . ProvidiMedical errors are also connected with the complex human cognitive task of planning . CBR appProviding that the privacy and confidentiality issues, which are even more stringent in this case, are dealt with appropriately, opening EHR to patients could benefit them . It is pMedicine has always and will always be a case oriented profession. Medical Informatics has recognized this early through the works of various researchers who pioneered the area of decision support systems . Relevan• The case base, the memory of past problem-solving instances• The case matching or pattern matching procedure which retrieves the relevant cases for a certain problemWhile humans seem to possess a natural support for these two components, there is still work to be done in order to make the computer support this kind of knowledge acquisition and processing. We envision four important challenges in advancing towards CBR enabled EHRs:1. Case record comprehensiveness2. Organization on similarity and associative principles (associative memory) and development of advance data visualization techniques3. Development of pattern recognition and similarity measures between heterogeneous records4. Solving ethical issues and provision of privacy and confidentiality measuresEHR comprehensiveness is required because the exhaustiveness of a case base is not only a function of the number of records but also of the richness of each case record. Current knowledge processing technology limits the acquisition and especially the processing of comprehensive EHR records which incorporate structured data, images, video-clips, bio-signals, genomic data, unstructured textual data covering clinical findings, detailed patient history, etc. However, as knowledge processing technology advances and knowledge acquisition bottlenecks are overcome, it might be possible to overcome the heterogeneity and sparseness of EHR and allow the creation of representative case-bases and the organization of knowledge on principles that facilitate similarity based retrieval.Temporal knowledge is also a good example of a heterogeneously represented type of knowledge in the form of potentially non-interoperable standards for dates and times and temporal knowledge of various degrees of precision, embedded in knowledge facts such as "soon after receiving the drug, the patient developed a rash." Currently, for many people, the problem may seems to boil down to devising yet another standard which encompasses all the different temporal representations of dates, times and temporal concepts into a unified, common representation. From a knowledge engineering standpoint, and again currently for many researchers, this may equate to the creation of a comprehensive ontology of temporal knowledge. However, the problem of representing time starts to look like a somewhat limited version of another burning problem of Medical Informatics: that of medical terminologies. The fact that all these issues remain largely unsolved, can only help the case for CBR and for adaptive, empirical methods and approaches to knowledge processing. We believe that such approaches have the potential to cope and overcome the problems with redundant, possibly ambiguous representations, which have arbitrary degrees of precision. Thereby we are specifying a goal towards which the development of EHR should proceed.Similarity based retrieval is difficult with current database technology. For example, queries to retrieve cases which are similar to a textual description of a given case are difficult to answer. The comprehensiveness of EHR must be complemented with the possibility of indexing its records on similarity principles. Conceptually, the functionality of EHR will be that of an associative memory of cases that will enable the CBR paradigm. The organization of a case-base must be complemented by the development of advanced data visualization techniques that comply with the principles of organization of information by similarity. One example of such data visualization techniques are self-organizing maps . These mCBR relies on the proper management of the case base and on appropriate mechanisms for matching and retrieval of these case records. All similarity retrieval mechanisms are based on some sort of distance calculation between the problem at hand and the records in the case base, followed by the retrieval of the most relevant ones. Clinical narratives and other EHR components containing unrestricted text represent a particularly difficult challenge for semantic similarity measures. The development of terminology systems based on less structured (e.g., latent semantic indexing, connectionist models) and data-driven approaches will provide the semantic richness, dynamicity and reusability needed for such complex tasks.Ayercillin, an item unknown to the system and hence not appearing in Figure A concrete example for the potential feasibility of such approaches, is the automated knowledge induction based on contextual similarity modeling ranging from morphological to sentential context  Figure . An expeHowever, despite such proof-of-concept applications and other progress in data mining and knowledge extraction from heterogeneous databases, case matching remains largely an open research question.We discuss this challenge last, not because it is less important but, on the contrary, because of its potential to become the most important obstacle to individual knowledge processing. The very fact that individual knowledge has the potential to contribute to solving future problems instances, raises the important ethical issue whether such knowledge should be made available to decision makers and researchers. Because the definition of individual knowledge implies the possibility to match it in time and space with an application context, i.e., with a patient, sharing individual knowledge is counterbalanced by the need for privacy and confidentiality. In addition, to further complicate matters, it may turn out that some of the most useful records for future instances of decision making are instances of medical errors or other unexpected events that are unique in their course of events and therefore easily identifiable together with their contexts of development (i.e., patients, providers, family members).The high complexity of individual knowledge renders explicit, manually controlled access to individual knowledge cases and their components unfeasible. The only solution to this problem seems to be of technological nature. Current privacy and confidentiality measures which include de-identification, de-nominalization and scrambling of the unique personal identifiers automated or semi-automated seem insufficient to counteract the potential to identify patients from unique, individual knowledge patterns.As a general approach, we propose that the accurate measuring of similarity of individual knowledge could form the basis of a confidentiality risk assessment. This could be intuitively understood by considering that:• very similar individual knowledge patterns which are in great numbers are a very low threat to the privacy and confidentiality infringement, and, at the other extreme,• stand-alone patterns which possess unique features or combinations of features, are at high risk of privacy and confidentiality breaches.knowledge about the proper use (e.g., access, modification, transfer) of individual knowledge. This potentially complex, particular case of meta-knowledge processing could be implemented and managed using the principles of CBR paradigm itself, by building case-bases with examples of both proper and improper  individual knowledge accesses and that can be compared with future access instances.In addition, the provision of privacy and confidentiality could be regarded as a special case of knowledge processing, which involves Overcoming this very important challenge hinges on the possibility to effectively measure the similarity between heterogeneous records and on the advancement of knowledge processing on CBR principles.If successful, CBR research might therefore fulfill a longstanding need for intelligent information processing and advance informatics towards the ideal of individual knowledge processing. This calls for further investigation of information processing models that are, similarly to human experts, capable to efficiently move across the knowledge spectrum. One class of such models is represented by artificial neural networks ,23, whicCBR provides potential solutions to important problems that, among other, stymy the usefulness of EHR. The natural integration of learning with reasoning and the CBR resemblance to the cognitive models of human decision-making hold the promise to overcome the "brittleness" and "knowledge acquisition bottleneck" of classical expert systems. The CBR applications to the medical field have the potential to offering the training and decision support needed by health professionals and the means towards a true patient-centered healthcare.With a CBR theoretical foundation still in its infancy and with limited medical applications in existence, more research is needed for providing proofs of the feasibility of practical CBR-EHR applications. Challenges in the way to accomplish this include the increasing complexity, ethical issues as well as the paradigm shift that our current computing devices must undergo in order to support the CBR principles of knowledge processing.1. Science is twofold and is driven by two opposite forces: that of creating theories , and that of applying theories to practical applications (applied sciences). Medical Informatics is fundamentally an applied science that should be committed to advancing patient-centred medicine through individual knowledge processing.2. Case-based reasoning is the technical solution that enables a continuous individual knowledge processing that could be integrated with the Electronic Health Records.3. Medicine is an information and knowledge intensive domain where time-constrained decision problems can only be solved effectively based on the recollection of similar problems and their solutions (i.e., a case-based reasoning strategy). The collective sharing of experiences is important for making future decisions as well as for learning how to make decisions.4. Unlike computers, human decision makers possess the components necessary to perform case-based reasoning naturally .5. Applying the CBR approach to EHR might be a way to overcome the important obstacles of EHR acceptance and use, providing that technical challenges and ethical issues arising are addressed appropriately.AI Artificial intelligenceAIT Algorithmic Information TheoryCBR Case Based ReasoningE Explicit knowledgeEHR Electronic Health RecordsG General knowledgeLSA Latent Semantic IndexingI Individual knowledgeNDM Naturalistic Decision MakingRCT Randomized Controlled TrialRPD Recognition-Primed DecisionU Implicit (Unobvious) knowledgeThe author(s) declare that they have no competing interests.SP researched the paper and provided a first draft. JA, JM critically revised the manuscript three times each and provided their own additions to the initial draft. JA provided more feedback on the cognitive aspects and decision-making as well as writing style and missing references. JM additions were with regard to the writing style, clarity, missing references and the overall organization of the paper.SP and JM worked on the responses to reviewers' comments. SP wrote a first revision of the paper. JM provided extensive feedback as well as new references and suggested a major revision that includes recent ideas. JA also commented and made suggestions on the knowledge spectrum model and on the meta-level view on Medical Informatics. SP overhauled the entire paper. JM revised the new version and provided feedback. SP operated the changes and proposed new modifications. JM revised the second draft. JA provided feedback on the second draft of the paper with regard to fundamental aspects of knowledge modalities.SP and JM incorporated the minor changes suggested by the last review.All authors read and approved the final version of the paper.The pre-publication history for this paper can be accessed here:


The human immunodeficiency virus Vif protein overcomes the inhibitory activity of the APOBEC3G cytidine deaminase by prohibiting its packaging into virions. Inhibition of APOBEC3G encapsidation is paralleled by a reduction of its intracellular level presumably caused by the Vif-induced proteasome-dependent degradation of APOBEC3G.In this report we employed confocal microscopy to study the effects of Vif on the expression of APOBEC3G on a single cell level. HeLa cells dually transfected with Vif and APOBEC3G expression vectors revealed efficient co-expression of the two proteins. Under optimal staining conditions approximately 80% of the transfected cells scored double-positive for Vif and APOBEC3G. However, the proportion of double-positive cells observed in identical cultures varied dependent on the fixation protocol and on the choice of antibodies used ranging from as low as 40% to as high as 80% of transfected cells. Importantly, single-positive cells expressing either Vif or APOBEC3G were observed both with wild type Vif and a biologically inactive Vif variant. Thus, the lack of APOBEC3G in some Vif-expressing cells cannot be attributed to Vif-induced degradation of APOBEC3G. These findings are consistent with our results from immunoblot analyses that revealed only moderate effects of Vif on the APOBEC3G steady state levels. Of note, viruses produced under such conditions were fully infectious demonstrating that the Vif protein used in our analyses was both functional and expressed at saturating levels.Our results suggest that Vif and APOBEC3G can be efficiently co-expressed. Thus, depletion of APOBEC3G from Vif expressing cells as suggested previously is not a universal property of Vif and thus is not imperative for the production of infectious virions. Replication of human immunodeficiency virus type 1 (HIV-1) in most primary cells and some immortalized T cell lines is dependent on the expression of a functional Vif protein. In the absence of Vif, virus replication is restricted by a host factor that was recently identified as CEM15 (now referred to as APOBEC3G)  but whosVif is a 23-kDa basic protein that is expressed late during infection in a Rev-dependent manner . ImmunocLike Vif, APOBEC3G is a cytoplasmic protein. In fact, co-immunoprecipitation analyses demonstrated an interaction of Vif and APOBEC3G in transiently transfected cells ,27,29-32The current study aims at characterizing in more detail the effects of Vif on the expression of human APOBEC3G on a single cell level. The study was initiated because of the apparent discrepancy between the drastic effects of Vif on APOBEC3G reported by Marin et al and our own finding of only moderate effects of Vif on APOBEC3G expression in transiently transfected cells. In our study, Vif was expressed from a subviral construct in a Tat- and Rev-dependent manner while APOBEC3G was expressed either in a Tat-dependent manner from an HIV-1-LTR-based vector or independently from a CMV-promoter-based expression vector. The Tat-dependent APOBEC3G expression vector was used to restrict APOBEC3G expression to cells also expressing Tat (and thus Vif).Confocal microscopic analysis of HeLa cells transiently transfected with Vif and APOBEC3G expression vectors revealed significant variations in the number of double-positive cells in identical samples ranging from as low as 40% to as high as 80% of transfected cells depending on fixation method and antibodies employed. Importantly, the appearance of cells expressing only Vif or APOBEC3G was observed both with wild type Vif and a biologically inactive variant and thus cannot be explained by Vif-induced degradation of APOBEC3G. Finally, despite the efficient co-expression of Vif and APOBEC3G, viruses produced in these cultures were fully infectious. We therefore conclude that the Vif-induced exclusion of APOBEC3G from virus-producing cells reported by Marin et al  does notA number of previous studies reported the efficient Vif-dependent degradation of APOBEC3G by cellular proteasomes ,8,28. HoAn earlier study investigating the coexpression of Vif and APOBEC3G by confocal microscopy concluded that APOBEC3G was virtually excluded from Vif-expressing COS7 cells . To veriIn the experiment shown in figure In addition of measuring the context-dependent expression of APOBEC3G, we also wanted to determine the influence of the fixation procedure on the efficiency of Vif:APOBEC3G co-staining. It is well known that the choice of fixative can affect the ability of a given antibody to recognize a specific epitope on its target protein. Frequently, epitopes are masked because of the folding properties of a protein in vivo or because of pre-existing protein-protein interactions that may compete for antibody binding. To test this possibility we compared a formaldehyde fixation procedure employed previously  with theHeLa cells were transfected with pNL-A1 and pHIV-APO3G at a 1:1 molar ratio. Cells were fixed 24 hr later either with methanol (MeOH) as in figure For a more quantitative analysis and to determine possible effects that arise from the use of different antibodies, we extended the experiment shown in figure To quantify the results, multiple optical fields were analyzed n = 5–10) with a total of at least 100 transfected cells for each parameter. As can be seen in figure –10 with Under optimal conditions, wild type Vif and APOBEC3G were coexpressed in about 80% of transfected cells . Experiments are ongoing to study the differential effects of Vif expressed from pNL-A1 and Vif expressed from a codon-optimized vector on APOBEC3G stability. However, these results could suggest that the effect of Vif on APOBEC3G steady-state levels may be influenced by the context in which Vif is expressed. At any rate, despite our inability to observe Vif-dependent cellular depletion of APOBEC3G, we were invariably able to recover fully infectious HIV under conditions were the intracellular levels of APOBEC3G were only moderately affected. We therefore conclude that (i) Vif has the ability to rescue viral infectivity even in the presence of APOBEC3G and (ii) that intracellular depletion of APOBEC3G and rescue of viral infectivity may be functionally separable activities of Vif.For now, the reason for the differences in the sensitivity of APOBEC3G to Vif noted by us versus other research groups remains unexplained. APOBEC3G can form oligomeric structures and is able to interact with Vif. It is therefore possible that such complexes undergo conformational changes that can mask epitopes thus limiting the access of antibodies used in the experiments. Thus, the discrepancy between our findings of the coexpression of Vif and APOBEC3G in the majority of cells and the virtual exclusion of APOBEC3G from Vif-expressing cells reported by Marin et al.  may be aThe inability of Vif expressed from pNL-A1 to deplete APOBEC3G is consistent with our previous inability to observe APOBEC3G degradation in kinetic studies . More reExpression of Vif and APOBEC3G in our experimental setup does not lead to the elimination of APOBEC3G from Vif expressing cells. In fact, more than 80% of successfully transfected cells efficiently co-expressed both proteins. Similar results were observed when a biologically inactive Vif variant was co-expressed with APOBEC3G suggesting that the absence of APOBEC3G in some of the Vif-positive cells is not due to Vif-mediated APOBEC3G degradation but reflects a general characteristic of the transient expression system. Moreover, APOBEC3G expression levels were very similar for Vif-positive and Vif-negative cells as judged from the immunostaining consistent with the only modest reduction in APOBEC3G steady-state levels observed in our immunoblot analyses. Nevertheless, viruses produced under such conditions were fully infectious in the presence but not in the absence of Vif attesting to the biological activity of all the proteins involved and demonstrating that Vif was expressed at saturating levels. We conclude that production of infectious viruses from APOBEC3G expressing cells is dependent on Vif but does not necessitate APOBEC3G exclusion from virus-producing cells.gag and pol products. A vif-defective variant of pNL-A1, pNL-A1vif(-) was constructed by deletion of an NdeI/PflMI fragment [The full-length molecular clone pNL4-3  was usedSerum from an HIV-positive patient (APS) was used to detect HIV-1-specific capsid (CA) proteins. A monoclonal antibody to Vif (MAb #319) was used for all immunoblot analyses and some of the immunohistochemical analyses as indicated in the text and was obtained from Michael Malim through the NIH AIDS Research and Reference Reagent Program ,35,37 FoHeLa cells were propagated in Dulbecco's modified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS). LuSIV cells are derived from CEMx174 cells and contain a luciferase indicator gene under the control of the SIVmac239 LTR . These c2 flasks to about 80% confluency. Cells were transfected using LipofectAMINE PLUS™ (Invitrogen Corp, Carlsbad CA) following the manufacturer's recommendations. A total of 5–6 μg of plasmid DNA per 25 cm2 flask was used. Cells were harvested 24 hr post-transfection. Transfection efficiency in our analyses was generally 30% to 40%.For transfection of HeLa cells, cells were grown in 25 cmVirus stocks were prepared by transfecting HeLa cells with appropriate plasmid DNAs. Virus-containing supernatants were harvested 24 hr after transfection. Cellular debris was removed by centrifugation (3 min, 3000 × g) and clarified supernatants were filtered (0.45 μm) to remove residual cellular contaminants. For determination of viral infectivity, unconcentrated filtered viral supernatants were used for the infection of indicator cells. For immunoblot analysis of viral proteins, virus particles (7 ml) were concentrated by ultracentrifugation through 4 ml of 20% sucrose in PBS as described before [7 cells), and mixed with an equal volume of sample buffer (4% sodium dodecyl sulfate, 125 mM Tris-HCl, pH 6.8, 10% 2-mercaptoethanol, 10% glycerol, and 0.002% bromphenol blue). Proteins were solubilized by boiling for 10 to 15 min at 95°C with occasional vortexing of the samples to shear chromosomal DNA. Residual insoluble material was removed by centrifugation (2 min, 15000 rpm in Eppendorf Minifuge). Viral proteins were obtained by boiling concentrated viral pellets in a 1:1 mixture of PBS and sample buffer. Cell lysates and viral extracts were subjected to SDS-PAGE; proteins were transferred to PVDF membranes and reacted with appropriate antibodies as described in the text. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences, Piscataway NJ) and visualized by enhanced chemiluminescence (ECL, Amersham Biosciences).For immunoblot analysis of intracellular proteins, whole cell lysates were prepared as follows: Cells were washed once with PBS, suspended in PBS (400 μl/105) in a 24-well plate total volume 1.2 to 1.4 ml. Cells were incubated for 24 hours at 37°C. Cells were then harvested and lysed in 150 μl of Promega 1x reporter lysis buffer (Promega Corp., Madison WI). To determine the luciferase activity in the lysates, 50 μl of each lysate were combined with luciferase substrate (Promega Corp., Madison WI) by automatic injection and light emission was measured for 10 seconds at room temperature in a luminometer (Optocomp II, MGM Instruments, Hamden CT).To determine viral infectivity, virus stocks were normalized for equal reverse transcriptase activity and used to infect LuSIV cells  for 10 min followed by two washes in PBS or fixed in FA buffer  for 20 min at room temperature followed by two washes in PBS. Coverslips were stored in PBS at 4°C until use. FA-fixed samples were permeabilized for 30 min at room temperature in permeabilization buffer (1% Triton X-100, 10% sucrose in PBS) prior to incubation with antibodies. For antibody staining, cover slips were incubated in a humid chamber at 37°C for 30 min with primary antibodies at appropriate dilutions in 1% BSA in PBS. Cover slips were washed once in PBS (5 min, room temp) and incubated with Texas-Red- or Cy2-conjugated secondary antibodies (diluted in 1% BSA in PBS) for 30 min at 37°C in a humid chamber. Cover slips were then washed twice with PBS and mounted onto microscope slides with glycerol gelatin (Sigma-Aldrich, St. Louis) containing 0.1M N-propyl gallate (Sigma-Aldrich, St. Louis) to prevent photo bleaching and were stored at 4°C in the dark until analyzed by confocal microscopy.For confocal microscopy, a Zeiss LSM410 inverted laser scanning microscope was employed. The microscope was equipped with a krypton/argon mixed-gas laser and was operated by the Microcosm Renaissance 410 (v2.3.4) software package. Images were acquired with a Plan-Apochromat 63x/1.4 oil immersion objective (Carl Zeiss, Thornwood). Additional optical magnification (up to 5-fold) was achieved using the zoom feature of the image acquisition software. For two-color analysis, objects were excited using 488/568 nm laser lines. Green and red emissions were recorded through appropriate filters (515–540 nm band pass filter for Cy2 and 590 nm long pass filter for Texas-Red) and stored in separate (red and green) image channels. At the same time, bright field images (Nomarski optics) were collected and stored in a third (blue) channel. Image quality was enhanced during data acquisition using the Renaissance 410 line average feature (8 or 16x).None declared.S. K. carried out immunoblot analyses, infectivity assays, and was involved in the construction of plasmids and the production of antibodies. E.M., M.A.K., H.T., S.O., and R.G. participated in immunoblot analyses, infectivity studies, sample preparations, data validation, and overall experimental design. K.S. conceived of the study, performed IFA analyses, and coordinated the study. S.K. and K.S. participated in the writing of the manuscript. All authors read and approved the final manuscript.


Cervical ripening is an inflammatory reaction. The glucocorticoid receptor (GR) mediates glucocorticoid anti-inflammatory reactions, whereas nuclear factor (NF)kappaB is a key pro-inflammatory transcription factor. Prostaglandins as well as platelet activating factor (PAF) are inflammatory mediators. Inducible nitric oxide synthase (iNOS) regulates the level of nitric oxide (NO) in response to various inflammatory stimuli. We hypothesize that a changed biological response to glucocorticoids could be a mechanism regulating the inflammatory events resulting in cervical ripening.We monitored GR and NFkappaB, prostaglandin synthases cyclooxygenase (COX)-1 and -2, iNOS, as well as the PAF-receptor (PAF-R) in the uterine cervix from term pregnant women (with unripe cervices) before the onset of labor (TP), immediately after parturition (PP), as compared to non-pregnant (NP), using immunohistochemistry and RT-PCR.The GR protein was detected by immunohistochemistry in the nuclei of stroma and squamous epithelium (SQ). Stromal GR staining was increased in TP as compared to the NP group and decreased again after parturition. GR staining in SQ was decreased after parturition as compared to term. NFkappaB was present in SQ and glandular epithelium (GE), stroma and vascular endothelium. Increased nuclear NFkappaB staining was observed postpartum as compared to term pregnancy in stroma and GE. Stromal immunostaining for COX-1 as well as COX-2 was increased in the TP and PP groups as compared to the NP, and GE displayed an intensely increased COX-2 immunostaining at term and postpartum. Stromal PAF-R immunostaining was highest at term, while it was greatly increased in GE postpartum.No difference in the immunostaining for iNOS was found between the groups. RT-PCR showed a predominance of GRalpha to GRbeta mRNA in cervical tissue. The COX-2 mRNA level was increased in the PP group as compared to the TP group.There is a decrease in GR levels in human cervix at parturition. Concomitantly there is an increase of factors such as NFkappaB, PAF-R, COX-1 and COX-2, suggesting that they may participate in the sequence of events leading to the final cervical ripening. The human uterine cervix undergoes biochemical changes resulting in softening, effacement and dilatation during pregnancy and labor. This remodeling, or ripening, is a prerequisite for parturition . It is cAmong the group of structurally related, ligand-inducible nuclear steroid receptor transcription factor proteins, GR was the first that was cloned and sequenced . GR and NFκB is a key pro-inflammatory regulator. GR and NFκB are both inducible transcription factors with diametrically opposed functions in inflammatory responses. A mutual negative direct and indirect cross talk between GR and NFκB has been well described in previous studies ,14.2 (PGE2) is widely implicated for cervical ripening in clinical practice [Prostaglandin Epractice . Among tpractice . The COXpractice .Platelet-activating factor (PAF) is a lipid pro-inflammatory mediator, involved in several reproductive processes, i.e. parturition . PAF is Nitric oxide (NO) is synthesized intracellularly from the amino acid L-arginine through the activity of specific synthase enzymes (NOS) . The ind2 release from human cervical tissue explants [2 concentrations in inflammatory tissues [2 in human cervix [NO stimulates PGEexplants , and is  tissues ,25. NO d tissues ,27, at t tissues  and in n tissues . BesidesOur hypothesis is that glucocorticoids exert a direct receptor mediated effect in the human cervix uteri, and that a changed biological response to glucocorticoids could be a mechanism behind the events resulting in cervical ripening at parturition. Since NFκB has opposed functions in inflammatory responses as compared to GR, we presume that NFκB could also be a regulator of the inflammatory events leading to cervical ripening. These inflammatory events could be mediated via factors such as the PAF-R, iNOS and/or COX enzymes.All women gave their informed consent and the Local Ethics Committee of the Karolinska Hospital approved the study. All were healthy, had uncomplicated pregnancies and were without medication prior to parturition. The non-pregnant (NP) women were hysterectomised due to benign disorders not involving the cervix. The women in the term pregnant (TP) group all had unripe cervices with a Bishop score <5 points and none of them were in labor. Biopsies were obtained during elective caesarean sections before onset of labor. The biopsies from the post partal (PP) women were taken after a normal vaginal delivery.For the immunohistochemistry study of GR and NFκB, cervical biopsies were obtained from one para and eleven primipara TP women (n = 12) with a mean age (range) of 33 (28–38) years, and a mean gestational age of 38 (37 to 40) weeks. The women of the PP group (n = 14) were all primipara and had a mean age of 31 (22–37) years, and a gestational age of 40 (39 to 42) weeks. The NP control group (n = 8) had a mean age of 43 (32–49) years, and a mean parity of II (I-III).The cervical samples available for the immunohistochemistry studies of COX-1, COX-2 and PAF-R were TP (n = 8), PP (n = 10) and NP group (n = 6). Biopsies for RNA preparations were not available for the RT-PCR study from all subjects, NP (n = 5), TP (n = 6) and PP (n = 5). The women in the NP group were significantly older than those of the other two groups. Since hysterectomies in young women are uncommon most biopsies in the NP group are from women in the middle of their 40s, but they were all menstruating regularly and did not receive any medication.Cervical biopsies were obtained transvaginally (for the TP and PP groups) from the anterior cervical lip at the 12 o'clock position, from 10–20 mm depth. The tissue samples from the hysterectomies were obtained directly after the uterus was removed during operation. The same physician (YSV) collected all the samples. The biopsies were immersion-fixed in 4% phosphate buffered formaldehyde at 4°C overnight, stored at 4°C in 70% ethanol and thereafter embedded in paraffin. From the biopsies that were large enough, a small piece was cut off prior to fixation, and frozen in -70°C until RNA preparation.2, 7.5 mmol/l dithiothretiol, 0.5 mmol dNTPs, 1 μg random hexameters, and 400 U of Moloney murine leukemia virus reverse transcriptase (Gibco-BRL, Paisley, UK).Total RNA from frozen cervical tissue samples was purified with the SV Total RNA isolation system (Promega, Madison, WI) according to a procedure recommended by manufacturer. One microgram of total RNA from each sample was reverse transcribed at 42°C for 45 min in a final volume of 40 μl with a reaction mixture containing 50 mmol/l Tris-HCl (pH 8.3), 75 mmol/l KCl, 3 mmol/l MgClOligonucleotide primers for the GRα gene were as follows : 5'-CCT ® master mix (Quiagen GmbH, Hilden, Germany) containing 2.5 μM of each oligonucleotide primer in a final volume of 20 μl. The reaction mixture was overlaid with mineral oil. After an initial incubation for 15 min at 95°C, the samples were subjected to 33 (GRα, COX-1 and COX-2) and 40 (GRβ) cycles of 30 s at 94°C, 40 s at 60°C, and 60 s at 72°C with a final extension step at 72°C for 10 min in the DNA Thermal Cycler 480 . The amount of PCR product for GRα increased linearly up to 36 cycles and for COX-1 and COX-2 it increased linearly up to 38 cycles (data not shown). Quantitative measurement of GRβ mRNA would require larger amounts of cervical RNA than were available, since GRβ showed very low expression with a visible band only after 40 cycles.For PCR, the cDNAs corresponding to 50 ng RNA were added to 10 μl of HotStarTaqTo standardize the quantification method, an endogenous 18S rRNA was used as an internal standard. The 18S rRNA primers and Competimers™ (modified at their 3' ends to block extension by DNA polymerase) were obtained from Ambion . The standard, and the GR and COX mRNAs were amplified in parallel and under the same conditions. A mixture of 18S primers and Competimers™ (1:9) was used to modulate amplification efficiency of 18S rRNA to the same linear range as GRα, COX-1 and COX-2 when amplified under the same conditions. The predicted size of the PCR product for 18S was 489 bp. The PCR products were run on 2% agarose gel and stained with ethidium bromide. Bands were captured and analyzed using ChemiDoc Gel Documentation System (Bio-Rad Laboratories, Hercules, CA). The levels of GR and COX PCR products were normalized against the 18S product. The RT-PCRs were repeated twice.Paraffin sections (5 μm) were used and a standard immunohistochemical technique (avidin-biotin-peroxidase) was carried out as described before  to visuaReplacing the primary antibody with non-immune goat IgG was used as negative control.A monoclonal mouse anti-iNOS antibody was used for detection of iNOS (N32020, Transduction Laboratories, Lexington, KY, USA). It recognizes the C-terminal domain of iNOS. The antibody was diluted 1:50 and incubated for 70 minutes in room temperature. Replacing the primary antibody with non-immune mouse IgG was used as negative control.A microscope and CCD video camera connected to a computer with an image analysis program (Leica Imaging System Ltd., Cambridge, UK) was used to assess quantitative values from GR and NFκB immunohistochemistry. The quantification of nuclear immunostaining was performed on the digitized images of systematic randomly selected fields of stroma and squamous epithelium. Ten fields were analyzed separately in each section of tissue, using the color-discrimination software. Positive staining is presented as a ratio of the area of positively stained nuclei (brown) to the total area of cell nuclei (brown and blue).Two observers blinded to the identity of the slides, performed all the assessments. The staining was evaluated semi-quantitatively using a grading system. The staining intensity was graded on a scale of (0) no staining, (1) very faint, (2) faint, (3) moderate and (4) strong staining.Statistical calculations for the data from the relative quantification of RT-PCR products, the immunohistochemistry results by image analysis and manual scoring were performed by ANOVA on Ranks  and significances were evaluated by Dunn's test. Values with different letter designations are significantly different (p < 0.05).By immunohistochemistry the GR protein was localized to the nuclei of cervical stroma (S), squamous epithelium (SQ), glandular epithelium (GE) and vascular endothelium Figure  in samplPositive NFκB immunostaining in cervix uteri was present in stroma, GE, SQ and vascular endothelium in NP (left column), TP (middle column) and PP (right column) groups Figure . Strong Immunostaining for COX-1 Figure  was founImmunostaining of COX-2 Figure  was founImmunostaining of PAF-R was found in stroma, neuronal ganglion (G) and GE Figure . The immFaint positive staining was found in SQ, stroma and GE Figure . There wThe GRα mRNA level was determined by RT-PCR Figure . GRα mRNVery low levels of GRβ mRNA were present in 4 out of 6 samples from the TP group, and in 2 out of 5 samples in the NP and PP groups Figure .There was no difference in the COX-1 mRNA level between the groups, as assessed by RT-PCR Figure  Figure , top.The COX-2 mRNA level was the highest in the PP group Figure  Figure , bottom,This study is, to our knowledge, the first reporting GR expression in human cervix uteri during pregnancy and at parturition. In a recent study on human endometrium , GR protGlucocorticoids exert their anti-inflammatory effects primarily by inhibiting the expression of cytokines e.g. IL-8, as demonstrated in human fibroblasts , tumour Maternal plasma levels of cortisol increase until term , but theOur data shows that the GR protein was present in cervical stroma, SQ and vascular endothelium in samples from non-pregnant, term pregnant and postpartum women. The decrease in GR levels in stroma and SQ at parturition, as compared to term, could be interpreted in terms of a GR mediated glucocorticoid anti-inflammatory activity during pregnancy that is ended at parturition.The NFκB protein was present in cervices from non-pregnant, term pregnant and postpartum women. This protein, ubiquitously expressed in a variety of cell types, is found in the cytoplasm in its inactive form, but translocates into the nucleus upon activation . TherefoPolymorphonuclear leukocytes and macrophages migrate from blood vessels and accumulate in the cervix uteri before parturition . GR and 2 in the human body [Cortisol is a potent inhibitor of COX-2 in myometrium, decidua and cervix , and ref. This woman body . We coulman body , but alsman body , and refman body , the act2 release from human cervical tissue explants [2 in human uterine cervix [Nitric oxide stimulates PGEexplants . Nitric explants . Treatmee cervix . The imme cervix ,48. Tsche cervix . LedinghPlatelet activating factor (PAF), like prostaglandins derived from the arachidonic acid precursor, is a multifactorial pro-inflammatory mediator, which has been implicated in parturition. Local application of PAF in rats induced cervical ripening , whereasIf the process of cervical ripening is disturbed, either resulting in a preterm delivery or to a prolonged delivery time, possibly ended by a cesarean section, it will lead to increased risks for both the mother and the child. Preterm delivery is the leading factor causing neonatal mortality and morbidity . An incrWe have demonstrated that the human uterine cervix is a potential target organ for glucocorticoids during pregnancy. The higher GR protein levels in cervical stroma and SQ before parturition may reflect a GR mediated anti-inflammatory effect of cortisol during pregnancy, with a subsequent decline of this activity at parturition. The concomitant increase in nuclear NFκB levels in the cervix suggests activation of this transcription factor at parturition. NFκB activity promotes pro-inflammatory events and could be responsible, at least in part, for the observed increase in COX-1, COX-2 and PAF-R levels.