Recent advances in our understanding of cell signaling have revealed assemblies of signaling components often viewed in fluorescence microscopy as very large, irregular "punctae". These punctae are often dynamic in nature, appearing to act as mobile scaffolds that function in integrating protein-protein interactions from large arrays of signaling components. The visualization of these punctae, termed "signalsomes" when applied to protein assemblies involved in cell signaling provokes the question, what is the physical nature of these structures made visible in live cells through the expression of fluorescently-tagged fusion molecules?Mr (0.5 and 2 MDa). Activation of the Wnt canonical β-catenin/LEF-Tcf-sensitive transcriptional response leads to an upfield shift of >5 MDa of the Dvl3-based signalsome. Fluorescence correlation spectroscopy (fcs) is a single molecule analysis performed in live cells that experimentally measures the diffusion coefficient and permits calculation of MW of the signalsome (0.2 and 30 MDa species in vivo), which also reveal an upfield shift in MW in response to Wnt3a. Proteomics provides for molecular dissection of the composition of the signalsome isolated from untreated and Wnt3a-treated cells.Steric-exclusion chromatography on wide-bore matrices, fluorescence correlation spectroscopy, and advanced proteomics permits the analysis of several important physical properties of signalsomes. Wnt canonical signaling is essential to normal cell development and dysregulation can lead to cancers in humans. Punctae/signalsomes have been reported based upon the study of fluorescently-tagged mammalian Dishevelleds. Dishevelleds are phosphoprotein scaffolds that demonstrate dynamic character and mobility in cells stimulated with Wnt3a. Recent studies have successfully isolated Dvl3-based signalsomes from mouse totipotent embryonic teratocarcinoma F9 cells in culture and sized by application of steric exclusion chromatography (SEC), displaying large discrete Mr/MW, molecular composition, and intracellular locale.Dvl3-based punctae/signalsomes made visible by fluorescent microscopy now can be interrogated by advanced physical means, defining such properties as signalsome  So, we might anticipate that for at least some of the time, Dvls operate as scaffolds that are supermolecular complexes.Dsh/Dvls are unique phosphoprotein scaffolds essential for normal cell signaling and development in the animal kingdom-5. Dvls  pathways,6-9. How pathways, casein  pathways, casein  pathways,13, Daam pathways, etc.) o pathways, for exa pathways-19. The in vivo, including: How large are the complexes?; Where are they localized intracellularly?; How many other DIP partners are found in Dvl-based supermolecular complexes?; and, How does the composition of these punctate structures change in response to Wnt stimulation? We now know much more about the nature of these punctae: they are large, are multi-protein (or supermolecular), are not lipid vesicle-associated [Florescence microscopy from several labs (including the authors') uniformly reports the presence of Dvls as "punctate" structures in cells (using immune-tagged and auto-fluorescent-tagged Dvls)-27. Dr. membrane), includerd of the Dvl[rd  is largely bereft on known DIPs[rd of Dvl3 is the region of highest non-identity among Dvls and remains a region deserving of detailed interrogation for high-value DIPs, such as K-homology splicing regulator protein (KSRP)[All three Dvls microscopically appear as "punctae",23,26,27f the Dvl,33. The nown DIPs. This C-in (KSRP) and Ras fcs) in live cells; and, proteomic interrogation of Dvl3-based punctae, assuming that such punctae would survive biochemical isolation by SEC.How are such punctae to be interrogated, their physical nature and composition established? The punctate structures revealed for Dvls by fluorescence microscopy in live cells are suggestive of supermolecular complexes of large size. Biochemical analysis of individual Dvls by sodium dodecyl sulfate-polyacrylamide gel mobility has revealed that they form Dvl-based oligomers ,22. HomoMr) the complexes as well as enabling subsequent proteomic analysis, although requiring significant amounts of starting material. The second operates at the cell level using a single (or scanning) 1.5 femtoliter voxel and conditions in which expression of an autofluorescently-tagged molecule are maintained very low (5-50 molecules/cell). Under such conditions, fcs offers unparalleled analysis of calculated mass (MW) of complexes based in autofluorescently-tagged Dvl3. Isolating such "super" molecular complexes offers new challenges that render most common SEC techniques irrelevant, incapable of sieving such large molecular structures. SEC matrices more commonly found in industrial settings were exploited for these analyses. Very long column (60-90 cm), commercially packed under stringent conditions, can be adapted to provide optimal separation of large supermolecular complexes  can be calculated with precision. After several years of testing instruments and capabilities elsewhere, an fcs core facility was created, featuring two high-grade fcs-equipped fluorescence instruments. For some technologies, investigator-based training and experience is sufficient for most research applications. Based upon our own experience, an fcs center requires close collaboration with biophysicists who use these instruments in their own research to provide advice and assistance in applying fcs to new problems in cell signaling. The results of several years of experience have been both unexpected and remarkably exciting. We succeeded in performing fcs of eGFP-tagged Dvl3 in F9 cells as well as HEK293 cells[fcs of cells absent stimulation by Wnt3a is very large, observed in discrete sizes that display MW ranging from 25-35 MDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), SEC (molecular dissection of isolated peaks), and fcs  uniformly detect Dvl monomers and the dimers with high precision and good agreement. Significantly, establishing the physical nature of the large  and very large  Dvl3-based complexes brings us back to our initial goal of interrogating large punctae of Dvls observed in cells undergoing activation with Wnt.The second technology exploited was fluorescence correlation spectroscopy, s Figure . The dwe293 cells. The masper se. Is it possible to interrogate the nature of the proteins found in the Dvl3-based complexes? Initially this seemed impractical, since the yield of supermolecular complexes through a standard preparation was limiting. By migrating to mega-preps derived from 2-3 liters cell cultures of F9 cells, molecular dissection of Dvl3-based complexes isolated as distinct peaks from SEC by modern proteomic strategies became a reality.Are these large/very large supermolecular complexes simply large polymers of Dvls? By no means. We have been able to use the SEC coupled to proteomics to identify the DIP constituents of the large Dvl3-based complexes. As others have reported , we findfcs analysis revealed complexes in untreated F9 cells with MWs greater than those detected by SEC[fcs readily identified smaller monomeric and oligomeric assemblies of Dvl3, established by SEC and SDS-PAGE alike [fcs reflects true higher-order complex structure . Many non-covalent associations, however, are labile to shear forces that can be generated in SEC. Significant differences in apparent masses can be anticipated between in situ between fcs and SEC analyses. Further testing of industrial, large-pore matrices likely will continue to extend the upper reach of SEC from 10 to 40 MDa-Mr.As noted, SEC analysis successfully identified Dvl3-based complexes of large MW that displayed a >5 MDa-upfield shift in mass in response to Wnt3a stimulation. Yet theed by SEC. The fcsGE alike , providifcs, what distinct advantages does it afford us? An initial worry with fcs might be that its sensitivity and precision might be too limited. The SEC and fcs analyses of Dvl3-based complexes evolved in tandem and it became clear fortunately that the size of the Dvl3-based supermolecular complexes were ideal for fcs determination. The strengths of fcs are that one can obtain calculated MW of eGFP-tagged Dvl3-based supermolecular complexes in live cells, under native conditions, sampling intracellular domains through precision placement of the very small voxel in which the targets are tracked .What about Mr peaks observed only when cells were stimulated with Wnt3a. One cardinal concern is always that of the potential presence of contaminating proteins/complexes of Mr similar to those that are Dvl3-based. The upfield shift in apparent mass observed in the Dvl3-based complexes isolated from Wnt3a-treated cells holds the key to this challenge. Operationally, unrelated proteins/complexes "contaminating" Dvl3-based supermolecular complexes isolated from untreated cells by definition would not co-migrate with the higher Mr Dvl3-based assemblies formed in response to Wnt3a. The upfield shift of >5 MDa moves the Dvl3-based complexes to a less populated region of the chromatogram. SEC and proteomics employed in tandem have born this out in early experiments, i.e., DIPs and some new classes of signaling proteins have been identified. Within this novel group, proteins involved in mRNA stability and AKAPs (AKAP5 and AKAP12) have emerged (unpublished data) and are of keen interest. The ability to contrast the proteomic profiles of Dvl3-based peaks isolated from untreated versus Wnt3a-treated cells thus becomes an enabler for focusing upon DIPs in the Dvl3-based complexes. Proteomic identification of complex constituents will enable determination of docked/undocked partners during Wnt activation.The third strategy necessary to address the initial hypothesis on the composition of the Dishevelled-based punctae is proteomics. Proteomic analysis offers an ideal strategy with which to interrogate the composition of Dvl3-based supermolecular complexes. Assuming sufficient starting materials from peaks of Dvl-based complexes isolated by SEC, the goal would be to interrogate both the peaks isolated from untreated cells as well as those upfield, higher-Mr peaks). If "contamination" of the lower Mr peaks is suspected, peaks can be pooled and subjected to field IEF in porous gels, to characterize, enrich, and identify  Dvl3-based complexes, segregated from other "contaminating" complexes. Such highly-enriched peaks can be subjected to proteolytic degradation and proteomic analysis by LQ-ESI-MS-MS, requiring a state-of-the-art proteomic center with staff experienced in probing low abundance targets in complex mixtures. Key molecules of known function have been readily identified in Dvl3-based 2 MDa-Mr peaks, assembled in response to Wnt3a and activation of the Wnt/β-catenin canonical pathway . Novel molecules, such as AKAP5 and AKAP12 that are key in cAMP-based signaling, were newly identified (unpublished observations). Positive identification is ascribed only to a protein for which at least 10 unique fragments have been fully sequenced, from multiple runs, and multiple samplings. What can be accomplished through such a marriage of SEC and proteomics is really impressive: identifying large order assemblies, discerning constituent partners and following dynamic assembly in response to signaling pathway manipulation.Dvl3-based supermolecular complexes subjected to such molecular dissection yield much new information. A typical mega-prep of cells yields about 0.5-0.7 mg of protein in the 2-5 MDa peaks. Ten to 20% of the pooled peak sample is consumed for each analysis subjected to fragmentation and to liquid chromatography electrospray ionization tandem mass spectrometry (LQ-ESI-MS-MS) analysis  were often observed as "puncta". These puncta were large, irregular in shape, and the subject of speculation as to their actual mass and composition. For heterotrimeric G-protein-based signaling, punctate structures were the rule, not the exception.Wnt signaling is heterotrimeric G-protein-linked in flies as it is frogs and zebrafish. Downstream of intrinsic membrane proteins, e.g., Frizzled-1 and LRP6, involved in canonical Wnt signaling are structures which integrate and propagate the upstream signals. Dishevelleds are core proteins in virtually all aspects of Wnt signaling and were shown early to form large irregular punctae by a variety of optical based analyses. Known to dock important protein kinases, adaptors, and phosphatases, Dvls provided an ideal test case in which to ask more detailed queries about the size, composition, and dynamic character of these phosphoprotein scaffolds, characterized earlier as simply punctate structures.Mr. This approach demonstrated that Dvl-based supermolecular complexes could be prepared and their hydrodynamic properties characterized by SEC. This advance demonstrated feasibility, which was the overarching challenge to establishing what these punctate signalsomes are physically.As recently reported, Dvl-based punctae have been shown in fact to be large supermolecular structures that can be prepared from cells and sized by steric exclusion chromatography. The Dvl3-based structures can be subjected to molecular sieving on wide-bore matrices packed in long columns calibrated and operated with state-of-the-art FPLC systems. The peaks identified are discrete, displaying highly reproducible Mr of >5 MDa. Furthermore, activation of the canonical pathway in the absence of Wnt3a which stimulates Lef/Tcf-sensitive transcription was preceded by formation of the Dvl3-based signalsomes. Blocking the ability of Wnt3a to activate the canonical pathway at several distinct levels likewise blocked signalsome formation. Thus the linkage between signalsome formation and function was established in this pathway.It was essential to gain insights into earlier observations that the Dvl-based punctae are influenced by treating cells with Wnt3a, activating the canonical beta-catenin, Lef/Tcf-sensitive transcriptional response. Interrogation of the Dvl3-based complexes by SEC revealed a marked upfield shift in Mr of 1-2 MDa of the chromatogram, the amounts of total protein present in that region of the profile has fallen off dramatically from that observed at 0.2-0.5 MDa (unpublished observations). In addition, the sharp large (>5 MDa) upfield shift provoked by Wnt3a alone would enrich the Dvl3-based signalsomes, moving these signalsome well away from "contaminating" complexes encountered downfield. Starting with mega-preps of cells in culture, sufficient material can be prepared to perform proteomic interrogation of peaks of supermolecular complexes like the Dvl3-based complexes, to find out something about these very large punctae/signalsomes. Constituent partners that might be expected  as well as unexpected, curious partners  were identified by LQ-ESI-MS-MS. The application of field isoelectric focusing (FIEF) of the signalsomes in large-pore gels matrices may provide an additional enrichment. By comparing the proteomic profiles from the 1 MDa-Mr complexes (absence of Wnt stimulation) versus >5 MDa-Mr complexes (assembled in response to Wnt3a), it will be possible to define the relative abundance of partners, assuming that the ionization of the fragments is equivalent. Through such a strategy other supermolecular complexes/signalsomes assembled in response to signaling become fully addressable. For any such supermolecular complex to assemble in a bona fide signalsome, the assembly must occur in advance of the propagation of the signal downstream. For Wnt canonical signaling, the assembly of the upfield Dvl3-based signaling complexes is largely completed by 30-60 min, well before the Lef/Tcf-sensitive transcriptional activation occurs.Physical isolation of the Dvl3-based signalsomes was an advance, but would the composition of resolved peaks of the SEC-sized supermolecular complexes be addressable? As noted above, these peaks migrated to upfield of much of the proteome/complex profile. Even at fcs capabilities offer many novel and powerful insights for the study of signalsomes. As a single molecule analysis that can be targeted for sampling in discrete locales in live cells, fcs is only beginning to be exploited for the study of signalsome mass and dynamic mobility. The Dvls, shown to display mobility in cells stimulated with Wnt3a, offer an ideal test case for sampling the diffusion coefficient of their signalsomes in perinuclear, cytosolic, and juxtamembrane regions of the cell. The hypothesis to be tested is that the apparent MW of these Dvl-based signalsomes will reflect changes in size as the signalsome is assembled and also where in the cell the assembly is occurring. Upon docking to the inner leaflet of the membrane, for example, the apparent MW would increase infinitely when the diffusion coefficient is derived from a molecule essentially immobilized at the membrane. But this scenario remains largely speculation, awaiting empirical analysis in cells undergoing activation of the Wnt canonical pathway using autofluorescently-tagged Dvl3  as the signal.The Mr derived from SEC and the MW calculated by fcs for the Dvl3-based signalsomes of unstimulated cells deserve some interrogation. The MW of the Dvl3-based supermolecular complex is several-fold larger than that reported on the bases of SEC analysis. The MW determined by the fcs data benefits from the fact that this single molecule analysis is performed in situ, in live cells. This is not to say that fcs estimates of MW are without bias, since the strategy does require assumptions about the hydrodynamic properties of the complex, such as axial ratio and partial specific volume, that potentially bias the calculations. On the other hand, the SEC analysis provides a very precise Mr for complexes subjected to molecular sieving. Thus, it must be kept in mind that the shear force created as a result of the size of the complex, the pressure under which the molecular sieving is being performed, and the viscosity of the fluid can be considerable. It is possible that the large and very large complexes under study may have some higher order structure of assembly that is lost to the shear force of SEC. Attempts either to minimize the shear force by reducing the SEC pressure to the point of just overcoming back diffusion or to change the matrix employed for SEC to an ever larger bore may provide an avenue by which to interrogate the role of shear force, if any, in jeopardizing the integrity of a much larger Dvl3-based complex. Alternatively, sampling fcs from numerous subcellular locales, e.g., cytosol, perinuclear, and juxtamembrane, may reveal Dvl3-based complexes in live cells with MW in closer agreement with those assigned by SEC.The differences between the apparent Mr of the large, supermolecular entities such as signalsomes. Testing the function of such complexes, if addressable once isolated from the cell, is rather challenging, even when the system of Wnt signaling is involved. There are numerous tools that can be applied to the Wnt canonical pathway to either stimulate or inhibit signaling at various levels. Used judiciously, these tools can probe the extent to which complex and formation and function are related. For Dvl3-based signalsomes the structure/function relationship seems quite strong. Key mutants of Dvl3 have been identified that block signalsome formation in response to Wnt3a. These have been shown to block downstream signaling and can be understood in the context of interrupting important protein-protein interactions of Dvl. Ideally, one would seek to identify mutations in either Dvl or Axin that interfere with assembly of Dvl3-based signalsomes, although scanning for such mutants randomly would be laborious. Structure-function studies of Dvl3  can yield insights, directing one where to look first for interesting protein-protein interactions essential to the formation of complex/signalsomes and downstream signaling. Higher-order assemblies, identified by fcs, may be based upon weak interactions that are susceptible to the shear force (or cell dissolution) generated by the SEC under current conditions.Researchers who study cell signaling do so from a broad array of disciplines and interests. For development and Wnt signaling in particular, the approaches are diverse, providing a collect synergy that has brought major advances to the study of early development. Dishevelled is a phosphoprotein scaffold that has a critical, but not fully understood, role in development, examined in the fly, frog, zebrafish, mouse and human species. Dishevelled has been under interrogation by microscopy for years, studies uniformly revealing this key signaling protein as dynamic "punctate" structures of relatively large dimensions. But just what in fact are such large signaling punctae observed in the context of cell signaling? A fundamental hypothesis from the microscopic analysis to the current day can be stated simply in the question, "what is the physical nature and functional significance of signaling punctae, often referred to as signalsomes?" Three strategies are briefly described herein that afford unprecendented ability to address the central question. Assuming one can break open the cell and retain the punctae, it is possible to subject these complexes to SEC to arrive at an approximation of the fcs data benefits from its ability to be extracted from rather small volumes in discrete regions of the cells in vivo. This benefit is offset, to some extent, by our ignorance of other cell constituents that may be bona fide docking sites for signalsomes. Even transient docking of signalsomes to other cellular entities can artificially increase the MW derived from fcs, especially for proteins with significant dwell times in docking. Docking to the inner leaflet of the cell membrane to LRP6 or Frizzled GPCRs would reduce the diffusion coefficient of the signalsome, which would artificially increase the apparent MW in the final analysis by fcs. Modern proteomic capabilities provide a portal to identify complex partners assembled into signalsomes. When activation of a pathway leads to a physically larger signalsome , samples derived from basal as opposed to stimulated cells can be subjected to proteomics to address what partners have been added/lost to the signalsome as it assembles to propagate the signal downstream? On the surface, the intersection of SEC, fcs, and proteomics would seem unbeatable in sorting out the structure/function of very large supermolecular complexes encountered in a variety of cell signaling pathway. Whether or not such cautious optimism will proves well-founded remains to be seen. Painstaking application of this powerful trio of techniques to a broader array of targets, not just signalsomes, will undoubtedly be required to test the central hypothesis. The Dvl3-based signalsome has taken on a decidedly new look based upon recent advances in technology; this outcome alone was worth the investment of time and resources.The Steric-exclusion chromatography, fluorescence correlation spectroscopy, and advanced proteomics afford biologists powerful new ways to interrogate structure/function relationships of very large supermolecular complexes, typified by Dishevelled-based signalsomes involved in Wnt signaling in early development.2. For transfection, plasmid DNA was introduced into cells by using LipofectAMINE and PLUS Reagent (Invitrogen) as per the manufacturer's instruction. Stable clones were selected in medium containing G418 (0.4 mg/ml).Mouse F9 teratocarcinoma cells were obtained from the ATCC collection . Cells were cultured in Dulbecco's modified Eagle's medium  supplemented with 15% heat-inactivated fetal bovine serum  at 37°C in a humidified atmosphere with 5%COExpression vector pcDNA3.1 harboring eGFP-tagged human Dvls  was constructed as described . Human Dfcs measurements were performed in the Fluorescence Correlation Spectroscopy Center at Stony Brook (Department of Physiology & Biophysics) using a Zeiss LSM 510 Meta/Confocor 2 apparatus  fitted with a 40X NA 1.2 C-Apochromat water-immersion objective.The D = 3 × 10-6 cm2/second) in water [fcs [Standard configurations and minimal laser powers are employed to avoid photobleaching. Routinely, the pinholes are adjusted at least daily and the detection volume calibrated by measuring the diffusion of Rhodamine  of diffusing complexes the complexes are assumed to be spherical and the data processed using the following equation to determine the hydrodynamic radius:where, k is Boltzmann's constant, T is the temperature, η is the viscosity of the solvent, and R is the hydrodynamic radius. MW is related to R by following equation:-7 cm2/s and the MW = 27 kDa.where, V is volume and http://www.osa.sunysb.edu/Proteomics/Instrumentation.html, accessed 12062010).The liquid chromatography electrospray ionization tandem mass spectrometry (LQ-ESI-MS-MS) analyses were performed at the Stony Brook Proteomic Center at the Health Science Center (Samples were first filtered (0.45 μm) and diluted with buffer. Typically 20 mg protein is applied to the Superdex 200 (or 400) gel filtration column , pre-equilibrated with 20 mM Tris-HCl, pH 8.0, 0.2 M NaCl, and 1.0% glycerol. The fractions collected were maximally 0.9 ml/each. Each fraction was analyzed by SDS-PAGE and immunoblotting. Protein concentration was determined using the Bradford assay. For more precise analysis of 1- 7+ MDa complexes, cell lysates were subjected to SEC on a Sephacryl S-400 gel filtration column (HiPrep Sephacryl S-400 high-resolution column) operated by fast-performance AKTA liquid chromatography .fcs: fluorescence correlation spectroscopy; KD: knock-down; KSRP: K-homology splicing regulator protein; LQ-ESI-MS-MS: liquid chromatography electrospray ionization tandem mass spectrometry; Rfz1: rat Frizzled-1; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEC: steric-exclusion chromatographyDMEM: Dulbecco's modified Eagle's medium; Dvl1: Dishevelled-1; Dvl2: Dishevelled-2; Dvl3: Dishevelled-3; Dsh: Fly Dishevelled; F9: mouse totipotent embryonal teratocarcinoma F9 cells; HEK 293: human embryonic kidney 293 cells; The authors declare that they have no competing interests.The authors contributed equally to the evolution of the study design; the authors contributed equally, performed the studies, gathered the data, and outlined a draft of the manuscript. HYW wrote the manuscript; CCM edited the drafts, read and approved the final version of this unpublished work. Each author read and approved the final manuscript.


High levels of the plasma peptides mid-regional pro-adrenomedullin (MR-proADM) and mid-regional pro-atrial natriuretic peptide (MR-proANP) are associated with clinical outcomes in the general population. Data in patients with chronic kidney disease are sparse. We therefore investigated the association of MR-proANP and MR-proADM levels with all-cause and cardiovascular (CV) mortality, CV events and peripheral arterial disease in 201 incident dialysis patients of the INVOR-Study prospectively followed for a period of up to more than 7 years. The overall mortality rate was 43%, thereof 43% due to CV events. Both baseline MR-proANP and MR-proADM were associated with higher risk of all-cause  and CV mortality  after adjustment for age, sex, previous CV events, diabetes mellitus and time-dependent type of renal replacement therapy. We then stratified patients in high risk (both peptides in the upper tertile), intermediate risk (only one of the two peptides in the upper tertile) and low risk (none in the upper tertile). Although demographic, clinical and laboratory variables were similar among the intermediate and high risk group, to be with both parameters in the upper tertile was associated with a 3-fold higher risk for all-cause  and CV mortality . In summary, among incident dialysis patients MR-proANP and MR-proADM were shown to be associated with all-cause and CV mortality, with the highest risk when both parameters were in the upper tertiles. Patients with chronic kidney disease (CKD) are at increased risk for death. Cardiovascular and non-cardiovascular mortality are 8 to 9 times higher in incident dialysis patients when compared to the general population There is evidence that peptides involved in maintaining the cardiovascular and renal homeostasis such as atrial natriuretic peptide (ANP) and adrenomedullin (ADM) may play a key role in the compensatory mechanisms of CKD. Both hormones are elevated in the early stages of CKD The aim of this study was therefore to investigate whether MR-proANP and MR-proADM plasma concentrations at the start of dialysis treatment are associated with risk of cardiovascular or all-cause mortality in a prospective cohort study of incident dialysis patients.The INVOR-Study Ethic statement: The study was approved by the ethics committee of the Innsbruck Medical University and all patients enrolled in the study provided written informed consent.st, 2000 and April 30th, 2006 were consecutively enrolled with the advantage that all patients of this region are treated by the same care provider. During this period of 6 years a total number of 235 incident dialysis patients were included and followed until the study endpoint was reached or follow-up was censored at December 31st, 2009. Ten patients having a malignant tumor at initiation of dialysis were not recruited defined by the exclusion criteria. Due to inappropriate or missing blood samples MR-proANP and MR-proADM was measured in 201 out of 235 patients. All data and analyses described in this manuscript are based on these 201 patients.All incident dialysis patients from this province starting chronic dialysis treatment between May 1Clinical, laboratory and medication data were collected prospectively starting at the time of initiation of dialysis. These data included age, sex, height, weight, body mass index, diabetes status and current smoking status. Type of and change in renal replacement therapy  were recorded and considered as time-dependent treatment status for data analysis. Vascular access procedures and the type of vascular access  for hemodialysis were also evaluated.Information on the following clinical events were collected before initiation of dialysis and during the entire observation period thereafter: coronary artery disease , cardiovascular disease , peripheral arterial disease .We used two novel commercially available fully automated sandwich immunoassays for the measurement of MR-proANP (B.R.A.H.M.S MR-proANP KRYPTOR) and MR-proADM (B.R.A.H.M.S MR-proADM KRYPTOR) according to the manufacturer's instruction manuals . The design of these assays is based on immunoluminometric assays described previously As described earlier, the limit of quantitation was 4.5 pmol/L for the MR-proANP assay with a within-run imprecision coefficient of variation (CV) of <4.5% between 10 and 20 pmol/L and <2.5% between 20 and 1000 pmol/L, and between-run imprecision CV of <6.5% between 10 and 1000 pmol/L The first outcome of interest was all-cause mortality and cardiovascular mortality. We also investigated other endpoints such as cardiovascular disease and peripheral arterial disease. Cardiovascular mortality was defined as death of myocardial infarction, heart failure, sudden death, ischemic or hemorrhagic stroke. Cardiovascular disease (CVD) events were defined as fatal and non-fatal myocardial infarction, percutaneous transluminal coronary angioplasty (PTCA), aortocoronary bypass (ACBP), angiographically-proven coronary stenosis ≥50%, ischemic cerebral infarction or transient ischemic attack. For peripheral arterial disease (PAD) at least one of the following events was existent: significant ultrasound- or angiographically-proven vascular stenosis, percutaneous transluminal angioplasty (PTA), peripheral bypass or amputation. An incident PAD event was only considered as a first time manifestation or a deterioration of PAD in terms of e.g. a change in PAD stage according to Fontaine. Two patients were lost to follow-up, one regained renal function and the other one moved away.2-test and continuous variables were analyzed using an unpaired t-test or the non-parametric Wilcoxon rank-sum test. Data are presented as mean±SD and as median and 25th and 75th percentiles for skewed variables where appropriate.At baseline, categorical data were compared using χTo investigate the influence of MR-proANP and MR-proADM on all-cause mortality, cardiovascular mortality, cardiovascular disease and peripheral arterial disease, multivariable adjusted Cox-proportional hazards regression models were performed. In all analyses a p-value of 0.05 was considered significant. Variables were chosen for the multiple Cox regression analysis since they were either well-known risk factors for all-cause or CV mortality or they showed correlations with MR-proADM or MR-proANP. The first model was adjusted for age, sex, previous cardiovascular events, diabetes mellitus and type of renal replacement therapy, which was modeled time-dependently (Model 1). The second model (Model 2) was additionally adjusted for albumin, C-reactive protein, current smoking status, native fistula and echocardiographic data (ejection fraction ≤60% versus >60%). Due to sparseness of the data and risk of overfitting, this fully-adjusted model should only be considered as a sensitivity analysis to Model 1, which is taken as the main model in this investigation. Linear relationship assumption of MR-proANP and MR-proADM in the Cox models was tested, as well as the proportional hazards assumption. One standard deviation (SD) was taken as the unit of measure for each of the continuous outcome variables to ensure comparability of Hazard Ratios. For ease of interpretation, additional fully adjusted Cox-proportional hazards regression models were calculated by dividing MR-proANP and MR-proADM into tertiles and also by stratifying patients in groups of high risk (both MR-proANP and MR-proADM in the upper tertile), intermediate risk (only one of the two parameters in the upper tertile) and low risk (none of the two parameters in the upper tertile). Adjusted survival curves are given for each of these risk groups and for each of the tertiles, holding all covariates fixed at their mean level. We also performed a sensitivity analysis with censoring at the time of transplantation. All analyses were conducted in SPSS version 16.0 software  and R using the package “survival”.2 = 0.62).When we analyzed the data according to tertiles of MR-proANP and MR-proADM , a non-sThis increased risk was clearly identified using a stratification based on the tertiles of the two peptides by combining the highest tertiles of MR-proANP (≥794 pmol/L) and MR-proADM (≥3.11 nmol/L). It might introduce some considerable bias to the data analysis when a patient is selected for transplantation and follow-up time is censored at the time of transplantation. For this reason the above main analysis was performed by a time-dependent modeling of the renal replacement therapy status as discussed earlier The study at hand investigated the two peptides MR-proANP and MR-proADM in a prospective long-term cohort study of incident dialysis patients. We observed that increased concentrations of both peptides are associated with an increased risk of all-cause as well as cardiovascular mortality and this risk was about threefold elevated when both parameters were in the upper tertile of the entire patient group. This elevation of both parameters discriminated especially patients with intermediate and high risk which were otherwise similar in terms of clinical and laboratory parameters. Therefore, the incremental benefit to identify high risk patients is achieved by the combined evaluation of these two biomarkers. If a patient had an isolated increase in one of the two parameters, the risk was similar compared to patients which did show an increase at all see . ObviousPresumably, ANP reflects the central volume overload and intrinsic heart disease, whereas ADM reflects the decompensated reaction to the multifactorial stress state in preserving the integrity of the cardiovascular system in ESRD (22). It is of interest that we observed a strong association with all-cause and cardiovascular mortality but not with cardiovascular events combining fatal and non-fatal events. The slight increase in risk for CVD events  if both peptides were in the upper tertile was driven by the fatal CVD events. If these fatal events were excluded, the estimates for non-fatal CVD events did not show in any direction . This underscores that the pronounced increases of both peptides might reflect more the hemodynamic disturbances related to cardiac dysfunction rather than atherosclerotic processes resulting in non-fatal CVD events without hemodynamic decompensation. The combined measurement of these two peptides might provide surrogate variables for volume status but also act as an early indicator of deteriorating hemodynamic changes.The diagnostic and prognostic utility of MR-proADM has been shown in various non-CKD cohorts and for different endpoints such as heart failure, cardiovascular events, and all-cause mortality It is currently unclear whether the systemic and renal haemodynamic effects are caused by MR-proADM itself or predominantly through its active peptide ADM since an active role of MR-proADM is currently unclear. However, the eligible measurement of the active hormone ADM was doubted largely mainly due to the short half-life of 22 minutes and the partial binding to complement factor H Our findings complement and extend previously reported cohort studies of end-stage renal disease for the biomarkers ANP MR-proANP concentrations were substantially increased in our cohort of dialysis patients when compared to patients with mild to moderate impairment of kidney function To date prognostic biomarkers which predict outcome in this high risk cohort of CKD patients are sparse but highly demanded. However, as we discussed recently The prospective recruitment of all patients starting dialysis treatment over a period of six years in a clearly described area allowed a complete recruitment of patients requiring renal replacement therapy with almost no loss to follow-up during a long observation period. We can therefore exclude the most important bias of cross-sectional studies with a mix of prevalent and incident cases and the resulting survival bias. We furthermore considered even the observation period following kidney transplantation by modeling the treatment status in a time-dependent model, but on the other hand excluded by sensitivity analysis, that this procedure has obscured our results.There are also some limitations of this study. A first limitation is the relatively small number of patients and events. We therefore could only adjust our analyses for a small number of variables (6 variables in Model 1). An extended adjustment with more variables  was provided but should only be considered as sensitivity analysis. Interestingly, the estimates for MR-proADM and MR-proANP remained widely stable in Models 1 and 2, the additional sensitivity analyses and a very simple model only adjusted for age and sex. This argues for a pronounced independence of the two investigated variables for the prediction of endpoints. Second, we examined patients at the time immediately before renal replacement therapy was started. Therefore, some patients might not have been in a steady metabolic or extracellular volume state of the parameters evaluated. Concentrations of these hormones might therefore not necessarily be applicable to other cohorts of patients with CKD. On the other hand, values measured after the start of dialysis treatment as done in other mostly smaller studies might represent dialysis dose or dialysis efficacy and thus answer a different question. Third, our study includes only Caucasian patients of a clearly described geographical region with almost complete ascertainment of incident dialysis patients over a defined period of time. It therefore lacks generalizability to other ethnic populations as well as other recruitment procedures. Finally, since the measurements of the two peptides are not rigorously standardized, concentrations measured by various assays are not necessarily comparable.Among incident dialysis patients MR-proANP and MR-proADM were shown to be associated with all-cause mortality and cardiovascular mortality, with the highest risk when both parameters were in the upper tertiles.Table S1The association of MR-proANP and MR-proADM as well as MR-proANP tertiles and MR-proADM tertiles and furthermore for patients with high risk (both MR-proANP and MR-proADM in the highest tertile) with different endpoints using multiple Cox-proportional hazards models.(PDF)Click here for additional data file.


EMBO Molecular Medicine, Cabezas-Wallscheid et al. bring us a step closer to this goal with an improved Tet-inducible mouse model of t-associated AML -associated human acute myeloid leukaemia (AML) have been difficult to develop, limiting insight into the malignancy associated with this frequent chromosomal translocation. In this issue of The t is found in approximately 10% of human AML and encodes for the leukaemia fusion protein AML1-ETO (RUNX1-RUNX1T1)  malignancy that appears to satisfy these conditions and will undoubtedly improve our understanding of AML1-ETO function  AML is generally associated with a good prognosis, with 60–70% of patients being cured of their disease in response to standard chemotherapy. Nevertheless, 30–40% of t patients relapse and the prognosis for relapsed AML is dismal , over 50% of the mice showed disease progression. Whether this results from cooperating events that substitute for the AML1-ETO signal or is due to incomplete loss of AML1-ETO expression upon doxycycline removal remains to be determined. This is an important question, given the efforts that are focused on targeting the AML1-ETO protein directly , with varying success and no accepted consensus as to which approach is both faithful to the human disease and relatively tractable as a model system…The authors performed a number of different gene expression comparisons to determine the nature of the signals associated with short-term expression of AML1-ETO as well as the signals present at the time of disease manifestation in the L-GMP. In essence, attempting to identify AML1-ETO-specific signals as well as cooperating signals important for disease progression. The new model faithfully recapitulated many of the findings that have been made in other model systems, and showed a gene expression pattern that mirrors the signature found in t blast samples expressing AML1-ETO. In addition, the nature of the cooperating events in the L-GMP, identified by comparing signatures from Day 10 AML1-ETO-induced GMP and L-GMP from overtly leukaemic animals, fit with patterns expected from numerous studies, implicating cellular signalling pathways associated with transformation, survival and proliferation as critical for cooperation with AML1-ETO signalling. Although this particular study utilized the GMP and L-GMP in comparative analyses, it will be of great interest to repeat these studies using HSC in place of GMP. While many human AML samples do in fact have populations with cellular phenotypes resembling progenitor cells  that demonstrate leukaemogenic activity in xenograft studies, the vast majority of human AML patient samples show highest leukaemic activity in cells that resemble HSC (CD34+CD38−) (Bonnet and Dick, Many questions remain unanswered regarding the nature of the signals downstream of AML1-ETO and the particular cooperating events that lead to leukaemia progression in the context of this oncogene. This novel inducible model represents a new tool in the arsenal for leukaemia researchers. In addition, it can serve as a powerful therapeutic screening model once the appropriate cooperating events are supplied to allow progression to a lethal AML disease.Conflict of interest statement: J.C.M. is a Scholar of the Leukemia and Lymphoma Society. The author declares that he has no conflict of interest.


We examined trends in AIDS-defining illnesses (ADIs) among individuals receiving highly active antiretroviral therapy (HAART) in British Columbia (BC), Canada to determine whether declines in ADIs could be contributing to previously observed improvements in life-expectancy among HAART patients in BC since 1996.HAART-naïve individuals aged ≥ 18 years who initiated treatment in BC each of the following time-periods 1996 - 1998; 1999 - 2001; 2002 - 2004; 2005 - 2007 were included. The proportion of participants with reported ADIs were examined for each time period and trends were analyzed using the Cochran-Armitage Trend Test. Cox proportional hazards models were used to examine factors associated with ADIs.A total of 3721 individuals  initiated HAART during the study period. A total of 251 reports of ADIs were received from 214 unique patients. These occurred in a median of 4 months (IQR = 1-19 months) from HAART initiation. The proportion of individuals with a reported ADI did not change significantly from 4.6% in the earliest time period to 5.8% in the latest period (p = 0.181 for test of trend). There were no significant declines in any specific ADI over the study period. Multivariable Cox models found that individuals initiating HAART during 2002-04 were at an increased risk of ADIs  in comparison to 1996 - 98, but there were no significant differences in other time periods.Trends in reported ADIs among individuals receiving HAART since 1996 in BC do not appear to parallel improvements in life-expectancy over the same period. The introduction of highly active antiretroviral therapy (HAART) in 1996 resulted in significant reductions in HIV/AIDS morbidity and improved survival among HIV-infected individuals compared to the pre-HAART era -9. This Life-expectancy of individuals initiating HAART in British Columbia (BC), Canada has continued to increase since the introduction of HAART . In 1996In this study we examined trends in reported ADIs among participants who initiated HAART in the BC HIV/AIDS Drug Treatment Program during the years 1996 to 2007.The BC HIV, Drug Treatment Program (DTP) provides free antiretroviral medications to all medically eligible HIV-infected individuals free-of-charge . Data foCD4 cell counts were measured using flow cytometry and fluorescent monoclonal antibody analysis , and HIV viral load was measured using the Roche Amplicor Monitor assay . Adherence to HAART was defined as the number of days for which HAART is dispensed divided by the number of days for which HAART is prescribed in the first year of treatment. Deaths are recorded through physician reports and through record linkages between the DTP and the British Columbia Vital Statistic registry. Physicians of DTP participants are mailed a form to assess the clinical stage of HIV disease each year, based on the CDC classification, and are mailed another form if their patient discontinues treatment.Physician reports of ADIs (CDC Stage C diseases) and patient characteristics were studied for HOMER participants who began treatment in each of the following time-periods: 1996-1998, 1999-2001, 2002-2004, and 2005-2007. We also conducted a data linkage with the provincial cancer registry in order to identify additional AIDS-defining cancers which were not reported by physicians. Patients were followed from the date of starting HAART until the date of ADI (if a condition was reported) or the later of date of death or last laboratory result, to a maximum of 36 months after beginning therapy. The overall reporting rate by physicians was calculated by the number of staging and discontinuation forms returned divided by the number of forms sent.We compared participant characteristics using Chi-square and Kruskal-Wallis tests. ADI trends and overall reporting trends over time were analyzed using the Cochran-Armitage Trend Test. We calculated 12-months ADI event rates using life tables and constructed Kaplan-Meier curves to examine the time to first ADI diagnosis. Cox proportional hazards models were used to examine independent factors associated with time-to-ADIs. The final multivariate model was constructed using a backward stepwise procedure, with era of HAART initiation forced into the model. To examine the effect of treatment adherence in each era we ran models with and without adherence measures to determine if this affected our results. All analyses were conducted using SAS version 9.1.3 .A total of 3721 individuals  initiated HAART during the study period. The median baseline CD4 count was 190 cells/μL (interquartile range [IQR] 90 - 310 cells/μL) and 644 (15%) participants had AIDS at baseline. Table Pneumocystis jirovecii pneumonia (17%) and Non-Hodgkin's lymphoma (15%) were the most commonly reported and/or diagnosed ADIs. The proportion of individuals with at least one reported ADI was 4.5% for individuals initiating HAART in 1996-98, 5.7% in 1999 - 2001, 7.8% in 2002-04 and 5.5% in 2005-07. We did not observe a statistically significant trend in the proportion of participants with any ADI over the study period , or any cause-specific ADI. The median CD4 cell count for those with ADIs were 140; 95; 70 and 80 cells/μL for each time period (p = 0.213). The 12-month probability of reported ADIs in each time-period was as follows: 1996-98 = 0.020 ; 1999-2001 = 0.038 (0.025-0.050); 2002-04 = 0.059 (0.042-0.076); 2005-07 = 0.047 (0.034-0.060). A similar trend was also reflected in the Kaplan-Meier analysis of ADI-free survival which found significant differences between the different periods of HAART (log-rank test p = 0.008), with the 2002 - 2004 period having the highest risk of ADI (data not shown).The median follow-up time for all patients was 53 months (IQR 24-101 months) during which there were 251 ADIs reported from 214 patients ; and the inclusion of NNRTIs in the first drug regimen . A multivariate model which included adherence to therapy also found no difference in risk for ADIs associated with the time-period in which participants initiated HAART.In the multivariable model Table , we founWe reviewed the number of physician's reports submitted for other programmatic reasons such as clinical staging or medication discontinuation forms to see if ADI reports could have been influenced by changes in overall physician reporting. Our results indicate that the number of other physician reports decreased significantly over the study period with 63% of physicians submitting at least one report in 96-98, 62% in 1999-2001, 44% in 2002-04 and 49% in 2005-07..The proportion of individuals with reported ADIs within 36 months of treatment initiation has not changed significantly among individuals accessing HAART in BC over a 12-year period. Considering that the baseline CD4 remained relatively constant, it was not surprising that the incidence of reported ADI's did not significantly change. However, this result is somewhat unexpected given the improvements in life-expectancy we have seen in the same period in this population [The importance of reductions in non-AIDS related conditions contributing to improvements in clinical outcomes has been previously highlighted by the SMART ,15 studyWe did find that individuals who initiated treatment during 2002-04, did have an increased risk of being diagnosed with an ADI. However this does not appear to be part of a trend towards an increased or decreased risk over time. It is noteworthy that this time-period was characterized by the lowest median baseline CD4 cell counts (150 cells/μL), but that this relationship persisted even after adjustment for baseline CD4 counts. Since this period was prior to the release of the SMART data, when medically supervised treatment interrupted were considered a reasonable part of clinical management, it is possible that such interruptions may have contributed to the increase incidence of ADIs during this period.Our results contrast somewhat with a recent examination of rates of ADIs among participants in the HIV Outpatient Study, which did find significant reductions in the incidence of ADIs between 2003-2007 in comparison to 1998-2002 . The HOPWhile we did not find significant changes in the rates of ADIs in later time-periods, a significant minority of HAART patients continue to experience serious illness even in the latest time period. Early linkage of HIV patients to care and better adherence to treatment plan have been shown to prevent the development of ADIs  and imprThere are several limitations to our study. Firstly, the number of ADIs reported in each time-period was quite small which limited our ability to detect significant changes in reported cases. Secondly, we expect that physicians underreport ADIs events, however, this underreporting appears to be greatest in the later time-periods which should have biased our results towards showing significant declines, and instead the ADI rate remained statistically unchanged. Conversely, it is also possible that physicians have become more astute or vigilant about reporting AIDS over time. Third, there is a possibility of variations in the quality of reports of cases with the ADI. This can be less of a problem for ADIs with clear diagnostic criteria  than with more subjective diagnoses (wasting). Lastly, as with all observational studies the lack of difference we have observed may be confounded by other factors which differ between the time-periods and we are unable to measure.The overall incidence of ADIs after HAART has not changed significantly after the introduction of the HAART in BC. These observations suggest that previously described recent improvements in the life expectancy among patients initiating HAART might have been because of reductions in the occurrence of other non-AIDS related clinical conditions. Further research is needed to examine this hypothesis.JSGM has received funding from Merck, Gilead and ViiV Healthcare to support research into Treatment as Prevention, consultancy fees from Merck, and speakers' fees from Clinical Care Options. RSH has received a research grant from Merck and a conference travel grant from GlaxoSmithKline. None of the other authors have any known competing interests.DMM, JSGM, RSH and SJ conceived of the study, and participated in its design and coordination. BY, VL and RSH supervised the data collection and the preparation of the dataset for analysis. KC conducted all of the data analysis. SJ wrote the first drafts of the paper, incorporated the comments of the other authors and was assisted by KA. All authors approved the final version of the manuscript for submission.


Pontastacus leptodactylus, in the context of short term hyperglycemia. Hence, two different chemically synthesized cHH enantiomers, containing either L- or D-Phe3, were injected to the circulation of intermolt females following removal of their X organ-Sinus gland complex. The effects triggered by the injection of the two alternate isomers were detected after one hour through measurement of circulating glucose levels. Triggered changes of the transcriptome expression profile in the hepatopancreas were analyzed by RNA-seq. A whole transcriptome shotgun sequence assembly provided the assumedly complete transcriptome of P. leptodactylus hepatopancreas, followed by RNA-seq analysis of changes in the expression level of many genes caused by the application of each of the hormone isomers. Circulating glucose levels were much higher in response to the D-isoform than to the L-isoform injection, one hour from injection. Similarly, the RNA-seq analysis confirmed a stronger effect on gene expression following the administration of D-cHH, while just limited alterations were caused by the L-isomer. These findings demonstrated a more prominent short term effect of the D-cHH on the transcription profile and shed light on the effect of the D-isomer on specific functional gene groups. Another contribution of the study is the construction of a de novo assembly of the hepatopancreas transcriptome, consisting of 39,935 contigs, that dramatically increases the molecular information available for this species and for crustaceans in general, providing an efficient tool for studying gene expression patterns in this organ.The crustacean Hyperglycemic Hormone (cHH) is a neuropeptide present in many decapods. Two different chiral isomers are simultaneously present in Astacid crayfish and their specific biological functions are still poorly understood. The present study is aimed at better understanding the potentially different effect of each of the isomers on the hepatopancreatic gene expression profile in the crayfish  Pontastacus leptodactylus, commonly called narrow-clawed crayfish, inhabits Western Asian and Eastern European lakes and watercourses. Its reproductive season includes mating from December to January and spawning in January. Eggs are incubated glued to the female pleopods till July P. leptodactylus maintained at 19°C, led to a premolt period of 17 days ending with ecdysis . Adults naturally shed their exoskeleton in summer just after hatching, even though some of them molt also in autumn The freshwater astacid Crustacean hyperglycemic hormones (cHHs) are a pleiotropic crustacean-specific neuropeptide family, functioning in a variety of physiological processes, recently reviewed by several authors Orconectes limosus have been clarified by Ollivaux and Soyez de novo sequenced through a multifaceted mass spectrometry approach by Jia and colleagues The structure of the cHH genes, the derived precursors and peptides have been recently reviewed by Webster and colleagues P. leptodactylus is not considered a model organism, it is one of the most studied species among decapods with about 143 published papers regarding its physiology O. limosus. He also identified the involvement of the second messengers: cAMP, cGMP and calcium ion and a participating adenylate cyclase. However, no effect on gene expression was examined and no distinction was made between the two O. limosus cHH isomers, discovered later P.leptodactylus even one hour post-injection is well documented de novo assembly of the hepatopancreas transcriptome, dramatically increased the molecular information available for this species and undoubtedly provides a useful tool for further gene expression experiments in this organ.Although P. leptodactylus were bought from a local dealer on July, 2011. They were kept for two weeks before the experiment in 120 L tanks provided with closed circuit filtered and thoroughly aerated tap water at ∼18°C and were fed by fish pellets  three times per week. Only females with no abdominally incubated ova were taken for the induction experiment. The females were at the end of the ova incubation period.Adult P. leptodactylus females were divided into four groups each composed of 8 females. Two of the groups were injected with D- and L-cHH, respectively . A control group was sham-injected by the hormone carrier (S). Bilateral ablation of the XOSG, aimed at prevention of any possible interference due to endogenous cHH was performed 48 hours before the injections, and a fourth group of naïve females (N) was added to the experiment as control just before injections. All 32 females were sacrificed one hour post-injection. The incubation period included ten minutes of anesthesia in ice before sacrifice and all efforts were made to minimize the suffering of the animal. Immediately prior to the hormonal injection the females were bled for the evaluation of the pre-induction hemolymphatic glucose level. A second bleeding was performed just before sacrifice. Hemocytes were pelleted from the sampled hemolymph and the serum was kept on ice for later glucose measurement, which was performed using a glucose oxidase method . The statistical analyses of glucose levels recorded in the four groups and females' morphometric parameters were performed using R software, version 2.14.1 The chemical synthesis of the peptides and the glucose level induction protocol were accomplished according to Mosco et al. The experiments comply with the current laws of Italy, the country in which they were done. No specific permits were required for the studies that did not involve endangered or protected species. Individuals were maintained in appropriate laboratory conditions to guarantee their welfare and responsiveness. After the experiments were completed, crayfish were sacrificed by hypothermia.Twelve out of 32 females were randomly chosen for the RNA sequencing. RNA was extracted from frozen tissues, homogenized in TriReagent RNA isolation solution  following the manufacturer's instructions. The resulted RNAs were further purified using the RNeasy kit . The RNA level was quantified by spectrophotometer and its quality was examined using capillary electrophoresis .RNA sequencing was carried out at the Applied Genomics Institute , on an Illumina sequencer HiSeq2000. The hepatopancreas reference transcriptome assembly was derived from a 2×100 bp paired-end sequencing performed on a cDNA library obtained from equimolar amounts of RNA taken from all experimental females. Gene expression was evaluated using the single-end 50 bp Illumina sequencing from the 12 distinct RNAs.P. leptodactylus hepatopancreas. We determined the presumptive amount of conceptual full length transcripts by using the Full-lengther Next webtool th aa and with an e-value below 1e-04. Filtered contigs displaying an average coverage lower than 25× of mapped 50 bp reads were discarded prior to the RNA-seq analysis for creating a robust set of contigs not subject to random expression fluctuations, corresponding to the 95th percentile of the genes expressed in the hepatopancreas throughout the experiment.The processing and analysis of the obtained raw sequences was carried out using the CLC Genomics Workbench 4.5 software . Raw sequence reads were trimmed according to base calling quality. The resulting 2×100 bp sequence reads were assembled assuming a paired reads distance between 100 and 600 base pairs, and setting the penalties for mismatches to 2, insertions and deletions to 3 and similarity and length fraction to 0.9 and 0.5, respectively. The minimum allowed assembled contig length was set to 200 bp. The obtained contig assembly served as a comprehensive reference for the functional genomics RNA-seq analysis The 50 bp sequencing reads from the 12 different samples  were individually mapped on the filtered reference set using the RNA-seq procedure, considering a maximum number of mismatches cost of 3 and a maximum of 10 hits for a read. The expression values were calculated based on unique gene reads. The Baggerly's test 6. The default Blast2Go similarity annotations were also checked manually against the list of resemblances obtained for each contig by BLASTx, to conform to the UniProt nomenclature guidelines and to select the annotations in a more educated manner, based on the GO terms and domain characterization described below. Blast2Go was used also to assign Gene Ontology (GO) functional terms Differentially expressed transcripts were characterized with the Blast2Go platform The morphometric characteristics and the molt stage of the experimental females as well as their hemolymphatic glucose levels before injection and at their sacrifice are presented in P. leptodactylus females, generated 296,112,296 nucleotide reads. The number was reduced to 289,731,590 after quality trimming. The de novo assembly by the CLC Genomic Workbench produced 42,187 contigs with an average length of 802 bp. This assembly was used as a reference transcript library for subsequent analyses. A total of 9,474 contigs longer than 1 Kb was obtained, while the longest contig almost reached 15 Kb in length. rd (32.2%) of the transcripts were putatively assembled to their full length, 1/3rd (32.6%) covered either the N-terminal or the C-terminal region, whereas the remaining 35.2% consisted of internal fragments.The Illumina 2×100 bp sequencing of the hepatopancreas of adult P. leptodactylus reference library. The two most represented species were Daphnia pulex (Crustacea) and Tribolium castaneum (Insecta). The first thirteen BLAST top-hits species are reported in Figure S1. To date, the only crustacean whose genome has been fully sequenced is the branchiopod Daphnia pulexCherax quadricarinatus, performed by 454 was simultaneously prepared by the present co-authors and their collaborators and will be soon released for the public (TSA: GADE00000000). At present, only 275 nucleotide sequences belonging to the genus Pontastacus are stored at NCBI, while only 67 originated from Pontastacus leptodactylus. Next generation sequencing greatly simplifies large-scale molecular studies of non-model organisms, and this transcriptome sequencing with the assembly of 39,935 contigs, represents the first large-scale sequencing approach in this genus, as well as a remarkable contribution to the genetic knowledge of decapods, paving the way to straightforward comparative molecular studies on non-model crustaceans.From the output of Blast2Go we checked the thirteen Top-hits species sharing similarity with the Table S1. The expression profiles of the various females, namely the list of mapped reads per each gene in each sample were clustered by two methods, hierarchical clustering  and a principal component analysis , which highlighted a large difference between D-cHH-injected animals and all the other groups, which in turn clustered together and did not display detectable differences in this preliminary analysis.An average of 12.7±5.2 million short reads were obtained from the sequencing of RNAs extracted from the hepatopancreas of each sampled female. An average of 50.8±6.9% reads were mapped to the reference transcriptome. The comparison between the S and N groups was useful to depict the effects derived solely from the XOSG removal procedure. XOSG extirpation resulted in the differential expression of 214 transcripts, with up-regulation  exceeding down-regulation . It is likely that the transcripts characterized by an increase of expression in respect to the naïve state are connected to two main events, the injury and the reduced production of several hormones caused by the XOSG removal. 63% of the up-regulated and 67% of the down-regulated genes showed no resemblance to known sequences. Among the genes which demonstrated similarity we identified in particular some transcripts connected to glycolysis process, such as transcript containing enolase domain (IPR000941), probably connected to the removal of cHHs production sites.The hypergeometric test highlighted the carbohydrate metabolism (GO:0005975) and the chitinase activity (GO:0004568) as the most relevant GO terms repressed by XOSG extirpation; and many associated Interpro domains, e.g. glycoside hydrolase, family 9 (IPR001701), six-hairpin glycosidase (IPR012341) and chitinase II (IPR011583) were significantly repressed. Many other GO terms and Interpro signatures were found to be up-regulated, even though most of them could not be easily linked to any reported physiological effect of the extirpation. The most studied process in many crustacean species triggered by XOSG extirpation is undoubtedly vitellogenesis P. leptodactylus we observed a significant repression of the IPR domain of the chitinase II (IPR011583) within the S group. This transcript pertains to the group 1 reported in Salma et al. P. leptodactylus the repression of six transcripts associated to metabolic process (GO:0008152) was observed in the S group: differently from Marsupenaeus japonicusAs for other genes, in pandalid shrimp, eyestalk ablation down-regulated the hepatopancreatic chitinase expression and also in Cherax quadricarinatus by Yudkovski et al. C. quadricarinatus experiment in contrary to the present experiment in which they were not differentially expressed. In general, the reduction in the hepatopancreas metabolic activity was more pronounced nine days after ablation than in P. leptodactylus two days after ablation.A similar although somewhat different experiment was carried out in the crayfish XOSG removal affects also the circulating levels of hormones other than the cHH. These include the cHH type II hormones The injection of the two cHHs, distinguished only by chirality, showed different effects on gene expression. The overall difference can be clearly demonstrated by the results of the clustering procedures revealing relatively different expression profile of the D-cHH-treated group in comparison to the other three groups, and in turn elucidated much more similar expression profiles with each other . More inTo examine a possible overlap in the effects exerted by L- and D-cHH we identified differentially expressed transcripts shared by the two treatments. Six transcripts elucidated differential expression caused by both treatments, but the majority of them had different trends of expression. No transcripts shared up-regulation caused by both treatments, while 2 non-annotated transcripts were mutually down-regulated (Pastle_hepa_c8804 and Pastle_hepa_c22565). Three transcripts were up-regulated after L-cHH, but down-regulated following D-cHH injection; among them a trypsin 4, a transcript containing chitinase II domain (IPR011583) and one without similarity. Only one non annotated transcript resulted to be up-regulated in D and down-regulated in the L group.In the light of the results described above, the analysis was mainly focused on the variations between the S and the D groups.As previously shown by the PCA analysis, the D-cHH administration caused pronounced alterations in comparison to the sham injected females, dominated by down-regulated genes . The hypergeometric test elucidated a few D-cHH up-regulated functions, and in particular several GO terms related to peptidases, including cysteine-type peptidase (GO:0008234) and endopeptidase activity (GO:0004197), the papain domain of peptidase C1A, (IPR013128) and the cathepsin propeptide domain of proteinase inhibitor I29 (IPR013201), which is usually found in the N-terminus of many proteinases.On the contrary, glycolysis (GO:0006096) appeared to be the GO term most significantly repressed in the D-cHH-injected female crayfish, which may be related to the return of the glycemic level to the native state, and suggests a modulation activity of the D-cHH on the glucose and energy metabolism in the hepatopancreas.th (50 out of 147) of the up-regulated transcripts in the S group were restored at lower level following the D-cHH injection , but also prevents the secretion of other cHH superfamily hormones (cHH type II), possibly causing a series of gene expression changes not solely related to the deprivation of cHH itself. Therefore, the administration of synthetic cHH was only expected to partially restore the gene expression profile observed prior to XOSG ablation. In fact, about 1/3+/D-glucose co-transport The L-configuration provides the peptide with a strictly hyperglycemic activity whereas the D-cHH may exhibit other functions, in addition to a strong hyperglycemic potency. The finding of several transcripts containing a sodium symporter domain (IPR002657) suggests that D-cHH may also regulate glucose absorption by the hepatopancreas, a process that occurs through a NaIt has to be emphasized that the experiment was not intended to mimic natural situation in which the two hormonal species may be present in different levels, not necessarily reflected the injected amounts, but to apply rather pharmacological dose aimed at distinguishing the effects of the two isoforms. The data all together suggest that, despite being apparently able to restore glycemia to almost normal levels in eye-ablated animals one hour post injection see Table 1,P. leptodactylus were examined here. The presence of both cHH isomers has been demonstrated only in Astacoidea P. leptodactylus sequenced transcripts required the construction of a comprehensive transcriptomic library of its hepatopancreas, comprising 39,935 contigs obtained through Illumina sequencing. This library was used as a reference gene assembly in a RNA-seq gene expression experiment. The short term effect of two enantiomers of the peptidic hyperglycemic hormone (D-cHH and L-cHH) of P. leptodactylus on hepatopancreatic gene expression was examined at two levels, the specific gene level and the functional group level.The effects of two cHH enantiomers on a multi-gene expression profile of The overall effect of the different treatments was demonstrated by clustering the 12 individual transcript profiles, which resulted with prominent difference of the transcription profiles of the D-cHH treated females in respect to all other samples.This overall effect is detailed as follows: The XOSG synthesizes and secretes multiple peptidic hormones, including D- and L-cHH. The effect of its removal on gene expression profiles revealed the differential expression of 214 transcripts, 147 up-regulated and 67 down-regulated. The carbohydrate metabolic activity was repressed as expected from the elimination of the glycemia inducing hormone.The L-cHH-injected individuals revealed an expression profile similar to sham injected individuals. In contrary, D-cHH caused a considerable short term change in the hepatopancreatic gene expression profile. 917 transcripts were responsive to this isomer: 857 out of them were down regulated and only 60 were up-regulated. The functions mainly affected were the up-regulation of the proteolytic activity and the down-regulation of glycolysis. L-cHH caused much less pronounced effects, with only 45 differentially expressed genes (30 up-regulated and 15 down-regulated), without the detectable alteration of any specific function.The hypothesized glycemia-related mechanisms of L- and D- cHH may involve protein activation with no changes in gene expression for both isomers and an additional D-cHH mechanism which involves changes in gene expression, which may be related or not to the increase of glucose levels in the circulation.The D-cHH effect on gene expression was also demonstrated by the hypergeometric test, which distinguished an opposite trend related to the glycolysis which was revealed in the XOSG ablated group (S). The later rend was characterized by the up regulation of the enolase (IPR000941) that resulted to be significantly repressed after the D-cHH administration. Moreover, this short-time study showed that D-cHH was apparently able to restore the expression of a certain number of transcripts to the level of the naïve state, and it was also able to affect molecular pathways not altered by the XOSG ablation.Figure S1P. leptodactylus contigs.The distribution of BLAST 13 Top Hit Species matching (TIF)Click here for additional data file.Figure S2A: Hierarchical clustering applying three clustering protocols, complete and single linkages and group average. (S) – sham injected, (N) – intact, (L) – L-cHH-injected, (D) – D-cHH-injected. B: principal component analysis. Green – native, Red – sham, Blue – L-cHH-injected and Yellow – D-cHH-injected.Clustering the 12 mapping profiles of individual females using hierarchical and principal component analyses. (TIF)Click here for additional data file.Table S1Please take a look at the excel document named as (XLSX)Click here for additional data file.


Whiplash injury affects 83% of persons in a traffic collision and leads to whiplash-associated disorders (WAD). A major challenge facing health care decision makers is identifying cost-effective interventions due to lack of economic evidence. Our objective is to compare the cost-effectiveness of: 1) physician-based education and activation, 2) a rehabilitation program developed by Aviva Canada , and 3) the legislated standard of care in the Canadian province of Ontario: the Pre-approved Framework Guideline for Whiplash developed by the Financial Services Commission of Ontario.The economic evaluation will use participant-level data from the University Health Network Whiplash Intervention Trial and will be conducted from the societal perspective over the trial's one-year follow-up. Resource use (costs) will include all health care goods and services, and benefits provided during the trial's 1-year follow-up. The primary health effect will be the quality-adjusted life year. We will identify the most cost-effective intervention using the incremental cost-effectiveness ratio and incremental net-benefit. Confidence ellipses and cost-effectiveness acceptability curves will represent uncertainty around these statistics, respectively. A budget impact analysis will assess the total annual impact of replacing the current legislated standard of care with each of the other interventions. An expected value of perfect information will determine the maximum research expenditure Canadian society should be willing to pay for, and inform priority setting in, research of WAD management.Results will provide health care decision makers with much needed economic evidence on common interventions for acute whiplash management.http://ClinicalTrials.gov identifier NCT00546806  Whiplash injury affects 83% of persons involved in a traffic collision . In the In the Canadian province of Ontario, health care resources used to manage traffic injuries within the automobile insurance system exceed those used by the Workers Safety and Insurance Board system for similar injuries . A recenThus, a major challenge facing health care decision makers is identifying cost-effective interventions for WAD. But there are no economic evaluations of WAD interventions in the published literature. The recently published systematic review of the neck pain literature by the Bone and Joint Decade 2000-2010 Task Force on Neck Pain and Its Associated Disorders did not identify a single cost-effectiveness analysis of WAD treatments . Our objThis paper describes the protocol of an economic evaluation alongside the University Health NetworkWhiplash Intervention Trial (UHN WIT). A complete description of the UHN WIT protocol hasbeen published elsewher . and is The UHN WIT is a three-arm randomized trial designed to compare the effectiveness of education and activation by a physician and two rehabilitation programs of care in patients with recent Grade I or II WAD Table . SelectiThe Education and Activation intervention is designed to promote self-efficacy and early return to normal activities of daily living. It reflects current guidelines and recommendations for the management of WAD ,19. A prIf follow-up visits are required, the participant is re-assessed by the same physician . The intervention above is repeated and adapted to the participant's status according to the physician's clinical judgment . Participants who do not recover six weeks post-collision, or improve but require more care, are referred for a multidisciplinary evaluation and enter the interdisciplinary rehabilitation intervention stream (described in the Soft Tissue Injury Care Model section below).The Pre-approved Framework is a clinical management guideline developed by the Financial Services Commission of Ontario, an arm's length agency of the Ministry of Finance which regulates traffic insurance in the province of Ontario. It is the legislated standard of care for Grade I and II WAD in Ontario. It focuses on the provision of interventions to manage pain and disability through functional restoration . The intParticipants with clinically important functional limitations are eligible for a functional assessment of their home, work, or school environment. Based on the assessment, an occupational therapist develops an intervention that may include: 1) recommendation to use aids or devices, 2) minor modifications at the work, home, or school environment, 3) instructions on adaptive strategies or alternate approaches to fulfill functional tasks, and 4) specific functional activities to increase tolerance. Participants who report clinically significant improvement in the first six weeks of care, but have not recovered, can receive up to four additional treatments over a two-week period. Participants who have not recovered in the first six weeks of care are re-evaluated and a new plan of management is developed by the physiotherapist.The Soft Tissue Injury Care Model is a staged multidisciplinary rehabilitation program developed by Aviva Canada  as an alternative to the Pre-approved Framework guideline. A physiotherapist leads the intervention which includes three integrated and sequentially-administered rehabilitation phases: 1) acute-sub acute care, 2) multidisciplinary evaluation, and 3) interdisciplinary rehabilitation. During the acute-sub acute phase (first six weeks post-collision), care is delivered by physiotherapists, and if necessary, kinesiologists and massage therapists. Care consists of: 1) reassurance, 2) education, 3) home exercises, 4) physiotherapy modalities, and 5) massage therapy. A maximum of 9 treatments (including massage therapy) during the first three weeks post-collision and an additional 8 sessions (including massage therapy) between the third and sixth week post-collision is provided. The limit on the number of visits is a restriction of the Soft Tissue Injury Care Model, not of the trial protocol. The type and frequency of treatment prescribed by the physiotherapist is based on his/her assessment. During this phase, the physiotherapist may recommend an in-home or job-site functional assessment.Patients who require treatment beyond the acute-sub acute phase are referred for a multidisciplinary evaluation. The purpose of the multidisciplinary evaluation is to identify barriers to recovery and recommend the appropriate treatment through an interdisciplinary rehabilitation program. The interdisciplinary rehabilitation program has three specific goals: 1) overcome psychosocial barriers to return to function, 2) physical restoration, and 3) functional restoration. The program includes up to five weeks of daily intervention (each session may last up to 51/2 hours). Frequency, duration and type of care are determined by the interdisciplinary team and may include a job-site assessment. Services provided during interdisciplinary rehabilitation may include: 1) education, 2) reassurance, 3) goal setting and advice on self-management, 4) psychological counseling and stress management, 5) relaxation therapy, psychotherapy and family counseling, 6) cognitive behavioral therapy, 7) instruction on pain management techniques, and 8) strength, endurance, flexibility or cardiovascular exercises.All physicians and physiotherapists are trained by a trial coordinator during a formal training session. The session consists of a description of the study and instruction on the standardized intervention protocols. Clinicians receive printed materials to refer to. A trial coordinator conducts intermittent file audits and protocol reviews with clinicians to ensure adherence with the protocol.Table Secondary outcomes include the following. A recurrence occurs when a participant who reports to have recovered subsequently reports that s/he is: 1) slightly worse, 2) much worse, or 3) worse than ever. Neck pain intensity is measured with an 11-point numerical rating scale . The numThe primary analysis will be conducted according to the intention-to-treat principle. It will compare time-to-recovery across intervention arms using the Kaplan-Meier method, reporting median time-to-recovery and 95% confidence intervals . The mixRecurrences will be reported as rates (number of participants that report a recurrence per 28 days). Rates will be used because differences in participants' time-to-recovery could create a bias that favors less effective interventions (those with longer average time-to-recovery) since, in participants randomized to these interventions, there will be less observation time to observe potential recurrences. Time-to-recurrences will also be compared in a Cox proportional hazards model as above, with an additional covariate to account for differences in average time-to-recovery across interventions.The primary objective is to compare the relative cost-effectiveness of: 1) the legislated standard of care in Ontario for acute WAD, 2) physician-based Education and Activation, and 3) the Soft Tissue Injury Care Program, for persons with recent WAD injury, using participant-level data from the UHN WIT and Aviva Canada administrative claim files. Secondary objectives including conducting sensitivity analyses, an expected value of perfect information analysis, and a budget impact analysis. A societal perspective will be adopte ,39. The Ontario automobile insurance regulations specify that extended health care benefits  are used first when paying for traffic injury treatment. When extended benefits are exhausted, automobile benefits are used. However in this trial, Aviva Canada pays for all health care, including those generally paid by extended care benefits.Relevant costs will include all health care goods and services, and benefits provided by Aviva Canada during the study's 12-month follow-up period Table . ParticiCost data will be extracted from participants' insurance claim file by a blinded research associate unaffiliated with Aviva Canada using a standard data extraction form. The research associate will pilot data extraction by extracting data twice from the files of the first 50 participants to test intra-rater reliability. A value of at least 0.8 will be considered adequate for the quadratic weighted Kappa statistic  and intra-class correlation coefficient (in the case of continuous data). Data extraction will be modified if problems are identified. Cost data will be linked with trial data using the unique study identification number assigned to each participant at enrollment. Costs will be reported in Canadian dollars and standardized for inflation to the most recent available rates after the study has ended, using the Bank of Canada Consumer Price Index and adjusted for censoring using the Kaplan-Meier sample average estimator -42.We will use the quality-adjusted life year (QALY) as the primary health outcome for our analysis. SF-6D scores - derived from SF-36 response data - will be used to estimate participants' preference-based quality-of-life (utilities) over the 12-month follow-up period . These sParticipant characteristics and health resources consumed will be reported by intervention group, using descriptive statistics .Costs and health effects will not be discounted for the primary analysis since the time horizon is 12-months. Following current recommendations . costs rIncremental cost-effectiveness will be computed as the ratio of the difference in mean costs  to the difference in mean health effects , and will provide incremental cost-effectiveness ratios (ICERs):ICERs will represent the additional cost per additional QALY gained, additional cost per additional unrecovered day averted, and additional cost per additional recurrence over a 28 day period of one intervention compared to another, from least costly to most costly. ICERs will be plotted on a cost-effectiveness plan ,46. withWhether the ICER is considered cost-effective by a decision maker depends on the maximum the decision maker is willing to pay for an extra unit of health effect . An intervention that is cost-effective is thus defined as ICER < λ. The Incremental Net Benefit (INB) equatio . computeWhen the INB is positive, the value of a new treatment's extra benefits (ΔE × λ) outweighs its extra costs (ΔC), implying that the decision maker values the extra effect more than the extra cost . Conversely, when INB is negative, the decision maker does not consider the extra benefit to be worth the extra cost.We will use the net-benefit approac ,51. to mi) and cost (Ci) data for each patient i:We will examine baseline characteristics across intervention groups to determine whether randomization was successful in balancing observed baseline covariates. If there is unbalanced allocation in baseline characteristics, we will use net-benefit regression  which alIn its simplest form, regression is then used to estimate:1 and β2). The regression estimate of β1 will equal the incremental net benefit of physician-based Education and Activation compared to the Ontario legislated standard of care, and the regression estimate of β2 will equal the incremental net benefit of the Soft Tissue Injury Care Program compared to the Ontario legislated standard of care. Additional covariates can be added to adjust for unbalanced allocation in observed baseline covariates that could confound results [where α is the intercept term, t is an intervention dummy term , and ε is the stochastic error term. The regression coefficient β provides the estimate of the standard incremental net-benefit statistic. As there are two new interventions to test, we will use two treatment dummies, t1 and t2  to 100  . FinallyObservations with incomplete baseline covariates will be deleted from the analyses. Participants with missing data on the primary outcome (self-perceived recovery) and preference-based quality-of-life (SF-6D) will have the result of their last observation carried forward. Sensitivity analyses will be conducted, where reasonably likely scenarios are considered . Cost and effect data will be analyzed with two approaches: 1) only participants with complete data are included , and 2) all study participants are included, where imputed values for costs and effects in participants with missing data will be used (multiple imputation) . ResultsExpected value of perfect information (EVPI) represents the maximum amount that a health care decision maker should be willing to pay for additional evidence to inform future decisions . It focuA budget impact analysis will be conducted to assess the cost of replacing the Pre-approved Framework Guideline (the current legislated standard of care for acute Grade I and II WAD in Ontario) with the physician-based Education and Activation and Aviva Canada's Soft Tissue Injury Care Model ,62. The This paper allows for the peer-review of the proposed methods and provides a transparent statement of the planned analyses. Results of this economic evaluation are expected to provide health care decision makers with necessary economic evidence on common interventions for acute whiplash.EVPI: expected value of perfect information; ICER: incremental cost-effectiveness ratio; INB: incremental net-benefit; QALY: quality-adjusted life year; UHN WIT: University Health Network Whiplash Intervention Trial; WAD: whiplash-associated disorders.The authors declare that they have no competing interests.AMB, EB, GvdV, HMS, JDC, MS, and PC participated in the conception and design of the study. The analysis was designed by AMB, EB, GvdV, JDC, and PC. All authors participated in drafting this manuscript and approved its final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/11/594/prepub


Integrins are the foremost family of cell adhesion molecules that regulate immune cell trafficking in health and diseases. Integrin alpha4 mediates organ-specific migration of immune cells to the inflamed brain, thereby playing the critical role in the pathogenesis of multiple sclerosis. Anti-alpha4 integrin therapy aiming to block infiltration of autoreactive lymphocytes to the inflamed brain has been validated in several clinical trials for the treatment of multiple sclerosis. This paper provides readers with an overview of the molecular and structural bases of integrin activation as well as rationale for using anti-alpha4 integrin therapy for multiple sclerosis and then chronicles the rise and fall of this treatment strategy using natalizumab, a humanized anti-alpha4 integrin. Multiple sclerosis (MS) is a devastating autoimmune disease that is characterized by inflammation in the brain and spinal cord that damages the myelin sheath, thereby causing demyelination of neurons –3. DemyeInfiltration of the brain by autoreactive immune cells that originate in the peripheral circulation plays a central role in the pathogenesis of inflammation in MS , 5. DuriAlpha4 integrin is among 24 integrin families of cell-adhesion molecules containing noncovalently-associated alpha and beta subunits –10. EighWhat makes integrins very unique in many cell adhesion molecules is their ability to transmit signals across the plasma membrane bidirectionally . ActivatThe adhesive and signaling activities carried out by integrins are vital to many of the cell-cell and cell-extracellular matrix interactions involved in immune responses . Along wA beta2 integrin alphaLbeta2 (a.k.a. leukocyte function-associated antigen-1: LFA-1) that binds to ICAM-1 on endothelial cells critically regulates adhesive leukocyte interactions on the luminal surface of the vasculature during extravasation to inflamed tissues, as well as during normal recirculation through the lymphoid tissues of lymphocytes , 16. In Alpha4 integrin is predominantly in hematologic cells in adults; however, it is highly expressed in the heart  during early development in embryo. This explains why nonconditional alpha4 integrin knockout mice exhibited embryonic lethality . This miThe physiologic importance of the ability to upregulate leukocyte integrins is illustrated by two rare genetic disorders: leukocyte adhesion deficiency type I (LAD-I) and type III (LAD-III). LAD-I is caused by loss-of-function mutations in the beta2 integrin subunit that result in the absence, or severely reduced expression, of all beta2 integrin heterodimers on the cell surface of leukocytes , 19. LADNot only beta2 integrins, but also alpha4 integrins are severely affected in LAD-III that is caused by a genetic defect in kindlin-3, a cytoskeletal protein that activates integrins by binding to integrin beta cytoplasmic tails. LAD-III patients manifest not only an increased susceptibility to bacterial infections, such as occurs with LAD-I, but also platelet dysfunction . The latPersistent accumulation of leukocytes is a hallmark of the chronic inflammation observed in the affected tissues of autoimmune diseases , 22. ForThe cascade of leukocyte-endothelial cell interactions was originally thought to contain three steps  before transmigration of leukocytes across the endothelial barrier could occur. A crawling step has recently been added to this cascade. Specifically, leukocytes crawl along the endothelial surface from the point of arrest to that of transmigration , 24. TheDuring transendothelial migration (TEM), leukocytes proceed through endothelial cells via two distinct routes, either paracellular or transcellular . In the Pericytes are located on the basolateral side of vascular endothelial cells, wherein they surround the endothelial monolayers. Pericytes were thought to play merely a house-keeping and scaffolding role to support vascular endothelial cells; however, a recent investigation has revealed that pericytes in fact play a novel role in regulating leukocyte migration into interstitial spaces. Transmigrated leukocytes have been found to crawl in the perivascular space between the endothelial basement membrane and underlying pericytes before they enter an interstitial space . Before Organ-specific homing of leukocytes is made possible primarily by unique combinations of cell-adhesion molecules and chemokine receptors , 32. Naïin vivo experiment conducted by Yednock et al. showed that a blocking antibody to integrin alpha4beta1 prevented the infiltration of leukocytes to the brain, thereby inhibiting the development of experimental autoimmune encephalomyelitis (EAE), an established mouse model for multiple sclerosis [in vivo studies [The concept that the organ specificity of autoreactive effector T-cell homing relies on specific integrins led to the development of anti-integrin therapies for the treatment of autoimmune diseases including multiple sclerosis. A seminal clerosis . Capital studies –36, two The first randomized, double-blind, placebo-controlled trial that showed natalizumab to have some clinical efficacy in multiple sclerosis was performed in the UK . In thisA subsequent randomized, double-blind, placebo-controlled trial involving 213 patients was performed in 26 hospitals across the USA, Canada, and UK . The triCapitalizing on these pioneering clinical trials, two randomized, multicentre, placebo-controlled, double-blind trials, AFFIM and SENTINEL, were subsequently carried out. The AFFIRM  study was designed to evaluate the effects of the antibody on the progression of disability and the rate of clinical relapses . In thisThe SENTINEL  study was designed to evaluate the effects of adding natalizumab to a treatment regimen with interferon beta-1a . In the Natalizumab was approved in 2004 through the FDA's accelerated First Track Program; however, the antibody soon suffered a major setback when two cases from the MS patients in the SENTINEL study and another from Crohn's disease (CD) patients treated with natalizumab developed progressive multifocal leukoencephalopathy (PML) –44. PML After two deaths (one in MS and another in CD) of the three PML cases that resulted from natalizumab treatment, Biogen Idec voluntarily withdrew the antibody from the market in February of 2005, 4 months after accelerated FDA approval. However, in June of 2006, after comprehensively reviewing all patients exposed to natalizumab during clinical trials and carefully considering both the risk of fatal PML and the benefits of the treatment, the FDA advisory committee reapproved natalizumab for the treatment of relapsing MS patients .The comprehensive reviews of the patients in postmarketing surveillance have identified three PML risks associated with natalizumab including positive serum anti-JC virus antibody titers that serve as a robust biomarker , prior oNatalizumab is currently regarded as the most potent treatment for relapsing-remitting MS; however, the PML risk restricts its indications , 50. In Alpha4 integrin-mediated trafficking of pathogenic effector T cells to the brain has been a validated therapeutic target for the treatment of MS. Building on the success of natalizumab, which validated this therapeutic target in patients, the effectiveness of firategrast, an orally active small-molecule antagonist to integrin alpha4 that is manufactured by GSK, has been investigated in patients with MS in a multicentre, phase 2, randomized, double-blind, placebo-controlled, dose-ranging study involving 343 patients . Oral ad