By definition, parasites harm their hosts. However, some forms of parasite-induced alterations increase parasite transmission between hosts, such that manipulated hosts can be considered extensions of the parasite's phenotype. While well accepted in principle, surprisingly few studies have quantified how parasite manipulations alter host performance and survival under field and laboratory conditions.Ribeiroia ondatrae causes particularly severe morphological alterations within amphibian hosts that provide an ideal system to evaluate parasite-induced changes in phenotype. Here, we coupled laboratory performance trials with a capture-mark-recapture study of 1388 Pacific chorus frogs (Pseudacris regilla) to quantify the effects of parasite-induced malformations on host locomotion, foraging, and survival. Malformations, which affected ∼50% of metamorphosing frogs in nature, caused dramatic reductions in all measures of organismal function. Malformed frogs exhibited significantly shorter jumping distances (41% reduction), slower swimming speeds (37% reduction), reduced endurance (66% reduction), and lower foraging success relative to infected hosts without malformations. Furthermore, while normal and malformed individuals had comparable survival within predator-free exclosures, deformed frogs in natural populations had 22% lower biweekly survival than normal frogs and rarely recruited to the adult population over a two-year period.By interfering with limb development, the trematode Our results highlight the ability of parasites to deeply alter multiple dimensions of host phenotype with important consequences for performance and survival. These patterns were best explained by malformation status, rather than infection per se, helping to decouple the direct and indirect effects of parasitism on host fitness. Cephalotes atratus) changes in color from black to a rich red, thereby resembling the ripe fruit consumed by frugivorous birds, which function as the parasite's final host Although typically hidden from view, parasites and pathogens are a ubiquitous component of almost all communities While well accepted in principle, surprisingly few studies have quantified how parasite-induced changes in phenotype affect hosts under natural conditions Ribeiroia ondatrae causes dramatic morphological changes in host body plan, such as missing, misshapen or extra limbs An intriguing class of infections ideally suited to address such challenges involves parasites that induce morphological rather than behavioral changes in their hosts. While many examples of host phenotypic change involve subtle alterations in host behavior, color, or size Ribeiroia-amphibian system to broadly evaluate the consequences of parasite-induced phenotypic changes on host performance. Specifically, we quantified the effects of limb malformations on hosts' jumping distance, swimming ability, foraging success, and physical endurance among field-collected amphibians. We chose to use field-collected animals with naturally induced malformations to maximize the ecological realism of measured responses, given that lab-raised hosts can exhibit altered or impaired responses to relevant tasks Ribeiroia often occurs, nearly all amphibian hosts are infected but there is substantial variation in malformation status and severity. We extended these results to a field-setting by comparing changes in host survival and body condition in naturally-occurring amphibians that varied in malformation status over both short-term (biweekly) and longer-term  time scales. We hypothesized that malformations would reduce host performance and survival and that these patterns would be best explained by malformation status rather than infection intensity.Here, we used the All work was conducted in accordance with the policies and protocols set forth by the University of Colorado's Institutional Animal Care and Use Committee (IACUC), for which protocol number 09-07-GOO-01 was approved in May 2009.Ribeiroia ondatrae (hereafter ‘Ribeiroia’) and its second intermediate amphibian hosts. Ribeiroia uses rams horn snails (Planorbidae) as its first intermediate hosts, amphibians or fishes as second intermediate hosts, and birds as definitive hosts Ribeiroia cercariae often causes reduced survival and an increased frequency of severe limb malformations, including skin webbings, bony triangles, missing limbs, and extra limbs or limb elements Our study system focuses on interactions between the trematode parasite Ribeiroia often causes malformations that are distinctive in form and severity, such that hosts with modified phenotypes can be quickly identified and collected. Limb malformations are typically rare (<3%) in most amphibian populations Ribeiroia is abundant. Second, in ponds with moderate to high Ribeiroia infection intensity, a large proportion (∼30%) of emerging frogs may exhibit severe malformations, particularly in sensitive hosts such as Pseudacris regillaRibeiroia is common, most if not all emerging frogs are infected  even though not all animals are malformed, helping to decouple infection from phenotype. Moreover, while the presence and abundance of Ribeiroia is a strong predictor of malformations at the wetland scale Several factors make this study system well suited to address questions surrounding disease and the extended phenoytpe. First, Pseudacris regilla) from two ponds  known to support both Ribeiroia ondatrae and a high frequency of malformed frogs Ribeiroia is complete; (2) we collected frogs shortly after metamorphosis before they had extensive time to diverge in body size or foraging behavior as a function of malformation status, and (3) we necropsied a subsample (n = 60) of normal and malformed hosts to quantify Ribeiroia and ensure there were no differences in other forms of infection . For all trials, animals were not fed on the day that performance tests were conducted, and only one type of performance trial was conducted on a given day. We alternated trials between randomly selected normal and malformed individuals used in each performance task.We examined frog performance using a series of ecologically relevant locomotor performance tasks To assess swimming performance, we placed an individual frog at one end of a polyvinyl chloride (PVC) raceway (1.5×0.12×0.1 m) filled with filtered water to a depth of 6 cm and allowed it to swim the entire length of the raceway and back again. Frogs were encouraged to swim using gentle taps by the observer's index finger and trials were halted once an individual ceased swimming following three taps by the observer. Prior to each trial, we compared the temperature of the water and the body surface of each frog using a handheld infrared thermometer (Fluke 62 Mini) to ensure no differences between groups. We timed frogs as they swam over lines placed at 10 cm intervals and estimated ‘maximum burst speed’ as the minimum time required to travel a 40 cm section on any of three replicate swims. Using similar methods, we quantified swimming endurance as the total distance (cm) and duration (s) that an individual frog swam within a circular channel  with a water depth of 10 cm. Lines at 10 cm intervals around the perimeter provided a measure of distance travelled during review of trial videos.We placed a single frog in the centre of a 2×2 m disc and encouraged it to jump by gently touching it on the back. Typically, frogs made a series of jumps in rapid succession. Following each jump, we marked the frog's landing point with a marker. We scored a total of six jumps for each frog and used the single longest jump as our measure of ‘maximum jump distance’. In cases where a frog did not make a subsequent jump, we gently touched it on the back to elicit another jump.We evaluated each frog's foraging ability by adding either 5 wingless fruit flies or 1 small cricket into a 12×12×10 cm arena and quantifying time to first capture (s), time to consume a prey once captured (s), number of capture attempts (lunges), and proportion of prey items consumed. Trials lasted until all prey items were consumed or for a maximum of 300 s (fruit flies) or 720 s (cricket). Frogs, which were randomly assigned to prey type, were given a 10 min acclimation period prior to beginning trials and individual frogs were used only once.P. regilla can be captured in large numbers, regardless of malformation status, they provide an ideal system for CMR studies. Following capture, we assessed each frog's malformation status and size . On each sampling date except the final survey, we batch-marked frogs using biologically inert Visible Implant Fluorescent Elastomer (VIE) tags , which were injected into the muscle tissue of the inner thigh. This technique, which has been used successfully on a variety of amphibian species and life history stages Every two weeks between 29 May and 5 August 2009, we monitored malformation frequency, frog abundance, and frog body condition at Sheep Pond , a pond with a high frequency of parasite-induced limb malformations but a small enough size (0.028 ha) to achieve a high capture efficiency of metamorphosing frogs. We captured all frogs  observed along linear transects running parallel to the shoreline. Because metamorphic As a complementary study, we compared the survival of field-collected normal and malformed frogs in two outdoor cages that excluded predators. Cages, which were built on the shoreline of the pond and contained a constant water supply, were constructed with a wooden frame and aluminum fly wire (1.0 mm mesh) covering the top and sides (0.9×0.9×1.5 m). The base of each cage was buried 0.15 m into the substrate to prevent unwanted emigration and immigration. We placed 20 normal and 20 malformed frogs into each cage, each of which were selected at random and received a fluorescent tag as described above. After one month, we dismantled the cages and assessed the survival of frogs in each cage.P. regilla generally achieve sexual maturity within 1 year Ribeiroia abundance.Finally, we revisited the pond to quantify malformation frequency in breeding adult frogs the following two springs. Because 10-transformed values of each performance response. Frog size (SUL) was included as a covariate in all analyses. To further decouple the relationships among infection, malformation status, and performance, we used a subset of the data for which we had information on all three components and (1) compared Ribeiroia infection abundance between normal and malformed frogs at the end of the study and (2) evaluated the relative explanatory power of a frog's infection level and its malformation status in predicting performance.We evaluated the effects of malformation status on frog performance using univariate ANOVAs with logp) t+1, while recapture probability is the likelihood that a living and marked frog is recaptured at time t+1, and depends on recapture data from subsequent time points . Owing to this feature, recapture probability cannot be calculated independently from survival for the final time interval. Apparent survival is not considered ‘true survival’ since it does not incorporate emigration. Metamorphosing P. regilla typically do not emigrate away from a pond until late in the season We used the program MARK c), and we calculated the distance of each model (ΔAICc) from the model with the lowest AICc value, and the Akaike weight (wi), which is the relative likelihood of a particular model given the data To analyze the influence of malformation status on survival, we used an information theoretic approach 1, 114 = 1.183, P = 0.279; SUL: F 1, 114 = 0.637, P = 0.427). Among the subsample of frogs collected prior to initiating performance trials, we also found no difference in R. ondatrae metacercarial abundance between normal and malformed frogs . We also found no differences in infection by other larval trematodes, including Alaria sp., Manodistomum sp. and kidney-infecting echinostomes . No nematodes, cestodes, or adult trematodes were recovered .Of the 114 frogs used in performance trials, 63 were normal and 51 exhibited one or more malformations. Malformations affected the hind limbs and were dominated by extra limbs or feet (52.1%) and skin webbings (21.1%)  . We foun1, 113 = 26.915, P<0.001; 1, 66 = 22.21, P<0.001; 1, 66 = 17.578, P<0.001; 1, 111 = 33.55, P = 0.006; Malformation status strongly influenced each of the measured performance responses. The mean burst swimming speed of malformed frogs was 37% slower than that of normal individuals , malformed individuals ultimately captured a lower total proportion of available prey . Correspondingly, inclusion of Ribeiroia abundance did not increase explanatory power for any of the performance measures relative to models with malformation status alone . Malformation severity, or the number of abnormalities per frog After concluding performance trials, surviving frogs (n = 104) were necropsied to quantify infection by 2 = 136.776, n = 1388, P<0.001), with a peak (>70%) in mid-June followed by a progressive decline . This model was strongly supported with a model weight of 0.98, and no other models were within 8 ΔAICc  exhibited minor abnormalities: three supported small outgrowths, four had abnormal skin developments near the tail resorbtion site, one was missing a digit and one was missing a hand  and 6.5 (±2.9), respectively.Despite the consistently high frequency (∼50%) of malformations in metamorphic g a hand . Only twth century humans and rinderpest in African ungulates While diseases can sometimes lead to epidemic mortality, including spectacular examples such as bubonic plague in 14Ribeiroia causes fundamental changes in host body plan by altering the number and structure of hind limbs. Our results demonstrate that parasite-induced changes in phenotype deeply altered every dimension of host performance measured in this study. Malformed frogs exhibited substantial (37 to 76%) reductions in burst speed, swimming distance, endurance, and jumping distance. Correspondingly, while both groups made similar numbers of attempts, malformed frogs were less successful at capturing prey and ultimately consumed 28% and 55% fewer fruit flies and crickets, respectively. These effects were as severe or greater than those reported in similar studies undertaken with other amphibian parasites Ribeiroia infection has been well established Parasite-induced limb malformations in amphibians offer a valuable opportunity to investigate implications of the extended phenotype and broadly quantify the effects of disease on host attributes. Unlike many well-studied examples of parasite manipulation, which involve changes in color or behavior see , RibeiroRibeiroia infection, and Ribeiroia abundance explained no additional variation in host performance after accounting for malformation status. Malformation severity, estimated as the number of abnormalities per host, also correlated negatively with performance. This novel result reflects the inherent non-linearities in the relationship between infection and disease; the risk of pathology varies not only with parasite dosage but also with host developmental stage and the exact position of invading parasites Ribeiroia on amphibian hosts occurred through changes in body plan.Our data also provide insight into the mechanism through which phenotypic changes influence host performance. While many experimental studies compare the behavior or performance of infected and uninfected hosts, all hosts in our study were infected but varied in parasite abundance and malformation status. Based on our analyses, observed reductions in performance were driven by malformation status rather than by variation in infection intensity. On average, normal and malformed frogs supported comparable and overlapping levels of The debilitating effects of parasite-induced changes in body plan observed in the laboratory translated into reduced survival of hosts in nature. Based on a capture-mark-release study involving 1388 frogs, malformed hosts exhibited ∼22% lower biweekly survival relative to normal individuals, despite similar levels of parasite infection. Malformation status was included as a strong, negative predictor of host survival in the best-supported model. Correspondingly, field-caught frogs with one or more deformities had significantly poorer body condition relative to normal frogs. Although the specific cause of mortality in these free-living frogs is open to conjecture, both normal and malformed frogs exhibited comparably high survival in outdoor exclosures that prevented predators, suggesting that differential vulnerability to predators is likely an important contributing factor. Thus, even while abnormal hosts were significantly worse at capturing prey in experimental foraging trials, in nature they appeared to be at greater risk of predation than of starvation. In a previous field study, malformed frogs were more likely to occupy high-risk habitats and, when approached by a simulated predator, waited longer to escape and escaped over shorter distances than did normal individuals Ribeiroia infection compared with metamorphs , although whether this is due to the resulting malformations or to other characteristics associated with this infection site  is complex, 2) shows signs of a purposive strategy, 3) has arisen in several lineages of hosts or parasites, and 4) increases the fitness of either the host or the parasite Ribeiroia-induced malformations will most likely increase parasite transmission because of (1) the extremely detrimental nature of the induced deformities, which impair the primary locomotory system of the host Ribeiroia, including >50 species of birds and mammals Ribeiroia and the large number of hosts it can affect. While the current example of phenotypic manipulation is novel in its distinctiveness, we suggest that parasite-induced changes in host behavior and morphology are more widespread than often appreciated owing to their typically cryptic nature.More challenging, however, are efforts to demonstrate conclusively that parasite-induced alterations cause increased predation (and therefore transmission) by appropriate definitive hosts rather than unsuitable predators. Indeed, owing to the logistical challenges inherent to quantifying manipulations and transmission under real-world conditions Table S1Results of the capture-mark-recapture study. Listed is the number of normal and malformed individuals captured and marked, the number recaptures, the total number of captured frogs, and the percentage of individuals that were malformed.(DOC)Click here for additional data file.


It has been well established that serotonin (5-HT) plays an important role in the striatum. For example, during levodopa therapy for Parkinson's disease (PD), the serotonergic projections from the dorsal raphe nucleus (DRN) release dopamine as a false transmitter, and there are strong indications that this pulsatile release is connected to dyskinesias that reduce the effectiveness of the therapy. Here we present hypotheses about the functional role of 5-HT in the normal striatum and present computational studies showing the feasibility of these hypotheses. Dopaminergic projections to the striatum inhibit the medium spiny neurons (MSN) in the striatopalladal (indirect) pathway and excite MSNs in the striatonigral (direct) pathway. It has long been hypothesized that the effect of dopamine (DA) depletion caused by the loss of SNc cells in PD is to change the “balance” between the pathways to favor the indirect pathway. Originally, “balance” was understood to mean equal firing rates, but now it is understood that the level of DA affects the patterns of firing in the two pathways too. There are dense 5-HT projections to the striatum from the dorsal raphe nucleus and it is known that increased 5-HT in the striatum facilitates DA release from DA terminals. The direct pathway excites various cortical nuclei and some of these nuclei send inhibitory projections to the DRN. Our hypothesis is that this feedback circuit from the striatum to the cortex to the DRN to the striatum serves to stabilize the balance between the direct and indirect pathways, and this is confirmed by our model calculations. Our calculations also show that this circuit contributes to the stability of the dopamine concentration in the striatum as SNc cells die during Parkinson's disease progression (until late phase). There may be situations in which there are physiological reasons to “unbalance” the direct and indirect pathways, and we show that projections to the DRN from the cortex or other brain regions could accomplish this task. The serotonergic system projects widely in the brain with targets including sensory, motor, and limbic systems , including the substantia nigra pars compacta (SNc) and the striatum Vertes, . While tIn this paper, we present a hypothesis about the role of 5-HT in the healthy basal ganglia, focusing on the effects of 5-HT on the so-called direct and indirect pathways. In the next two sections we describe the direct and indirect pathways and our hypothesis. In the following section, we will present a computational model that we use to test the plausibility of our hypothesis. We will then describe the results of computational experiments on the model.The BG are a group of subcortical nuclei including the striatum, subthalamic nucleus, internal and external globus pallidus, and substantia nigra. Cortical-BG-thalamic circuits are critically involved in many functions including sensorimotor, emotion, and cognition  in the striatum. The names reflect the fact that the direct pathway proceeds from the striatum directly to either the internal portion of the globus pallidus (GPi) or the Substantia Nigra pars reticulata (SNr), the two output nuclei of the BG. The indirect pathway, on the other hand, also involves a subcircuit that includes the external portion of the globus pallidus (GPe) and the subthalamic nucleus (STN) before reaching the output nuclei. The two pathways have opposing effects on the thalamus: the indirect pathway has an inhibitory effect, while the direct pathway has an excitatory effect  · (SN) that expresses the release of DA in the striatum as function of SNc firing rate and of 5-HT concentration in the striatum. Most of the equations have decay terms that guarantee that firing or concentrations go to zero in the absence of input. Of course, the real physiological situation is much more complicated. We have left out important nuclei in the BG and there is no representation of the important dynamics within various nuclei. The purpose of this simple model is merely to allow us to investigate and illustrate the hypothesis about the role of 5-HT in maintaining homeostasis between the direct and indirect pathways.The differential equations satisfied by the variables are given below. They are the simplest equations one can imagine that express the positive and negative influences between the variables shown in Figure The values of the parameters in the differential equations and their meanings are listed in Table The values of the parameters were adjusted so that the steady state values of the variables are in ranges that correspond to experimental observations. For example, the steady state concentration of DA in the striatum is 2.72 nM  and this causes a 58% rise in striatal 5-HT that partially compensates for the drop in G. This is true despite the fact that the SNc firing rate declines because of inhibition from the DRN.Suppose that the gain, a7, which represents the amount of 5-HT released per DRN spike in the striatum. The new steady states are given in column C of Table a7. As a result, the indirect and direct pathways change by only 7.5 and 15%.Suppose that we decrease by 50% the coefficient d7 in equation (7) then we expect that the steady state value of 5-HT in the striatum would increase, so we decreased d7 from 2 to 1. The results can be seen in column D of Table The mechanisms by which chronic doses of selective serotonin reuptake inhibitors (SSRIs) relieve depressive symptoms in some patients remain largely unknown. Since many proposed mechanisms involve increases in extracellular 5-HT in projection regions of the raphe nuclei, it was of interest to ask what changes in the circuit in Figure d8 from 10 to 5 which should increase SNc firing. The results can be seen in column E of Table Since deep brain stimulation is used to treat a variety of neurological disorders, we asked how stimulation of the SNc would affect the circuit in the model. To do this we lowered the constant The simulations in A–E show how the network in Figure a5, to the DRN. If a5 is increased by 50%, the new steady states are as given in column F1 of Table The role of 5-HT in increasing DA release and the circuit in Figure decrease a5 by 50% the new steady states are as given in column F2 of Table Conversely, if we a2, to the direct pathway for one second between t = 1 s and t = 2 s. The result can be seen in Figure Suppose that there is a burst of cortical input to the direct pathway such as might occur when a particular action is selected and a particular group of striatal neurons in the direct pathway is stimulated. Now we are thinking of Figure There is a substantial increase in firing in the direct pathway that decays in about 4 s. Notice that this is followed by several seconds in which the direct pathway is inhibited and the indirect pathway is increased. This is a result of the feedback pathway via the DRN depicted in Figure G, in our model and also provide homeostasis of extracellular DA.The purpose of the simple model in this paper is to illustrate and investigate various homeostatic mechanisms arising from the DRN projections to the BG. It is clear that the model is highly simplified and that many important physiological details have not been considered. This paper is based on the fact that direct pathway MSNs express D1 receptors and are stimulated by DA, while MSNs in the indirect pathway express D2 receptors and are inhibited by DA. There are other sub-circuits of the BG, such as the hyperdirect loop  that increasing 5-HT in the striatum, as caused by an SSRI for example, would depress firing in the DRN. And, similarly, deep brain stimulation of the SNc would also depress DRN firing (Results E) and may explain the transient depression seen in some DBS patients.There are times when it might be important to overcome one of these homeostatic mechanisms, for example by changing the balance between the direct and indirect pathways for a subset of neurons involved in choosing a particular action. We showed in Results F how this can be done by changing the input to the DRN, in our example by excitatory or inhibitory input from a hypothesized nucleus X. It is tempting to think that this gives a possible explanation of the plethora of descending projections to the DRN Monti, .Our intention was to use our simple model to discuss possible roles of 5-HT in the striatum. Though our results are suggestive, the model depends on the circuitry that we hypothesize in Figure The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Health Quality Measures Format (HQMF) is a Health Level 7 (HL7) standard for expressing computable Clinical Quality Measures (CQMs). Creating tools to process HQMF queries in clinical databases will become increasingly important as the United States moves forward with its Health Information Technology Strategic Plan to Stages 2 and 3 of the Meaningful Use incentive program (MU2 and MU3). Informatics for Integrating Biology and the Bedside (i2b2) is one of the analytical databases used as part of the Office of the National Coordinator (ONC)’s Query Health platform to move toward this goal.Our goal is to integrate i2b2 with the Query Health HQMF architecture, to prepare for other HQMF use-cases (such as MU2 and MU3), and to articulate the functional overlap between i2b2 and HQMF. Therefore, we analyze the structure of HQMF, and then we apply this understanding to HQMF computation on the i2b2 clinical analytical database platform. Specifically, we develop a translator between two query languages, HQMF and i2b2, so that the i2b2 platform can compute HQMF queries.We use the HQMF structure of queries for aggregate reporting, which define clinical data elements and the temporal and logical relationships between them. We use the i2b2 XML format, which allows flexible querying of a complex clinical data repository in an easy-to-understand domain-specific language.The translator can represent nearly any i2b2-XML query as HQMF and execute in i2b2 nearly any HQMF query expressible in i2b2-XML. This translator is part of the freely available reference implementation of the QueryHealth initiative. We analyze limitations of the conversion and find it covers many, but not all, of the complex temporal and logical operators required by quality measures.HQMF is an expressive language for defining quality measures, and it will be important to understand and implement for CQM computation, in both meaningful use and population health. However, its current form might allow complexity that is intractable for current database systems (both in terms of implementation and computation). Our translator, which supports the subset of HQMF currently expressible in i2b2-XML, may represent the beginnings of a practical compromise. It is being pilot-tested in two Query Health demonstration projects, and it can be further expanded to balance computational tractability with the advanced features needed by measure developers. In 2010, the US Congress enacted a 10-year, $27 billion dollar incentive program to promote adoption and meaningful usage of electronic health record systems (EHRs) .MU1 involved a small number of computationally simple CQMs . The finQuery Health was convened by the Office of the National Coordinator for Health Information Technology (ONC) in 2011 to develop a standard approach for distributed population health queries. Query Health has defined a standards-based methodology using the Health Level 7 (HL7) Health Quality Measures Format (HQMF) for queries and the Quality Reporting Document Architecture (QRDA) for responses. Additionally, Query Health has developed a reference implementation using their selection of three best-of-breed technological components ,10.Query Health has three current pilots, which each use parts of this reference implementation . Two of Two of the pilots use the Informatics for Integrating Biology and the Bedside (i2b2) analytical platform for processing HQMF. i2b2 is a flexible, componentized clinical analytics and data warehousing platform that now enjoys widespread adoption as a research data repository, warehousing clinical data alongside the EHR at over 80 sites nationwide. It is part of an NIH-funded center charged with developing a national computational infrastructure for biomedical computing . i2b2 haHQMF is becoming the lingua franca for defining population health queries. A draft standard of HQMF was released by HL7 in 2010  and was Throughout the remainder of this manuscript, HQMF will refer to the revised HQMFr2 developed for the ballot now in progress, which is used by the Query Health pilots. Areas under active discussion in the ballot are noted.Our goal is to integrate i2b2 with the Query Health HQMF architecture, to prepare for other HQMF use cases (such as MU2 and MU3), and to articulate the functional overlap between i2b2 and HQMF. Therefore, we analyze the structure of HQMF, and then we apply this understanding to HQMF computation on the i2b2 clinical analytical database platform. Specifically, we develop a translator between two query languages, HQMF and i2b2, so that the i2b2 platform can compute HQMF queries.HQMF is a language for defining quality measures expressed as logical combinations of clinical variables, intended to return an aggregate count or percentage. For example, HQMF can express, in a computable manner, the following query: “the number of diabetes patients who have had a hemoglobin A1C test greater than 9% in the past year”.HQMF is derived from the HL7 v3 Reference Implementation Model (RIM), which is also the basis for Clinical Document Architecture (CDA) documents. Therefore CDA bears some resemblance to HQMF. Articulating all of the details of the HQMF format is beyond the scope of this article. Our purpose here is to highlight the structure of HQMF necessary to develop an implementation. For complete documentation, refer to the balloted HQMFr2 , the balWe present HQMF through two examples, both based on the NQF0059 measure to detect poorly controlled diabetes . In the The anatomy of a basic HQMF query is shown in 1, unit a). Individual criteria can further restrict the time search.Measure Period defines the time period the overall query covers, which in this case is 1 year , but the HgbA1c test is a code and its result is a value.The Data Criteria section defines the clinical variables in the query. Each variable is a Code can be either a coded value or value set. A coded value is a numeric code and a code system  represented by an HL7-registered Object Identifier (OID). A value set is a set of coded values referred to by a single OID. The coded values are OR’d together and are convenient shorthand when defining measures where an observation like “diabetes” could be recorded as multiple specific codes . At the time of this writing, the National Library of Medicine has begun hosting a small number of value sets at their Value Set Authority Center (VSAC) repository [pository ,23.Value can be any HL7 data type. When it is not a coded value, it is frequently one of: (1) a numeric value, when a specific value is desired, such as an HgbA1c of exactly 9%; or (2) an interval, seen in the age range and HgbA1c test in extension attribute) as well as other lists. Each list begins with one of six logical “combining operators”, three of which are shown in the figure. The others are atLeastOneFalse, onlyOneTrue, and onlyOneFalse.The Population Criteria section defines the calculations on these data elements needed to compute the quality measure. were seen at least twice within the two years before the end of the measure period; are between 18 and 75; and within the measure period have had either a diabetes diagnosis or have taken a diabetes medication, had an abnormally high value for their most recent HgbA1c test result, and are not documented to have polycystic ovaries or steroid induced diabetes.” Note that this still is not quite as complex as the actual measure, but it does use all of the HQMF features present in that measure. HQMF corresponding to the two modified data criteria and the new population criteria section can be seen in Example 1 presented a pedagogical simplification of the NQF0059 measure. In this second example, we introduce additional features present in the actual measure. The updated query  is: “Find all the patients who: code elements in the data criteria now reference value set OIDs, rather than defining a coded value. These are published by the NQF, and some are available for download from the NLM’s VSAC repository [The pository .value element with an excerpt element with a subset code of “RECENT”. This means, “find all HgbA1c test results within the measure period, filter out any results not greater than 9%, and report the most recent.”Excerpting applies a filter to a data criterion and reports a summary value of the filtered results . This can been seen in the first data criterion in repeatNumber element specifies that at least two ambulatory encounters must have occurred.The second data criterion in temporallyRelatedInformation element specifies that these encounters must have occurred within 2 years (pauseQuantity) of the end (typecode=SBE—“starts before end”) of the measure period (observationReference). Multiple temporal relationships can occur within a single criterion, and 17 types of relationships are defined. In addition to the measure period, temporal relationships can reference other data criteria.Also in the second data criterion in These two examples cover all the major features in NQF0059. However, to support very complex measures, HQMF implementations should also include the following features.Normally, results are reported as one aggregate number, but stratifiers allow reporting to be broken down by other criteria .An additional population group, this describes criteria for patients who should be excluded from the denominator only if they do not meet numerator criteria .This might include, for example, the average emergency department wait time.interpretationCode element. Others, such as details of medication route, admitting physician, or problem status require more complex XML structure using RIM data elements. Implementation Guides (IGs) will define how such elements should be expressed. The only available resources at the time of this writing are the Query Health IG draft and standards developer Keith Boone’s blog [Data criteria can have other modifiers beyond those discussed here. Some of these are simply data elements, eg, the clinician interpretation code for a vital sign goes in an e’s blog ,24. .i2b2 consists of a flexible relational data model and a somewhat more restricted set of Web services.The data model stores observations as “facts” associated with a date, patient identifier, and encounter identifier. Each fact has a key that follows a hierarchical structure  and a basecode that defines the source code and coding system . Optional modifier facts can supplement each primary fact . Facts can have a value, stored in numeric or text formats. The set of possible facts (the ontology) are user-defined, though several standard ontologies exist.The web services define an easy-to-understand query language expressed in an XML format. Queries are built in a web-based query tool and executed as highly optimized SQL statements by a “data repository” web service. A query consists of items dragged into a set of panels. The items in each panel are logically OR’d together, and all panels are logically AND’d. The NOT operation can optionally be applied to a panel. Each item can also have date constraints, though the only other temporal constraint currently supported is “all items in panel must be in the same encounter.” Results can be stratified on any observation , and queries can be combined through query-within-a-query , which allows deep nesting and more complex combinations of elements. Furthermore, ontology items can have arbitrary SQL statements embedded, for added flexibility.This query XML is logically equivalent to a subset of HQMF, and it is therefore possible to translate between these two formats, provided that queries are restricted to this subset.We first illustrate our translation method between i2b2-XML and HQMF by describing the conversion of Example 1 to an i2b2 query. Next, we analyze the HQMF subset supported by i2b2-XML, and we describe a web service we have implemented for i2b2 to both generate and consume HQMF conforming to this subset.First, each data criterion is converted to an i2b2 basecode. For age ranges (panel 1), age is expanded to the OR of every age in the range using the AGE basecode.  For all other values, an OID-to-i2b2 lookup table determines the basecode, which is implementation-configurable.Second, i2b2 basecodes are sent to the i2b2 ontology cell for conversion to associated i2b2 keys. An i2b2 key represents a position in the ontology tree, so the ICD diagnosis codes in panels 2 and 3 include not only the listed basecode  but also all subcodes . This nonstandard HQMF interpretation allowed us to specify an implicit diabetes value set with single coded value, prior to standardization of NQF value sets. Therefore, we feel this behavior  is useful at the present time, and we will revisit it once the NLM VSAC is more complete.Third, value constraints are added, such as the numeric interval “>9%” for HgbA1c.Fourth, time constraints are added. When no specific time constraint is specified, a time constraint is added to match the measurement period.allTrue, every item or list following is placed in a separate panel. For atLeastOneTrue, every item following is placed in the same panel. For allFalse, every item following is placed in the same panel, which is set to the exclude type .For HQMF features currently supported by i2b2-XML are shown in atLeastOneTrue and allFalse groups inside an allTrue group.We have implemented a bidirectional translator that can convert between HQMF-coded values and i2b2 keys see . For traStratifiers, excerpts, and features requiring query-within-a-query are not currently supported. Stratifiers were a low priority for the Query Health pilots, so they have been tabled. Query-within-a-query features are waiting on the more portable implementation in i2b2-XML planned for i2b2’s next release. Excerpts will be supported in the future through ontology definitions containing custom SQL code.Modifiers and value sets are partially supported, but they also require an appropriate HQMF i2b2 ontology . Dr. Michael Buck in the New York Department of Health is developing this ontology, which is an expression of CDA and QDM data elements in i2b2. We have tested his ontology’s ability to support HQMF modifiers and value sets by successfully implementing support for health care providers and clinician interpretation codes and by testing conversion of his NQF0059-compatible diabetes value set. When completed, this ontology will significantly aid i2b2 in consuming complex CQMs.To validate the translator’s accuracy, we developed four query libraries in i2b2-XML, one for each supported ontology configuration of the translator . Each library utilizes all features of the translator .Our translator supports a superset of SHRINE’s features, and it is able to generate and process HQMF corresponding to the i2b2-XML feature set in The translator is by default configured for diagnoses, labs, procedures, medication administration events, and the demographics in SHRINE . Non-SHRINE ontologies are supported through a configuration file. The translator is open source and presently available from the Query Health repository . It willWe have achieved significant interoperability between HQMF and i2b2 using only transformations of the two XML languages. Our web service successfully transforms queries bidirectionally between i2b2-XML and HQMF, supporting a superset of all queries possible in SHRINE. This HQMF translator has been integrated into the reference implementation of Query Health and is currently being used at two Query Health pilot sites. The first, through the New York State Department of Health, utilizes the bidirectional translator with Dr. Buck’s ontology, allowing researchers to create custom HQMF queries and execute them at i2b2 sites throughout New York. The second pilot, at the Beth Israel Deaconess Medical Center, connects their i2b2 instance (using the SHRINE ontology) to the FDA Mini-Sentinel network and demonstrates the FDA’s ability to send custom HQMF queries to i2b2 instances. i2b2 1.7, to be released in summer 2013, will extend the i2b2-XML language to support a larger subset of HQMF, including improved query-within-query and new temporal constraints. We hope this, along with Dr. Buck’s ontology, will give us the tools needed to compute CQMs for MU2 and MU3 using the XML translation approach.HQMF is an elegant but complex and challenging query language. It separates the definition of data elements from the logical operations used to combine them, and the format for clinical variables is similar to CDA, making basic understanding less difficult for HL7 developers. However, even the partial support we have achieved has been a massive effort. The work involved was more than 6 months of FTE time by an experienced software architect, and we had a preexisting sample transform from standards developer Keith Boone, support from Query Health team members, and the i2b2 analytical database system with a schema-defined XML language. Even with i2b2 1.7 and Dr. Buck’s ontology, full translator support will involve much additional effort. Although future implementers will have both the advantage of our experiences and an open-source, HQMF-enabled version of i2b2, software development groups should carefully consider the required effort. Furthermore, the current HQMF specification allows some query constructions that are computationally challenging. Multiple time relationships on a single criterion, the possibility of nesting time relationships and excerpts, arbitrarily deep nesting of population criteria groups, and the fact that not all population criteria operators are equivalent to logical operators suggests to us that running very complex queries could tax even powerful servers. Also, HQMF allows unrealistic query constructions. In particular, supporting 16 temporal relationships between criteria does not reflect the limited temporal granularity we have seen in existing clinical data warehouses. Finally, HQMF has limited ability to specify behavior when data are missing or noisy. Revisions to implementation guides and the HQMF standard will be needed to address these issues. Some of these difficulties  are being discussed in the current ballot reconciliations.HQMF is a powerful language for developing and computing measures of population health. Despite complexity concerns, we believe this format can be supported, which will be important in stage two of meaningful use and possibly required in stage three. HQMF is also a key component in Query Health and will likely play important roles in other health informatics initiatives. We have created a fully bidirectional HQMF translator, which converts between HQMF and i2b2-XML formats, supporting a superset of SHRINE’s features. This translator is freely available and has been integrated into the reference implementation of Query Health. Areas that we have been unable to implement will either be addressed in the next release of i2b2 or will be brought before appropriate decision-makers and standards developers. Expanding i2b2’s HQMF support through this XML translation approach will allow i2b2 to fully support the requirements of CQM processing in MU. The work lays a foundation for dynamic, distributed queries across diverse clinical systems with disparate data models.


Head and Neck Parapharyngeal space tumors are rare. Pleomorphic Adenomas are the most common Parapharyngeal space tumors. The purpose of this study was to define preoperative criteria for enabling full extirpation of parapharyngeal space pleomorphic adenomas via the transcervical approach while minimizing functional and cosmetic morbidity.The surgical records and medical charts of 19 females and 10 males with parapharyngeal space pleomorphic adenomas operated between 1993 and 2012 were reviewed.Fifteen patients were operated by a simple transcervical approach, 13 by a transparotid transcervical approach, and one by a transmandibular transcervical approach. Complications included facial nerve paralysis, infection, hemorrhage and first bite syndrome. There were three recurrences, but neither recurrence nor complications were associated with the type of surgical approach.A simple transcervical approach is preferred for parapharyngeal space pleomorphic adenomas with narrow attachments to the deep lobe of the parotid gland and for pleomorphic adenomas originating in a minor salivary gland within the parapharyngeal space. The parapharyngeal space (PPS) is often described as an inverted pyramid-like space whose base is at the sphenoid bone and apex at the greater cornu of the hyoid bone. We reviewed the medical records of all patients with PPS tumors referred to the senior author (D.M.F) between 1993 and 2012. Follow-up data were obtained for all patients from their clinical record notes, physical examinations and imaging studies. This study was approved by the institutional review board (IRB). Since only medical files were obtained, the IRB has approved this study without the need to obtain a signature on a patient consent form as long as all personal information has remained discrete including facial features and disclosing markings (IRB: TLV-0406-13).A computer-assisted search performed by the institutional tumor registry identified 108 patients with PPST. The eligibility criteria for inclusion in this study were confirmation of the diagnosis of PA of the PPS and surgery having been performed as the primary modality of treatment. Excluded were patients with deep lobe parotid tumors with minimal PPS involvement. Twenty-nine patients met the inclusion criteria and their medical charts were retrospectively reviewed to retrieve the data on age at operation, gender, tumor location, history of prior resections, surgical approach, additional treatment modalities received, outcome, and histological confirmation of the diagnosis and surgical pathology.This study was approved by the Tel-Aviv Sourasky institutional review board (IRB: TLV-0406-13). Since only medical files were obtained, the IRB has approved this study without the need to obtain a signature on a patient consent form as long as all personal information has remained discrete including facial features and disclosing markings. Patient records and information have been coded prior to analysis to blind researchers of all personal data.All cases of PPS tumors are preoperatively evaluated by the surgical and radiological teams in order to assess tumor origin and formulate a strategy for the surgical approach. According to our departmental protocol, patients with pre-styloid tumors suspected as being PPS PAs with a narrow attachment (elaborated in the discussion) of the tumor to the deep lobe of the parotid gland (DLPG) or those appearing with a separating plane of fat between the tumor and the DLPG on imaging studies, meet the criteria for a simple transcervical approach. Patients with broad attachments of the tumor to the DLPG are operated via the transparotid transcervical approach.The transcervical approach begins with a curvilinear incision within a natural skin crease in the neck, approximately 3 cm underneath the lower border of the mandible, and continues with an elevation of a subplatysmal flap to the height of the mandible. The submandibular gland is gently retracted medially, taking care not to damage the marginal mandibular branch. The sternocleidomastoid muscle is retracted posteriorly, and the accessory nerve and the digastric muscle are identified. The hypoglossal and lingual nerves are identified and preserved, and the facial artery is ligated if this is essential for a safe extirpation of the tumor, although this will not be necessary in most cases. We occasionally transect the tendon of the digastric muscle along with the stylohyoid muscle and the stylomandibular ligament in order to gain wider exposure of the operative field in cases of large or high-positioned tumors. In those cases not indicating transection of the digastric muscle, the approach to the PPST is made superior to the posterior digastric and posterior to the mandible. Extra-capsular dissection (ECD) is used to carefully detach the tumor from the surrounding tissues, bearing in mind that the carotid artery and jugular vein are located nearby. Using a right angle clamp, the tumor together with its stalk is transected from the parotid gland, thus leaving most of the deep lobe of the parotid gland intact. Generous irrigation with hot saline is used for homeostasis and starch cleansing in order to avoid tumor residue.The transparotid transcervical approach starts with the attachment of the patient to a facial monitor. A standard modified Blair incision is performed. Superficial parotidectomy is carried out next. After full exposure of all facial nerve branches, the deep lobe is dissected. The neck incision can be elongated as necessary in cases that the initial incision does not provide adequate exposure to the PPS. Since this approach endangers the facial nerve and is associated with adverse affects such as Frey's syndrome, we reserve it as mentioned for cases with broad attachments as pre determined by imaging studies.The transmandibular approach is rarely used, but is rather reserved for tumors necessitating wide exposure of the PPS, such as very large lesions, malignant transformations, revisions and masses that were exposed to radiotherapy. The transoral approach is reserved for very small tumors since it has some salient disadvantages, one of which is harboring a major risk in cases of major bleeding since control of the external carotid artery traversing the PPS is extremely difficult. Additionally, the fact that the surgical field is narrow can limit the extent of dissection and lead to the possibility of an incomplete tumor resection as well as to spillage. In contrast, the Da Vinci system offers the advantage of wide vision and superior precision that may overcome those drawbacks and enable a safe and efficacious transoral excision. Statistical analysis was performed using SAS software version 9.2 . Comparison between groups of patients was performed using the Mann-Whitney test for continuous variables and the Fisher's exact test for categorical variables. Time to first recurrence was plotted using Kaplan-Meier charts and compared between groups using the Log-Rank test.Twenty-nine patients underwent surgical resection for PPS PA between 1993 and 2012. There were 19 females and 10 males whose mean age at surgery was 46.4±14.6 years  and mean follow-up was 41.7±10.5 months . Three surgical approaches were used. A simple transcervical approach was chosen for 15 cases (52%), a transparotid transcervical approach for 13 cases (45%), and a transmandibular approach in one case (3%).The surgery-related complications included one permanent marginal mandibular nerve paralysis, one wound infection and one first bite syndrome in the transparotid transcervical group, and one episode of hemorrhage in the transcervical group .Twenty-six of the patients with PPS PAs (89.6%) are currently free of disease. There were three recurrences (10.3%), however two patients had recurred after being initially treated in other institutions and therefore they are not considered as being our failures. All three of those patients eventually underwent multiple resections as well as radiotherapy : one undp = 0.054 for recurrences).Comparison between the simple transcervical and transparotid transcervical approach revealed no significant differences in recurrences and complications (Many authors favor the transparotid transcervical approach for extirpating PPS PAs arising in the DLPG, By not excising the submandibular gland but rather gently retracting it, the surgeon shall not only preserve the gland but also protect the marginal mandibular branch. In the rare occasion where further exposure of the operating field is necessary, it can be facilitated by transection of the digastric muscle, stylohyoid muscle, stylomandibular ligament and ligation of the facial artery.There is close agreement among surgeons regarding the full excision of PAs as opposed to simply enucleating them, with mounting new evidence for ECD as being the preferred technique both in superficial parotid gland tumors and PPST.Another important issue concerns PPS PAs not originating from the DLPG but rather minor salivary glands occupying this space. This important distinction could also be recognized preoperatively by high-resolution imaging that reveals a fat plane between the deep lobe of the parotid gland and the tumor. Since even the best modality with the highest possible resolution may miss a small insertion, it is essential to make every effort to determine whether an attachment does exist and possibly excise a small cuff of the DLPG, regardless of negative preoperative imaging findings, in order to circumvent small overlooked attachments during ECDs.One major drawback aside from known limitations of retrospective analysis is the rather short follow-up time as PA's are notorious for their tendency for long-term recurrences. Nevertheless, it is safe to generalize that as far as PPS PA's, they tend to recur before the five year mark.Our results encourage us to prefer the described simple transcervical technique for excising PPS PAs with narrow attachments originating in the deep lobe of the parotid gland.


However, these circulating levels of β-amyloid in cerebrospinal fluid or plasma may not reliably reflect the actual degree of amyloid present in the brain. Therefore, we investigated the relation between WMLs and β-amyloid plaques and amyloid angiopathy in brain tissue. WML on MRI or CT were rated in 28 nondemented patients whose neuroimaging was available prior to death. β-amyloid in plaques and arterioles were immunohistochemically stained and quantified in postmortem brain necropsies. WMLs were present in 43% of the total population. Both cortex and periventricular region showed no differences for β-amyloid deposition in either plaques or blood vessel walls in patients with WMLs compared to those without WMLs. Thus, our results indicate that there is no relation between the degree of WMLs and β-amyloid deposition in the brain.Population-based studies have investigated the relation between  Cerebral white matter lesions (WMLs) are commonly found on cerebral magnetic resonance imaging (MRI) and computerized tomography (CT) in elderly subjects , 2. The From an etiological perspective, heterogeneity exists with respect to the presence of WML: on the one hand, they are related to cardiovascular risk factors, including hypertension and atherosclerosis –8, but oβ-amyloid in cerebrospinal fluid or plasma has been established [β-amyloid in cerebrospinal fluid and WML [β-amyloid pathology in brain tissue in these particular studies. There are a few neuropathological studies which encountered a relation between the degree of CAA, β-amyloid plaques, and the severity of WML in patients with clinically confirmed AD, albeit that both the amyloid and white matter pathology were assessed on a microscopic level and not with neuroimaging [On a population level, there is also evidence for such heterogeneity since a relation between WMLs on MRI and levels of ablished , 10. How and WML . Furtheroimaging , 13.To investigate the relation between cerebral amyloid pathology within white matter lesions and the degree of white matter lesions on neuroimaging, one should preferably look for amyloid pathology in a sample of the white matter. This may have important implications for our understanding of the etiology and found heterogeneity of presumed vascular white matter lesions and provide a model for interaction between vascular and amyloid pathology in the pathophysiology of WMLs. To our best knowledge, there are no studies relating the presence of WMLs on neuroimaging with neuropathologically assessed amyloid pathology.β-amyloid deposition in microvessels and plaques in necropsies from the cerebral white matter and WMLs and WMLs assessed on MRI in an autopsy-based study of nondemented patients.We therefore investigated the relation between the degree of The study population consisted of 28 consecutive patients without any age restriction from whom neuroimaging was available on average 2.8 months prior to death. No significant difference exists in time of imaging prior to death between the WMLs groups. Neuroimaging was performed for routine investigations because of a suspicion of a neurological disorder not related to dementia . None of the patients suffered from dementia according to NINDS-AIREN  and NINC3 in size, were taken from the frontal grey matter and the underlying periventricular region in a standardized way, guided by the obtained CT or MRI scan. The periventricular region was defined as the region from the ventricle wall extending 1 cm into the white matter.Within 16 hours after death, necropsies were taken from the frontal lobe region of the hemisphere which was predominantly affected by the WMLs. When trauma or stroke occurred in this hemisphere, the necropsy was taken from the other, unaffected hemisphere. Samples, each about 2 cmTissue was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and embedded in paraffin according to a standard protocol.β-amyloid was demonstrated immunohistochemically on 4 μm thin paraffin sections using monoclonal antibodies against the βA4-amyloid .β-amyloid, sections were digitalized and quantified, using a Zeiss Axioskop microscope equipped with hardware and software of Microbrightfield, . Quantitative analyses were performed with a computer-assisted analysis system (Stereo Investigator) using Cavalieri's probe. Total β-amyloid staining in both plaques and cerebral blood vessel walls was measured by two independent raters in a random 25 × 106 μm2 cortex and periventricular white matter area. The β-amyloid deposition in plaques was expressed as the area of β-amyloid positive plaques divided by the total area measured, times 100 in order to obtain a percentage of the affected area. Subsequently, β-amyloid deposition in cerebral blood vessels was quantified in a similar manner. Immunohistochemistry and determination of β-amyloid positivity was measured without knowledge of rating of the WMLs or any other clinical data.To determine the amount of WMLs were rated with the age-related white matter changes (ARWMCs) rating scale by one single experienced rater (FEdL) . The useTotal degree of WMLs was calculated by adding the region-specific scores of both hemispheres (range 0–30).β-amyloid load in plaques and in cerebral blood vessels in patients with WMLs (ARWMC > 0) or without WMLs (ARWMC = 0) with the Mann-Whitney U test because of a nonparametric distribution of the amyloid deposition. To identify differences in population characteristics between groups, the Mann-Whitney U test and Chi-square test were used.We compared the β-amyloid, adjusting for age and gender.To assess whether any observed differences were influenced by age, partial Spearman's correlations were calculated between WMLs and β-amyloid plaques was 0.08% (range 0.0 to 1.02%) in the cortex and 0.0003% (range 0.0 to 0.003%) in the periventricular white matter region. Mean coverage of the area by vascular amyloid was 0.005% (range 0.0 to 0.040%) in the cortex, with no amyloid found in vessels in the periventricular white matter.β-amyloid plaques than those without WMLs (0.17% (SD 0.33) versus 0.01% (SD 0.03); P < 0.05, β-amyloid coverage and WMLs. After adjusting by age, no correlation could be found between cortical β-amyloid coverage and WMLs .Patients with WMLs had significantly more cortical β-amyloid plaques in the periventricular area and vascular amyloid in both the cortical and periventricular area did not differ between the WMLs groups also not after adjustment for age and gender.The amount of β-amyloid deposition in the brain of elderly nondemented persons.We found no association between WMLs and β-amyloid in the brain, rather than an indirect measure of amyloid pathology .To our best knowledge, this study is the first to investigate a possible relation between amyloid pathology and the presence of WMLs on MRI in nondemented elderly by directly assessing the load of β-amyloid and WMLs are related.In accordance with a recent study by Jonsson et al. , our resβ42, following the rationale that CSF Aβ42 reduction is seen in AD due to deposition in β-amyloid plaques. They found that WMLs and hypercholesterolemia explained only 9% of the variability in CSF Aβ42, suggesting that other factors must play a role in the development of white matter lesions. Having hypercholesterolemia raised the probability of low Aβ42 levels by 20%, while for each point increase in WMLs score , the probability of low CSF Aβ42 levels only increased by 3%. In contrast to our findings, Aβ42 levels did not correlate with age. However, it remains controversial whether amyloid markers either in CSF or blood plasma reflect actual amyloid pathology in the brain [Our results are not completely in agreement with a study by Stenset et al. . They inhe brain –19.A possible weakness of our study was the small number of patients. Since not even a trend towards a relation between amyloid pathology in the brain and WMLs on neuroimaging could be detected in our sample, we expect this relation to be weak, especially compared to the strength of the association with other common factors like age. However, one has to keep in mind that a type II error may have lead to false negative findings in our study.Neuroimaging was performed on average 3 months prior to death. WMLs might have progressed in this limited time interval, but this is very unlikely, as the progression rate of WMLs is known to be very slow .Theoretically, staining of the tissue might vary by reduced immunoreactivity due to different postmortem times, but in this study, there were no differences in postmortem time between groups (data not shown).β-amyloid plaques in the cortex are already present [Before clinical signs of AD are perceptible, both WMLs assessed on MRI and  present , 21 and  present .As hypothesized by others, this suggests that cerebrovascular disease may be in the causal pathway for development of AD or interacts synergistically with AD pathology.β-amyloid plaques has been considered the defining characteristic of AD, and it forms the core of the most studied theoretical framework for AD: the amyloid cascade hypothesis [The presence of pothesis .On the other hand, WMLs might be more supportive of the vascular hypothesis , 25. WMLAs a result, some researchers are in favor of the amyloid cascade hypothesis, while others believe in the vascular hypothesis. However, the association between vascular risk factors and AD does not rule out degenerative mechanisms underlying pure AD. It can be hypothesized that vascular and degenerative mechanisms actually develop in parallel. Vascular brain injury could act additively or synergistically with concomitantAD pathology to produce more severe cognitive dysfunction than either process alone .β-amyloid load in the brain of elderly nondemented persons. Although they might have causal factors in common, WMLs and β-amyloid load in the brain could represent separate pathways to AD.To conclude, our results show no association between WMLs and Further research is necessary to clarify these complex relationships. Prospective, multimodal imaging studies in disease-free individuals that both visualize the degree of amyloid pathology   and the


Performance was reduced (−2.1%) in DEP-PLA vs CON (421.0±12.3 vs 412.4±9.7 s). However, performance was restored in DEP-CAF (404.6±17.1 s) compared with DEP-PLA, while no differences were found between DEP-CAF and CON. The anaerobic contribution was increased in DEP-CAF compared with both DEP-PLA and CON , and this was more pronounced in the first 3 km of the trial. Similarly, total anaerobic work was higher in DEP-CAF than in the other conditions. The integrated electromyographic activity, plasma lactate concentration, oxygen uptake, aerobic contribution and total aerobic work were not different between the conditions. The reduction in performance associated with low CHO availability is reversed with caffeine ingestion due to a higher anaerobic contribution, suggesting that caffeine could access an anaerobic “reserve” that is not used under normal conditions.The purpose this study was to examine the effects of caffeine ingestion on performance and energy expenditure (anaerobic and aerobic contribution) during a 4-km cycling time trial (TT) performed after a carbohydrate (CHO) availability-lowering exercise protocol. After preliminary and familiarization trials, seven amateur cyclists performed three 4-km cycling TT in a double-blind, randomized and crossover design. The trials were performed either after no previous exercise (CON), or after a CHO availability-lowering exercise protocol (DEP) performed in the previous evening, followed by either placebo (DEP-PLA) or 5 mg.kg The importance of endogenous carbohydrate (CHO) availability for high-intensity exercise performance has been well described in the literature 2max) after caffeine ingestion (5 mg.kg−1). Using the same exercise intensity and caffeine dose, Bell et al. 2peak was also found by Simmonds et al. −1). Although all of the aforementioned studies have investigated the effects of caffeine on performance during time-to-exhaustion tests, some studies have also found a positive effect of caffeine on performance during time-trials While low CHO availability seems to reduce the anaerobic contribution and impair performance during high-intensity exercise, acute ingestion of caffeine seems to have the opposite effect The mechanisms by which caffeine increases the anaerobic contribution and performance during high-intensity exercise is not fully understood, but it has been proposed that caffeine intake would promote an inhibitory action on adenosine receptors, which would increase the activity of the enzyme phosphofructokinase, thereby increasing anaerobic glycolysis tour de France), where the time to replenish endogenous CHO stores between sessions or races may not be sufficient. Furthermore, most studies with either caffeine Although several studies have investigated the isolated effects of both CHO availability and caffeine intake on anaerobic contribution and performance Therefore, the purpose of this study was to examine whether caffeine ingestion promotes an increase in the anaerobic contribution and performance during a 4-km cycling TT, when athletes begin the trial after reduced endogenous CHO availability 2peak 58.1±6.3 mL.kg−1.min−1) participated in this study. The participants had been training for at least 4 years, performed a weekly training volume of 12.1±4.3 h.wk−1 (250±101.3 km.wk−1) and competed regularly (∼20 competitions per year). The sample size required was estimated from the equation n = 8e2/d2, as suggested by Hopkins n, e, and d denote predicted sample size, coefficient of variation, and the magnitude of the treatment effect, respectively. Coefficient of variation was assumed to be 0.9% Before beginning the study, participants answered a questionnaire on readiness for physical activity (PAR-Q) and performed a cardiac screening test, including electrocardiogram. Seven amateur cyclists  ingestion one hour before the trial and 2) 12–14 h after a full-rest, followed by placebo ingestion one hour before the trial (CON). In the evenings before DEP-PLA and DEP-CAF trials, two additional visits were required and the athletes came to the laboratory to perform a validated protocol for reducing the endogenous CHO availability. All of the experimental trials were performed in the morning to avoid the impact of circadian variation Each athlete visited the laboratory on seven different occasions. On the first visit, athletes underwent an anthropometric assessment and an incremental test. During the second visit (after ∼48 h), athletes performed a 4-km cycling familiarization trial. During the next visits, the athletes performed three 4-km cycling TT in a randomized, double-blind and repeated-measures crossover design, separated by 7 days for washout, under the following conditions: 1) 12–14 h after a validated exercise-protocol designed to reduce endogenous CHO availability, followed by placebo (DEP-PLA) or caffeine (5 mg.kg2 and carbon dioxide production (VCO2) were measured breath-by-breath throughout the test with a gas analyzer . The gas analyzer was calibrated before each test in accordance with the manufacturer’s recommendations. The HRmax and VO2peak were taken as the highest value reached in the last stage and as the mean value obtained during the last 30 s of the test, respectively. The PPO was determinate as highest PO maintained during a complete stage. When the last stage was not completed, the PPO was determined in according the methods of Kuipers et al. The incremental test was performed on a cycle ergotrainer  and consisted of a 3-min warm-up at a power output (PO) corresponding to 100 W, followed by increments of 30 W every 3 min until voluntary exhaustion or when the athletes were not able to maintain pedal frequency between 80–90 rpm Forty-eight hours after the incremental test, the athletes performed a familiarization session with the TT procedures . The seat was fully adjustable vertically and horizontally for each cyclist before the TT, and cycling shoes were used to secure the feet to the pedals. The seat position was recorded and replicated during all subsequent experimental sessions. The athletes were instructed to perform the TT in the shortest possible time and to remain seated throughout test. The gear ratio was standardized at the beginning of each TT (53×16). However, immediately after the TT had started, participants were free to change the gear and pedal frequency as desired. The athletes were instructed to record all foods  consumed in the 48 hours before the familiarization test 2, respiratory exchange ratio (RER), HR, electromyographic activity (EMG), aerobic and anaerobic mechanical power output , work total (Wtot), total aerobic (Waer) and anaerobic work (Wan) were determined every 200 m and pooled for each 1 km. Blood samples (25 µL from an ear lobe) were obtained before the warm-up (rest), immediately before (Pre-TT) and 1 min after the TT (post-TT) to determine the plasma lactate concentrations [La]. The blood samples were transferred into micro-tubes containing sodium fluoride (NaF 1%) and then centrifuged at 3000 rpm at 5°C for 10 minutes. Plasma [La] was measured through enzymatic colorimetric reactions in a spectrophotometer  using commercial kits .The TT sequence began ∼96 hours after the familiarization test. Before each trial, the cycle ergotrainer was calibrated in accordance with the manufacturer’s recommendations. The TT was preceded by three maximal voluntary contractions (MVC) of the quadriceps muscles and a 5-min warm-up at 100 W (90 rpm) followed by a 5-min rest period . The dis2 and using gross mechanical efficiency estimated during the warm up, in accordance with Hettinga et al. 2 with the oxygen equivalent using the following equation:The Pan and Paer were calculated from RER, VOWe assumed a RER equal to 1.00 during the time trials Participants arrived at the laboratory at ∼8∶00 PM, at least two hours after their last evening meal. The protocol employed for reducing the endogenous CHO availability has previously been validated and has been shown to reduce endogenous CHO availability to ∼30% of pre-exercise values During the morning and afternoon of the CHO availability-lowering exercise protocol, participants followed, up to the beginning of the exercise, the same dietary pattern contained in their food record. However, after the exercise protocol was finished (∼ 10∶00 PM), the athletes received a low-CHO diet . Furthermore, the athletes received the same standardized, low-CHO breakfast  one hour before the trial in the next morning (∼ 8∶00 AM). In the CON trial, the athletes were asked to replicate the diet recorded 24 hours before the trial, and ate a standardized meal derived from their diet record . The breakfast consumed an hour before the CON trial was also derived from the diet recorded and consisted of 55.8±21.4% CHO, 26.5±14.7% fat and 17.7±7.8% protein .Before the MVC, participants performed a standardized warm-up, in a chair with the trunk-thigh angle at 90° and the knee at 60° from full leg extension (0°), consisting of four 5-s contractions of the knee extensors, interspersed by 30-s rest periods, at intensities corresponding to 50, 60, 70 and 80% of the maximum subjective force During the MVC and time trials, EMG signals of the vastus lateralis (VL) muscle of the right leg were recorded via bipolar Ag-AgCl surface electrode  at an interelectrode distance of 20 mm. We chose the VL muscle because it has been reported as the most appropriate to monitor EMG activity in the lower limb during a 4-km cycling TT The data are presented as means ± SD. Data distribution was analyzed using the Shapiro-Wilk test. two-way ANOVA with repeated-measures were used to verify the effect of condition and distance on the PO, Pan and Paer response. When a significant effect was found, the main effect was analyzed using the least significant difference test for pairwise comparisons. The Effect size (ES) and 95% confidence intervals (95% CI) were calculated to evaluate differences between conditions for mean values of Wtot, Waer, Wan, PO, Pan, Paer and time to cover the TT. The ES was calculated by dividing the difference between mean values of the conditions by the pooled SD Mean and individual values for performance are shown in Likewise, the mean PO was 7.0 and 10.8% lower (moderate effect), respectively, in DEP-PLA than in CON and DEP-CAF . A small effect was found in mean PO between DEP-CAF and CON . The Wtot  performeWhen values were expressed every 1-km interval , the PO Mean Pan during DEP-CAF was moderately greater than in CON  and DEP-PLA , and slightly lower  in DEP-PLA than in CON . The Pan in DEP-CAF for the first 3 km was significantly higher (P<0.05) than for DEP-PLA, but it was not significantly different from CON . No effeTotal Waer  generate2 during the 4-km cycling TT between the conditions , reported an improvement (3.1%) in 1-km cycling TT performance compared to a placebo. This improvement was accompanied by a 3.6% increase in the mean PO Previous studies have reported an increase in high-intensity exercise performance after caffeine ingestion Athletes adopted a more conservative starting pacing strategy when CHO stores were depleted. It is not fully clear if this conservative starting was caused by an intramuscular effect of the muscle glycogen depletion and/or a psychological strategy. However, despite the subjects being aware that they were depleted, they were not able to identify which capsule (caffeine or placebo) had subsequently been ingested, and, even then, adopted a more aggressive pacing strategy in DEP-CAF than DEP-PLA. Both iEMG and RPE at the beginning of the DEP-PLA trial were also similar to the DEP-CAF trial, even with a lower PO, suggesting that any effect of manipulation may have happened in the muscle. Furthermore, caffeine supplementation restored the PO in the first two kilometers and it was not associated with a reduced PO in the rest of the time trial.In contrast to the results of the present study, Hettinga et al. Even though mean PO was higher in the first 2 km of DEP-CAF compared with DEP-PLA, the RPE was not significantly different between the conditions, suggesting that participants were able to perform the first 2 km of a 4-km cycling TT with a higher PO/RPE ratio when caffeine was ingested. This is in accordance with previous results suggesting that, independent of the physiological or metabolic status, athletes normally adopt a pattern of increase in RPE proportional to the exercise distance completed 2max was ∼ 48% less in the leg that started the exercise with low muscle glycogen (99±18 µmol.kg dry wt−1) than the leg that began with normal muscle glycogen levels (207±22 µmol.kg dry wt−1). When analyzing each km, we observed an increase in the Pan during the first 3 km for DEP-CAF compared to the DEP-PLA. This large anaerobic production early in the trial was accompanied by a significant increase in the PO, supporting the idea that power distribution during a TT appears to be regulated primarily by changes in the anaerobic contribution According to some studies Several studies have suggested that the total amount of anaerobic energy that can be produced during a TT is fixed In the present study, we found that the total amount of anaerobic energy was higher in DEP-CAF than in both DEP-PLA and CON, indicating that caffeine exerts a more potent effect on the anaerobic contribution than low CHO availability. Furthermore, the greater Wan during the 4-km cycling TT after caffeine ingestion indicates that the total amount of anaerobic energy expenditure during TT exercise may not be fixed and that caffeine allows access to an anaerobic “reserve” that is not used under normal conditions. The existence of an anaerobic reserve has been demonstrated recently by Corbett et al. b to it more active form a, which would lead to an acceleration of glycogenolysis. However, this last mechanism seems unlikely in vivo, since a greater mobilization of calcium from sarcoplasmic reticulum has been observed only at high concentrations of caffeine that would be toxic to humans The mechanism by which caffeine ingestion exerts its effect on the anaerobic contribution remains unclear. Simmonds et al. It has been hypothesized that caffeine intake improves performance during high-intensity exercise through an increase in electromyography activity A potential limitation is that we did not include a control session (without CHO depletion) with caffeine ingestion. Despite the fact that this could have provided a more complete knowledge about the effects of caffeine on pacing strategy and TT performance with different levels of endogenous CHO availability, the inclusion of more experimental sessions had the potential to reduce the motivation of participants to participate in so many trials, potentially affecting our performance results. Thus, we focused the present study on verifying if caffeine could reverse the deleterious effect of low CHO availability on performance. In addition, the standardisation of the starting gear and the prevision of verbal instead visual distance feedback should also be acknowledged. However, the hard gear ratio as the used in the present study (53×16) has been described in the literature and justified due the high-intensity, fast-start nature of the 4-km cycling TT In conclusion, the results from the present study have shown that acute ingestion of caffeine induced a higher mean PO and anaerobic contribution during the first 2–3 km of the 4-km time trial. Furthermore, caffeine seems to attenuate the decrement in power output observed early in CON and DEP-PLA conditions, and preserves the ability to optimally perform the second half of the trial. In addition, caffeine ingestion reversed the impairment in time-trial performance caused by low CHO availability to levels found when endogenous CHO availability is normal. This improvement in performance was associated with a greater total anaerobic energy contribution with caffeine ingestion compared to both DEP-PLA and CON, indicating that caffeine exerts a more potent effect on the anaerobic contribution than low CHO availability. Therefore, we suggest that the total amount of anaerobic energy expenditure during TT exercise may not be fixed and that caffeine ingestion (even following a protocol to reduce endogenous CHO availability) allows access an anaerobic “reserve” that is not accessed under normal conditions. Our results suggest that caffeine may have an effect on intramuscular metabolism, i.e. increase on anaerobic contribution and total anaerobic work, rather than any significant effect on muscle recruitment.


Multicontrast MRI with T1, T2 and Ultrashort TE (UTE) sequences is used to image atherosclerotic plaque in human coronary arteries. MRI classification of the plaques is compared with their histological classification and found to correlate extremely well. The addition of UTE MRI adds significant value to the imaging of human coronary artery plaque by MRI.The differentiation of atherosclerotic plaque components in the carotid arteries with MRI has been successfully demonstrated. The detection of plaque calcification by MRI, however has been challenging. In addition, few studies have evaluated the ability of MRI to characterize atherosclerotic plaques in human coronary arteries (1). Here we use a combination of T1, T2 and ultrashort TE (UTE) MRI to evaluate atherosclerotic plaques in fixed post-mortem human coronary arteries. We hypothesized that the addition of UTE to T1and T2 weighted MRI would allow both calcified and lipid rich plaques to be accurately detected and distinguished from one another.Twenty eight plaques from human donor hearts with proven coronary artery disease were imaged on 9.4T horizontal bore MRI scanner . The specimens were imaged with a T1W 3D FLASH sequence , a T2W Rare sequence , and an UTE sequence . Plaques showing selective hypointensity on T2W MRI were classified as lipid rich. Areas of hypointesnity on the T1W images but not the UTE images were classified as calcified. After MRI, the plaques were sectioned for histological characterization with a pentachrome stain, which was used as the gold standard readout. The histological sections were digitized and co-registered with the MR images for analysis.Plaque classification by MRI was performed by 4 readers who were blinded to the histological plaque classification. The sensitivity and specificity of MRI for the detection of plaque calcification were 100.0% and 90.0% respectively. The sensitivity and specificity of MRI for the detection of lipid rich necrotic cores were 90.0%, 75.0%, respectively. Good inter-observer agreement was found for plaque classification by MRI . More importantly, MRI categorization of coronary artery plaques agreed well with the histological classification . An example of a lipid-rich necrotic plaque with calcification is shown in Figure MRI with T1, T2 and UTE contrast is able to accurately classify atherosclerotic plaques in human coronary arteries. This underscores the need to develop techniques to image the coronary artery wall in patients in vivo.R01 HL093038 and NCRR P41RR14075


Oxidized low-density lipoprotein (oxLDL) plays a key role in the formation of atherosclerotic plaques. However, its localization in human coronary arterial wall is not well understood. The present study was performed to visualize deposition sites and patterns of native oxLDL and their relation to plaque morphology in human coronary artery.Evans blue dye (EB) elicits a violet fluorescence by excitation at 345-nm and emission at 420-nm, and a reddish-brown fluorescence by excitation at 470-nm and emission at 515-nm characteristic of oxLDL only. Therefore, native oxLDL in excised human coronary artery were investigated by color fluorescent microscopy (CFM) using EB as a biomarker.(1) By luminal surface scan with CFM, the % incidence of oxLDL in 38 normal segments, 41 white plaques and 32 yellow plaques that were classified by conventional angioscopy, was respectively 26, 44 and 94, indicating significantly (p<0.05) higher incidence in the latter than the former two groups. Distribution pattern was classified as patchy, diffuse and web-like. Web-like pattern was observed only in yellow plaques with necrotic core. (2) By transected surface scan, oxLDL deposited within superficial layer in normal segments and diffusely within both superficial and deep layers in white and yellow plaques. In yellow plaques with necrotic core, oxLDL deposited not only in the marginal zone of the necrotic core but also in the fibrous cap.Taken into consideration of the well-known process of coronary plaque growth, the results suggest that oxLDL begins to deposit in human coronary artery wall before plaque formation and increasingly deposits with plaque growth, exhibiting different deposition sites and patterns depending on morphological changes. Oxidized low-density lipoprotein (oxLDL) plays a key role in the initiation, progression and destabilization of atherosclerotic plaques 125I-MDA2, 99mTc-MDA2, and 99mTc-labeled anti-LOX-1 mlgG Trafficking of oxLDL has been performed using radiotracers such as Imaging of native oxLDL in human coronary artery was performed immunohistochemically We succeeded in two-dimensional imaging of oxLDL in human coronary plaques by means of a color fluorescent angioscopy (CFA) system using Evans blue dye (EB) as a biomarker of oxLDL, both in vitro and in vivo The color fluorescence of oxLDL and other major substances that constitute atherosclerotic plaques  [27] wasThe intensity of fluorescence was categorized as strong, weak or absent when the exposure time required for imaging was taken to be within 1 s, >1 and ≤5 s, and >5 s, respectively.−5 M (the maximum concentration that does not precipitate) at 37°C and then mixed with each of the major substances in atherosclerotic plaques. The evoked fluorescence was photographed at ×40.In the present study, EB  was diluted in distilled water to a concentration of 10In the present study, conventional angioscopy using white light as a light source was used to classify the coronary plaques and normal segments because their angioscopic appearances represent their macroscopic pathological changes, and this technology is widely used clinically for detection of vulnerable coronary plaques Plaque is defined as a nonmobile, protuberant or lining mass that is clearly demarcated from the adjacent normal wall and the shape, location and color of which do not alter under the influence of a saline solution flush. Plaque is further classified as white or yellow based on its surface color. A normal segment is defined as milky-white and smooth-surfaced without any protrusions Images of the plaque obtained by conventional angioscopy were classified as white or yellow using an AquaCosmos image analyzer , which sets a window on an appropriate portion of an image and separates the color within the window into three primary colors, namely red, green and blue. Plaque was defined as “white” when the intensity ratio of red : green : blue was 1.0∶ 0.9∼1.1∶ 0.9∼1.1, respectively, and as “yellow” when it was 1.0∶ 0.8∼1.2∶ 0.3∼0.6, respectively The study was carried out with the approval of the Ethical Committees of the Japan Foundation for Cardiovascular Research, Ethical Committee of Toho University, Ethical Committee of Funabashi-Futawa Hospital, Ethical Committee of Chiba-kensei Hospital, and after obtaining written informed consent from the families concerned on the use of excised coronary arteries for pathological study to clarify mechanisms of coronary atherosclerosis.Fifty-five coronary arteries  were detached from 19 cadavers .A Y-connector was introduced into the proximal portion of each coronary artery for perfusion with saline solution at a rate of 10 mL/min and then the angioscope was introduced through the connector into the artery for observation. Initially, conventional angioscopy was carried out to detect plaque and because the light from the angioscope’s tip was visible through the coronary wall, the site of the targeted plaque could be confirmed.A total of 150 coronary segments which contained plaques and 79 normal segments were isolated by transecting their proximal and distal ends along the shorter axes to avoid any damage to the plaque. The isolated segment was then cut longitudinally to open the lumen.The 40 white plaques, 33 yellow plaques and 38 normal segments were mounted on deck glass in such a way that the luminal surface faced the deck glass. The surface was then scanned by CFM to investigate the fluorescence of the substances listed in In the remaining 42 white plaques, 35 yellow plaques and 41 normal segments, the center of each plaque or segment was transected and one half was mounted on a deck glass such that the transected surface faced the glass. After obtaining control picture, 0.1 mL EB solution was dripped onto the specimen. By this maneuver, the EB solution diffused into the transected surface and stained oxLDL.The plaques were divided into two layers, namely superficial  and deep , and relationships between the deposition site of oxLDL and plaque morphology studied by conventional angioscopy were examined.The fluorescent color, that was elicited by EB, was analyzed by AquaCosmos image analyzer. The fluorescent color obtained by CFM was divided into red, green and blue, and the fluorescent color was defined as “violet” when the ratio of red, green, and blue was 1.0∶0.65∼0.85∶0.75∼0.95, respectively, and it was defined as reddish-brown when it was 1.0∶0.35∼0.55∶0.20∼0.30, respectively.After CFA and CFM scanning, each sample was cut into slices along the shorter axis and stained with Oil Red O and methylene blue (MB), which stain lipids red, calcium black, and collagen fibers and smooth muscles blue 2 formulae. A value of p<0.05 was considered to be statistically significant. The differences in % incidence, deposition sites and patterns oxLDL were compared among normal segments, white plaques and yellow plaques.The data obtained were tested by χIn the presence of EB, oxLDL exhibited a violet fluorescence with “A”-imaging and a reddish-brown fluorescence with “B”-imaging . This coOn scanning the luminal surface, the % incidence of oxLDL deposition in 38 normal segments, 41 white plaques and 32 yellow plaques was 26, 44 and 94, respectively, indicating higher (p<0.05) incidence of oxLDL in yellow plaques than normal segment and white plaques. .On scanning the transected surface, the % incidence of oxLDL deposition in 41 normal segments, 42 white plaques, 14 yellow plaques without necrotic core (NC), and 18 yellow plaques with NC was 17, 45, 93 and 61, respectively, indicating higher incidence of oxLDL in white plaques and yellow plaques without NC than normal segments. There was a tendency that the % incidence in yellow plaques with NC was lower than that of yellow plaques without .The location on the scanned surface that exhibited combined violet and reddish-brown fluorescence was considered to be the site of deposited oxLDL.Thus, deposition pattern was classified as patchy, diffuse and web-like. Although the former two deposition patterns were observed in normal segments, white plaques and yellow plaques, web-like pattern was observed in yellow plaques but not in other groups .As shown in There was no significant difference in the incidence of oxLDL between yellow plaques with NC and those without. Deposition in superficial layer was predominantly observed in normal segments, deep layer deposition was not different among the three groups. Deposition in both layers in white plaques and yellow plaques with or without NC was significantly higher than normal segments . These dThe lipids that are stained by Oil Red O are cholesterol and cholesteryl esters and ceramide (unpubl. obs.), so in the present study the substances that were stained red in the coronary specimens were considered to be these lipids. There were normal segments and white plaques in which oxLDL deposited but the lipids did not. The lipids deposited in all yellow plaques but there were plaques in which oxLDL did not, indicating that it did not necessarily co-deposit with the lipids .We investigated low-molecular-weight substances that do not autofluoresce but elicit fluorescence when conjugated to oxLDL and discovered that EB elicits a reddish-brown fluorescence that is specific for oxLDL with “B”-imaging. Consequently, we used it with CFA to image oxLDL in human coronary plaques in both in vitro and in vivo The mechanism by which fluorescence of oxLDL is evoked is considered to be that EB deoxidizes oxLDL or that they conjugate each other to form an adduct eliciting the fluorescence colors. Because there are no other known major substances that show this combination of fluorescence colors, we considered this combination to be characteristic of oxLDL and that EB can be used as its biomarker.Localization of oxLDL has been studied mostly by immunohistochemical techniques; Kayo, et al observed deposition of oxLDL in NC (lipid core) but not in other portions of ruptured human coronary plaques that were obtained by endoatherectomy In the present study, the incidence of oxLDL increased in the order of normal segments, white plaques and yellow plaques without NC, but tended to decrease in yellow plaques with NC.White plaques are formed by intimal thickening induced mainly by proliferated collagen fibers; the white plaques grow into yellow plaques by accumulating lipids in themselves; acquire NC; and finally become vulnerable to disruption Although oxLDL is considered to be essential for formation of yellow plaques with necrotic core, a small number of yellow plaques with necrotic core did not contain oxLDL in the present study. It is known that chemical changes occur in the oxLDL that is accumulated in macrophages Both white plaques and normal segments were classified as with or without oxLDL deposition. No obvious differences for this result were found by histology or the sex and age of the patient. Growth factors such as fibroblast growth factor-2 and osteoprotegerin Deposition patterns of native oxLDL in vascular wall, especially in human coronary arterial wall, has been unclear because of a lack in systematic examinations on them.In the present study, the pattern of oxLDL deposition was classified as patchy, diffuse or web-like on scanning of the luminal surface. Deposition patterns were closely related to plaque morphology; in a patchy pattern in normal segments and white plaques, in a diffuse pattern in white plaques and yellow plaques without NC, and in web-like pattern in yellow plaques with NC. Although definite evidence is lacking, it is possible that the patchy and diffuse patterns indicate free deposition of oxLDL in the tissues, whereas the web-like deposition may indicate oxLDL accumulated in macrophages/foam cells, because this distribution pattern was observed solely in yellow plaques with a NC and because macrophages are known to exist in the fibrous cap covering the NC and accumulate oxLDL in themselves In most reports on molecular imaging by spectroscopy, oxLDL and other substances in atherosclerotic lesions showed dot-like distribution patterns There are many hard lines for clinical application of immunohistochemical techniques and radiolabelled antibodies or nanoparticles for imaging oxLDL in patients in vivo.EB has been clinically used for staining fibrin and the damaged vascular endothelial cells for many years and its clinical safety has been proven By using EB as a biomarker, deposition sited and patterns of oxLDL within coronary plaques and normal coronary segments of humans was successfully imaged by CFM. The results suggest that oxLDL begins to deposit in the wall of the human coronary artery before plaque formation and increasingly deposits with plaque growth, but tends to decrease after NC formation.The present experimental study of the sites and patterns of oxLDL deposition within human coronary plaques using CFM and EB as a biomarker seems to be the first of its kind. However, because atherosclerotic plaques contain a number of substances, there may be other unknown substances that exhibit the same fluorescence color in the presence of EB.