Adhesive capsulitis or frozen shoulder is a common condition characterized by shoulder pain and stiffness. In patients in whom conservative measures have failed, more invasive interventions such as arthrographic or arthroscopic distension can be very effective in relieving symptoms and improving range of movement. However, absolute contraindications to these procedures include the presence of neoplasia around the shoulder girdle. We present five cases referred to our institution where the diagnosis of shoulder joint malignancy was delayed, following prolonged, ineffective treatment for frozen shoulder. These cases highlight the importance of careful review of the radiology and the need for reconsideration of the diagnosis in refractory "frozen shoulder". Frozen shoulder was first described by Codman in 1934, as an idiopathic painful restriction in the range of shoulder joint movement, in the presence of normal plain radiographs . It is uTumours around the shoulder girdle are uncommon causes of shoulder pain and stiffness, but often present with symptoms and a clinical history identical to that of a frozen shoulder. A strict contraindication to arthrographic or arthroscopic distension of the shoulder is the presence of a local oncological process. Such procedures may change the surgical management from being a limb-preserving resection to a forequarter amputation. In the past month, five patients have been referred to us with malignant tumours around the shoulder joint, all previously diagnosed as having a frozen shoulder. All patients had undergone prolonged conservative management and hydrodilatation, with persistence of symptoms. Two of the patients had also undergone arthroscopic surgery. The following cases illustrate the importance of reconsidering the diagnosis in refractory frozen shoulder and the value of a detailed clinical history and examination and careful consideration of radiologic imaging in assessing recalcitrant "frozen shoulder".A 60 year old woman presented to her local medical officer with an eighteen month history of worsening right shoulder pain and stiffness. She was initially treated with oral analgesia followed by a cortisone injection without improvement. Two months later she had a hydrodilatation of the shoulder but her symptoms persisted. MRI was then performed, which demonstrated a large permeative tumour arising from the scapula Figure . She wasA 42 year old man was referred to an orthopaedic specialist with a history of sudden onset left shoulder pain following a work related activity. He was initially diagnosed with rotator cuff tendinopathy and subacromial impingement, and had a course of intensive physiotherapy followed by arthroscopic shoulder surgery, without improvement in symptoms. Two months later a minor incident involving his left shoulder led to an increase in pain and swelling and reduction in movement. Hydrodilatation was then performed but pain and function of the shoulder continued to worsen. On retrospective review of plain x-rays of the shoulder, a destructive lesion at the metaphysis with a cortical breach medially in the region of the surgical neck of the humerus was realized (Figure A 50 year old women was referred to an orthopaedic specialist with a 6 month history of episodic pain in the right shoulder, with associated decreased range of movement. Initial plain x-rays were unremarkable. She then underwent a variety of procedures, which included repeated subacromial corticosteroid injections, arthrographic distension, manipulation under anaesthetic, and arthroscopic debridement and acromioplasty. On arthroscopy, a marked synovitis was observed, to which her ongoing symptoms and the development of a palpable mass on the anterior aspect of the shoulder was initially attributed. Repeat plain radiographs two years after the onset of her symptoms demonstrated a large lesion extending from the glenoid cartilage into the base of the coracoid process Figure . She wasA 68 year old man had previously had a squamous cell carcinoma of the upper back excised, after which he had a three year history of shoulder pain and stiffness. He was treated for a frozen shoulder and received intensive physiotherapy and multiple subacromial corticosteroid injections, followed by hydrodilatation. Initially this seemed to settle his symptoms, although a month later pain and stiffness recurred, with marked reduction in shoulder function, and he was referred to us. An MRI was performed, which showed lesions in the supraspinatus and trapezius muscles, which were consistent with metastatic deposits Figure . The patA 55 year old female with a past history of malignant fibrous histiocytoma of the left thigh resected five years previously, presented to her local medical officer with right shoulder pain. Plain films were performed at the time which appeared normal (Figure et al. [Adhesive capsulitis or frozen shoulder is a common condition that may affect up to 5% of the general population in their lifetime. Although the aetiology of frozen shoulder is unknown, it has been associated with diabetes mellitus, thyroid disease, ischaemic heart disease and various autoimmune conditions . Other cet al.  demonstret al. [Tumours of the shoulder girdle are uncommon causes of shoulder pain and restricted movement. In most cases, they are diagnosed based on the presence of a soft tissue mass on clinical examination, as well as characteristic radiographic changes. Robinson et al.  suggesteet al. . MisdiagMRI: magnetic resonance imaging, CT: computed tomography, TSE: turbo spin echo, STIR: short tau inversion recovery.


The Menopause Rating Scale is a health-related Quality of Life scale developed in the early 1990s and step-by-step validated since then. No methodologically detailed work on the utility of the scale to assess health-related changes after treatment was published before.We analysed an open, uncontrolled post-marketing study with over 9000 women with pre- and post-treatment data of the MRS scale to critically evaluate the capacity of the scale to measure the health-related effects of hormone treatment independent from the severity of complaints at baseline.The improvement of complaints during treatment relative to the baseline score was 36% in average. Patients with little/no complaints before therapy improved by 11%, those with mild complaints at entry by 32%, with moderate by 44%, and with severe symptoms by 55% – compared with the baseline score. We showed that the distribution of complaints in women before therapy returned to norm values after 6 months of hormone treatment. We also provided weak evidence that the MRS results may well predict the assessment of the treating physician. Limitations of the study, however, may have lead to overestimating the utility of the MRS scale as outcome measure.The MRS scale showed some evidence for its ability to measure treatment effects on quality of life across the full range of severity of complaints in aging women. This however needs confirmation in other and better-designed clinical/outcome studies. The Menopause Rating Scale (MRS) was initially developed in the early 1990s  to measuThe validation of the MRS began some years ago -6 with t.Development and standardization of the scale were published elsewhere . In brieThe scale was defined as a menopause-specifc, health-related quality of life scale (HRQoL), because the profil of complaints in this scale importantly determines the HRQoL of women in this age span. Moreover, a good correlation between the results obtained with the MRS scale and the generic QoL scale was observed .).The MRS scale became internationally well accepted as far as the usage many countries is concerned. The first translation was into English . Other tLike in other health-related QoL scales, it is a challenge to satisfy the demands of a clinical utility and outcomes sensitivity. A comprehensive overview regarding conventional psychometric requirements of test reliability and validity were recently published elsewhere . It is t® = 2 mg estradiol valerate/2 mg estradiol valerate + 1 mg cyproterone acetate) using the MRS scale as outcome measure under routine conditions of office-based gynaecologists. The study was described in detail elsewhere [A multicenter, open post-marketing study was conducted with a product for hormone therapy .The statistical analyses were performed with the commercial statistical package SAS 8.2.Altogether, data of 9311 women were available for most of our analysis. However, the sample size varied slightly depending on the variables used because we had also missing information in a few variables.The mean age was 49.8 years (SD 6.4). About half of the participating women were still perimenopausal (51.9%) or were already in the postmenopausal phase (48.1%). The mean body mass index was not eye-catching with 24.7 (SD 3.7).The improvement of the health-related quality of life (HRQoL) – measured with the MRS scale – is described in Table Apart from the comparison of means, we calculated the relative improvement compared with the situation before therapy (baseline) to better understand the magnitude of change after therapy Table , i.e. inThe scale is able to measure an improvement in patients starting with "no/little complaints" , "mild" (5–8), "moderate"(9–15), and "severe" (16 + points) before therapy (= baseline). This is presented in Table It is interesting to compare the HRQoL before and after hormone treatment with the norm values of MRS obtained in an average population of aging women, i.e. not patients as in our post-marketing study. To this end, we compared only the MRS total scores in patients with the average female population Table . It becasuccessful (very effective and effective) and not successful . This alternative variable was then used for the comparison with the alternative "success-variable" based on MRS : "successful" (5 and more points reduction after therapy compared with baseline test) and "not successful" (less than 5 scoring points reduction after therapy compared with baseline test).The treating gynaecologist  assessed individually the efficiency of the hormone treatment in the above mentioned intervention study. The gynaecologist's expert opinion regarding treatment efficiency was categorized into two categories for the purpose of this analysis: The prediction of the expert opinion of the treating gynaecologist with the MRS data seems to be good: sensitivity (correct prediction of a positive assessment by the physician) 70.8% and specificity (correct prediction of a negative assessment by the physician) 73.5% Table .The MRS scale was developed (a) to assess symptoms of aging/menopause (independent from those that are disease-related) or HRQoL between groups of women under different conditions, (b) to evaluate the severity of symptoms over time, and (c) to measure changes pre- and post hormone replacement therapy. The aim of this paper was to empirically demonstrate that the latter claim is evident.Reliability measures  were found to be good across countries [validity it was shown that the internal structure of the MRS across countries was sufficiently similar to conclude that the scale really measures the same phenomenon [Reliability and validity are important to show the usefulness of the scale as a clinical utility in monitoring treatment effects – once all other methodological requirements are successfully demonstrated before. ountries . Regardienomenon .The comparison with another scale for aging women – although not a validated HRQoL scale (Kupperman) – showed sufficiently good correlations of the total score, which is compatible with the notion of a good criterion-oriented validity. The same is true for the comparison with the generic quality-of-life scale SF36 where also high correlation coefficients have been shown . AnotherHaving the above-mentioned psychometric data available, a point was reached to critically evaluate the capacity of the scale to reliably measure health-related effects of hormone treatment independent from the severity of complaints and – in addition – to the comparison of treatment effects measured by the MRS scale and the subjective assessment by the treating physician. To this end, many clinicians use the term "validity" and mean high utility for clinical work or research.The only hormone treatment study with the MRS scale as outcome measure in women during menopausal transition we could get data for methodological analysis was the above described postmarketing study. We hope to repeat/confirm this analysis with data of a more stringently designed clinical trial. But even on the basis of a methodologically weak dataset, in absence of other data, we got re-assuring methodological information about the MRS scale.It is a well-established experience that women with menopausal complaints respond to hormone therapy with a marked improvement of the HRQoL. This is what the MRS scale should be able to detect.We saw that the increased mean MRS total score at baseline (before treatment) markedly decreased after 6 months under treatment indicating a significant improvement of complaints & HRQoL. This was also the case for the mean scores of the three subscales. These data cannot disentangle the effect of treatment and "natural variation" of complaints over time. This however was not the point: It was not the intention of this paper to evaluate effectiveness of hormone therapy in an uncontrolled post-marketing study.The absolute improvement of symptoms during treatment was 9.3 points of the MRS total score on average. This is equivalent to 36% of the baseline score, and similar also for all three subscales. In other words, the MRS scale was shown to be successful in detecting treatment effects. The impressive magnitude of the therapy-related improvement of HRQoL should be obviously discussed in the context of selection of women with complaints susceptible for this kind of treatment by the participating gynaecologists. Another critical remark is that we cannot comment as to what extend the MRS scale is able to measure true or placebo treatment effects. But this is more a question concerning efficacy and the study draw any conclusions in this regard by definition of the study design.To answer the question whether the sensitivity of the MRS scale is good enough to detect even treatment-related changes in women with only little or mild symptoms as compared with severe ones, the analysis was stratified. An improvement of complaints/QoL was seen in an increasing degree in patients with little, mild, moderate and severe symptoms at baseline. The relative improvement increased with the degree of severity of symptoms at baseline, which is consistent with the general expectation. It seems to be important to underscore: The MRS scale seems to detect also a positive treatment effect in women with little complaints – although to a lesser degree.Moreover, we showed the capacity of the MRS scale to determine therapeutic efficiency with another approach: a face-value-comparison with norm values of the population ,3. The lsuccessful" and "not successful" for both the subjective opinion of the physician and the result of the MRS scale: The sensitivity (correct prediction of a positive assessment by the physician) was 70.8% and specificity (correct prediction of a negative assessment by the physician) 73.5%. In other words, the MRS scale fits well with the subjective assessment of the treatment effect estimated by the physician. However, conclusions have to be drawn very carefully because of a possibly inherent bias that may have inflated the positive result: The subjective assessment of "success" by the treating physician was obviously not as independent from the assessment by the MRS scale as desirable because the physician applied the scale to the patient. Even without being able to recall the result of the MRS six month ago or to calculate and compare the total score of both administrations, the interaction with the patients is likely to have introduced this bias in the direction of a higher compatibility between both assessments.The MRS scale was also tested whether it predicts the therapeutic assessment of the treating physician. At face value, the individually assessed efficiency of hormone treatment by the treating gynaecologists was comparable with the assessment by the MRS scale, i.e. using a simple dichotomization of the treatment effect in "Although the result may too positive compared with a blinded, really independent assessment, it permits to generate the working hypothesis of a sufficiently good prediction of the therapeutic effect by means of the MRS scale. This needs to be confirmed with better data, i.e. data from a blinded, independent comparison, i.e. with the currently used, self-administered MRS scale.The aim of this exercise was only to demonstrate that the MRS scale may well predict the clinical opinion about efficiency of hormone therapy, what was not empirically shown before. We recommend the MRS as standardized/validated "objective" scale for use in clinical studies, although some aspects discussed above need confirmation in a new study. Moreover, since the scale is already broadly used at the international level, it is important to sensitise users about some lacking information or weak evidence.The limitations of this study should be shortly summarized. First of all, this study was performed in a dataset where an earlier version of the MRS scale was used, i.e. the scale was not self-administered but completed in an interview of the physician with the patient. This could have influenced the magnitude of the absolute scores of the total and sub-scales. As far as pre-/post-treatment changes are concerned, the magnitude of the absolute changes may have been more influenced than the relative changes of the HRQoL assessment of the patients as discussed in this paper. Another problem along the same line is that we had to transform the old coding system into the new one. This was done with a simple linear transformation and is not likely to have introduced any bias. Another limitation is that this is the first study we are aware of for this kind of assessment of the validity to measure therapeutic intervention.It is not likely that the main conclusions of the study are materially biased. However, the results should be cautiously used  as long as not confirmed with data obtained with the currently used self-administered MRS scale without potential influence of the physician. It can be assumed that a new study with the currently recommended MRS scale – in the sense of "patient-reported-outcome" – would demonstrate positive results but to a lesser degree.The MRS scale showed some evidence for its ability to measure treatment effects on quality of life across the full range of severity of complaints in aging women. This however needs confirmation in other and better-designed clinical studies.The authors FS and SG are employees of the company that produces HRT products.We cannot however see any conflict of interest as far as the methodological aspects of the validation of the MRS scale are concerned.LAJH: responsible for the collection and evaluation of the data, and involved in writing of the paper. DMT: responsible for building the database of this publication, responsible for the statistical evaluation, and contributed to writing of the paper. FS: responsible for the post-marketing study and designing this paper, contributed to the manuscript. SG: responsible for designing and overseeing the post-marketing study of Climen, contributed to writing and revising of the paper JS: responsible for the field work of the post-marketing study, setting up the initial database, and for the preparation of the subset of data used for this publication. HPGS: Major responsibility in developing the MRS scale, contributed to writing/revision of the manuscript.


Mental fatigue, with decreased concentration capacity, is common in neuroinflammatory and neurodegenerative diseases, often appearing prior to other major mental or physical neurological symptoms. Mental fatigue also makes rehabilitation more difficult after a stroke, brain trauma, meningitis or encephalitis. As increased levels of proinflammatory cytokines are reported in these disorders, we wanted to explore whether or not proinflammatory cytokines could induce mental fatigue, and if so, by what mechanisms.+) in humans suffering from mental fatigue. At present, this is not possible for technical reasons. Therefore, more knowledge of neuronal-glial signaling in in vitro systems and animal experiments is important.It is well known that proinflammatory cytokines are increased in major depression, "sickness behavior" and sleep deprivation, which are all disorders associated with mental fatigue. Furthermore, an influence by specific proinflammatory cytokines, such as interleukin (IL)-1, on learning and memory capacities has been observed in several experimental systems. As glutamate signaling is crucial for information intake and processing within the brain, and due to the pivotal role for glutamate in brain metabolism, dynamic alterations in glutamate transmission could be of pathophysiological importance in mental fatigue. Based on this literature and observations from our own laboratory and others on the role of astroglial cells in the fine-tuning of glutamate neurotransmission we present the hypothesis that the proinflammatory cytokines tumor necrosis factor-α, IL-1β and IL-6 could be involved in the pathophysiology of mental fatigue through their ability to attenuate the astroglial clearance of extracellular glutamate, their disintegration of the blood brain barrier, and effects on astroglial metabolism and metabolic supply for the neurons, thereby attenuating glutamate transmission. To test whether our hypothesis is valid or not, brain imaging techniques should be applied with the ability to register, over time and with increasing cognitive loading, the extracellular concentrations of glutamate and potassium (KIn summary, we provide a hypothetic explanation for a general neurobiological mechanism, at the cellular level, behind one of our most common symptoms during neuroinflammation and other long-term disorders of brain function. Understanding pathophysiological mechanisms of mental fatigue could result in better treatment. Diagnostic and Statistical Manual of Mental Disorders, 4th edition ec and neurodegeneration characteristic of excitotoxicity, while the loss of neuronal glutamate transporter does not elevate [Glu]ec smission  and also neurons . In fact neurons . Even mo [Glu]ec .A large number of factors have been shown to affect the activity and expression of the glutamate transporters GLT-1 and GLAST. For example, GLT-1 is stimulated by phosphorylation by protein kinase C (PKC), while GLAST is inhibited by PKC at a non-PKC consensus site . The synProinflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 have since long been known to impair astroglial glutamate uptake even if the mechanisms are not fully understood. The inhibitory function of TNF-α was established as early as the 1990s, when TNF-α was shown to inhibit astroglial glutamate uptake  reported2+) signaling between BBB endothelial cells by reducing gap junction coupling and inhibiting triggered ATP release  reduced . There i reduced , with lesyncytia -33, and syncytia  could insyncytia ,36. Thissyncytia  and leadsyncytia .ec would also lead to astroglial cell swelling, with a resulting decrease in the extracellular space volume, and locally further increased [Glu]ec [+]ec [+]ec levels have been shown in experimental systems to inhibit glutamate release [Increased [Glu] [Glu]ec -42. The ec [+]ec ,44. Even release .Recent data indicate a dynamic and fine-tuning regulation of the glutamatergic transmission. One mechanism by which neurons regulate excitatory transmission is by altering the number and composition of glutamate receptors at the postsynaptic plasma membrane. This has been shown for the NMDA receptor in experimental systems and could have prominent importance for dynamic processes as learning and memory . Of greaFurthermore, in states of decreased astroglial glutamate uptake capacity, even astroglial glucose uptake, and consequently the supply of metabolic substrates to the neurons, has been reported to decrease -50 and tec in the basal frontal cortex could lead to a decrease in the noradrenaline and serotonin (5-HT) release in the cerebral cortex, which would also decrease glucogenolysis There is an extensive literature on inflammatory response with microglial activation and the production of proinflammatory cytokines  in neuroinflammatory/infectious and neurodegenerative diseases as well as in stroke and trauma ,54. The Several groups have also described enhanced production of proinflammatory cytokines in major depression [see ] and sicIn states of anxiety and stress, often experienced as secondary to mental fatigue, increased glucocorticoid levels have been demonstrated. Interestingly, long-term increases in glucocorticoids have been demonstrated to result in the production of both TNF-α and IL-1β .In the search for pathophysiological correlates to fatigue in MS, Roelcke and co-workers  demonstrec. The initial consequence would be slightly increased [Glu]ec, with less precision in glutamate transmission. This would disintegrate the "filter", which normally selects information and prevents it from reaching the cerebral cortex. We can take the sound from a low-frequency fan as an example. This sound is normally sorted out after hearing it for a while. If this sound is handled with less precision by auditory recognition systems, it will continually be recognized by brain centers as "new" information and be processed in the cerebral cortex as long as the sound is on. The "filter" that normally restrains already recognized information from reaching higher brain centers, has been "opened". From a physiological point of view, it seems appropriate that the individual, and not the brain at the synaptic level, should determine which information should reach, and be processed by, the cerebral cortex. The decreased attention, increased loudness and light sensitivity, and irritability could be physiological ways of avoiding overstimulation of higher cortical centers. In case the individuals cannot protect themselves from too much sensory stimulation, the filter's opening leads to overstimulation of the cerebral cortex. Here, the final shutdown of the glutamate transmission could be one mechanism underlying mental exhaustion  could be studied in animal models. Effects of astroglial dysfunction with regard to glutamate transport capacity would be of special interest. Even clinical studies with different treatment strategies could be important in casting some light on the accuracy of the hypothesis. Of utmost importance in all such studies would be test batteries making it possible to objectify and even quantify the degree of mental fatigue.It is not possible at present to ultimately prove whether or not the altered neuronal-glial interactions in glutamatergic transmission induced by proinflammatory cytokines could serve as a model to explain cellular mechanisms underlying mental fatigue. Brain imaging techniques able to determine and follow [Glu]Normally, mental fatigue and the associated symptoms disappear when the brain dysfunction is over. In some patients, the symptoms persist. We have at present no explanation for this, but if our hypothesis is correct, there could be a genetic failure preventing astroglial glutamate transporters from upregulating. Another explanation for why the symptoms persist could be that the pathological stimulation by brain plasticity creates new neuronal networks ,36.Providing information about mental fatigue, its cause and the prognosis, is of utmost importance for breaking the vicious circle, which comes with the risk for secondary anxiety and depression. Furthermore, it is important for the patient to imagine and learn how much sensory stimulation they can tolerate prior to feeling too exhausted. Due to recent results on changes in cell signaling and neuronal plasticity ,36, it mADHD attention deficit hyperactivity disorderAMPA alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionateATP adenosine triphosphateBBB blood brain barrier2+ calciumCaEc extracellularGLAST glutamate aspartate transporterGLT-1 glutamate transporter-1ec extracellular glutamate concentration[Glu]5-HT 5-hydroxytryptamineth revisionICD-10 International Classification of Diseases, 10IL-1/-6 interleukin-1/-6+ potassiumK+]ec extracellular potassium concentration[KLC locus coeruleusLTP long term potentialMS multiple sclerosis+ sodiumNaNA noradrenalineNFκB nuclear transcription factor kappaBNMDA N-methyl-D-aspartateNO nitric oxidePACAP pituitary adenylate cyclase-activating polypeptidePI3K phosphatidylinositol-3-kinasePKC protein kinase CSiv mac simian immunodeficiency virus macaquesTNF-α tumor necrosis factor alphaThe author(s) declare that they have no competing interests.Equal contributions by both authors.


Xenopus laevis.Rho GTPases and their downstream effector proteins regulate a diverse array of cellular processes during embryonic development, including reorganization of cytoskeletal architecture, cell adhesion, and transcription. Changes in the activation state of Rho GTPases are converted into changes in cellular behavior by a diversity of effector proteins, which are activated in response to changes in the GTP binding state of Rho GTPases. In this study we characterize the expression and function of one such effector, XCEP2, that is present during gastrulation stages in Xenopus laevis, a gene encoding a Rho GTPase effector protein  was isolated, and found to be highly homologous, but not identical, to a Xenopus sequence previously submitted to the Genbank database. These two gene sequences are likely pseudoalleles. XCEP2 mRNA is expressed at constant levels until mid- to late- gastrula stages, and then strongly down-regulated at late gastrula/early neurula stages. Injection of antisense morpholino oligonucleotides directed at one or both pseudoalleles resulted in a significant delay in blastopore closure and interfered with normal embryonic elongation, suggesting a role for XCEP2 in regulating gastrulation movements. The morpholino antisense effect could be rescued by co-injection with a morpholino-insensitive version of the XCEP2 mRNA. Antisense morpholino oligonucleotides were found to have no effect on mesodermal induction, suggesting that the observed effects were due to changes in the behavior of involuting cells, rather than alterations in their identity. XCEP2 antisense morpholino oligonucleotides were also observed to cause complete disaggregation of cells composing animal cap explants, suggesting a specific role of XCEP2 in maintenance or regulation of cell-cell adhesion in early embryos. This loss of cell adhesion could be rescued by co-injection with a morpholino-insensitive version of the XCEP2 mRNA.In a search for genes whose expression is regulated during early stages of embryonic development in Xenopus laevis. XCEP2 is involved in maintenance of cell-cell adhesion, and as such may constitute a regulatory component that could help to balance the need for tissue integrity and plasticity during the dynamic cellular rearrangements of gastrulation.XCEP2 appears to be an essential component in the early developmental program in  Xenopus laevis coordinated changes in cell shape and motility guide the initial formation of the dorsal lip, the initial site of mesodermal involution [Vertebrate gastrulation depends upon exquisite regulation of diverse morphogenetic processes including changes in cell shape, cellular adhesion and migration. For example, in volution ,2. Latervolution . Convergvolution . The cooXenopus laevis and zebrafish, non-canonical Wnt signaling on the dorsal side of developing embryos directly initiates the cellular rearrangements and migration that contribute to convergent extension of involuting mesoderm [Xenopus, resulting in severe gastrulation defects [Drosophila [The molecular basis for initiating and regulating the complex cellular movements of vertebrate gastrulation is only beginning to be discerned. Recent work has shown Wnt proteins as key upstream regulators in gastrulation movements. In mesoderm ,6. Othermesoderm ,8. Inactosophila , vertebrosophila ,11, and osophila ,12-14.Xenopus [Experiments perturbing the activity of Rho GTPases have suggested important regulatory roles for these proteins during gastrulation in Xenopus ,10,12,14Xenopus , cell adXenopus , cell miXenopus , vesiculXenopus  and signXenopus -21. ReguXenopus, when the integrity of the elongating mesodermal sheet must be maintained at the same time that major cellular rearrangements are occuring. Downregulation of C-cadherin-mediated cellular adhesion has been observed to correlate with convergent extension movements of mesodermal cells [Xenopus embryos suggests that analogous upstream regulatory mechanisms may operate during Xenopus gastrulation [Regulation of cell-cell adhesion is thought to be of particular importance during gastrulation movements in al cells . This chal cells . In addial cells -27. Recerulation .Xenopus gene encoding a member of the recently characterized CEP/BORG family of RhoGTPase effector proteins [In this study, the embryonic expression pattern and function of a proteins ,30 is exproteins ,30, and proteins ,32.Xenopus Cdc42 Effector Protein 2, hereafter referred to as XCEP2, is shown here to be developmentally regulated during early embryonic stages, with diffuse expression of mRNA in the animal region that diminishes during late gastrula to neurula stages. Experiments employing morpholino-mediated inhibition of translation suggest that expression of this protein is essential for normal gastrulation movements, and that its activity is required for maintenance of cell-cell adhesion between blastomeres of animal cap explants.Xenopus embryo. The sequence of one of these fragments was found to be highly homologous to the translational start site region of database cDNA sequences encoding Cdc42 effector protein 2. A full length cDNA corresponding to our isolated fragment was amplified using 3' RACE PCR, cloned and sequenced. An alignment of the predicted amino acid sequence for our isolated full length cDNA sequence with human [Xenopus (accession number BC045241) CEP2/BORG1 genes from Genbank revealed significant homology . These differences were found to occur almost exclusively in regions of the protein outside the three conserved domains  that define the CEP family of effector proteins. Given the allotetraploid genetic ancestry of Xenopus laevis, and the degree of similarity between the sequences, it is likely that these two sequences constitute pseudoalleles of the same gene. We choose to refer to our isolated pseudoallele as XCEP2A and the database allele as XCEP2B.A differential display approach was employed to reveal genes that are developmentally regulated during the transition from blastula to neurula. This screen allowed for the isolation of numerous cDNA fragments corresponding to differentially regulated mRNAs present in the th human  and XenoIn situ hybridization revealed that XCEP2A mRNA  is distributed uniformly over the animal hemisphere of the embryo at blastula and early gastrula stages , maintenance of this expression level through mid-gastrula stages, with a downregulation of mRNA expression starting at stage 10.5 and becoming more marked by stage 12.5 , MO1 antisense oligonucleotides pseudoalleles elicited significant gastrulation delay as compared to an identical dose of control morpholino oligonucleotide . Given this observation, it is possible that injection of either antisense XCEP2 morpholino significantly reduces the endogenous expression of both pseudoalleles. In addition to the dosage issue in the combined condition (noted above), cross-inhibition of this type could contribute to the observed lack of additive effects in the combined MO1 + MO2 condition.The antisense morpholino oligonucleotides used in these studies were found to elicit specific translational inhibition of XCEP2. Injection of antisense morpholino against our isolated pseudoallele resulted in largescale inhibition of translation of a myc-tagged fusion protein from co-injected mRNA . The absence of effects may not be unexpected, given current models of RhoGTPase effector protein activation. RhoGTPase effector proteins are activated by direct binding to GTP-bound RhoGTPases. As such, the number of activated RhoGTPase molecules may constrain the number of effector proteins bound and activated, regardless of the heightened expression levels of the effector protein.Xenopus embryos suggests that XCEP2 may play a direct role in directing the morphogenetic movements of gastrulation. This interpretation would be consistent with the limited characterization that have thus far been conducted on functional properties of this class of proteins [The observed effects of antisense XCEP2 morpholinos in proteins ,30. HoweIn order to assess the mechanism by which XCEP2 influences gastrulation, the effects of XCEP2 morpholinos on cultured animal cap explants were evaluated. While the initial intent of these experiments was to discern whether XCEP2 morpholinos prevented normal activin-induced convergent extension of animal caps, it soon became clear that morpholino-treated explants had a more fundamental deficiency. Within a 24 hour period, animal cap explants derived from XCEP2 antisense morpholino (MO1) -injected embryos were found to completely lose their integrity . CurXenopus Cdc42 effector protein 2 (XCEP2) in gastrulation movements. XCEP2 is a member of the recently characterized CEP family of Rho GTPase effector proteins [The data presented here indicate a role for the proteins ,30, whicproteins . Effectoproteins ,30. By aproteins ). The exproteins ,30.Consistent with a potential role for this class of proteins in embryonic morphogenesis, we have shown that XCEP2 expression is temporally regulated at gastrulation stages, when major modulations of cellular morphology, cytoskeletal organization, and cellular adhesion are occurring. mRNA for XCEP2 is present prior to mid-blastula transition, persists through mid gastrulation, and is strongly down-regulated by the time the blastopore closes and neurulation begins. This pattern would suggest that XCEP2 protein would be present through the period when active gastrulation movements are occurring. The diffuse spatial pattern of mRNA and, presumably, protein expression of XCEP2 may suggest that XCEP2 functions broadly in cells of the animal and equatorial regions. However, the observed broad spatial distribution of XCEP2 mRNA does not preclude the possibility that the XCEP2 protein may be functionally activated or inactivated at discreet times and locations during early embryonic development.Xenopus gastrulation, delay the closure of the blastopore and inhibit embryonic elongation. The observed rescue with XCEP2 mRNA is strong evidence for the specificity of the antisense morpholino effect. These effects are not due to a loss of mesodermal induction, as brachyury and goosecoid expression do not change in response to antisense XCEP2 morpholinos. This is particularly relevant given recent reports demonstrating a direct link between brachyury expression and control of cellular migration [Furthermore, we show that antisense morpholino oligonucleotides capable of blocking translation of the XCEP2 message interfere with igration .The effects we report require relatively high, although not unprecedented, doses of morpholino antisense oligonucleotide. This dosage requirement may reflect the difficulty inherent in morpholino-mediated translational knockdown of maternally expressed genes. XCEP2 mRNA and (presumably) XCEP2 protein are present prior to midblastula transition. Given this situation, the timing morpholino induced protein downregulation is dependent both upon the effectiveness of translational blockade, and the half life of the protein in the cytoplasm. In this context, it may be essential to impose close to complete translational inhibition in order to reduce protein levels rapidly enough to affect early embryonic events, such as gastrulation.Clearly, specific probes to assess endogenous XCEP2 protein expression will be necessary for fuller characterization of the role of this protein during gastrulation. For this reason, antibodies are currently being raised against the XCEP2 protein. These antibodies will be important in the characterization of the developmental time course of endogenous XCEP2 protein expression, assessment of the subcellular localization of the XCEP2 protein, isolation of potential XCEP2 binding partners, and in assessing and further optimizing the extent of protein down-regulation in morpholino injected embryos.Currently, the specific mechanism by which XCEP2 exerts its role in gastrulation is unknown. However, our preliminary data suggest that XCEP2 may either contribute to a required "ground state" of cellular adhesion or play a role in modulations in the strength of cadherin-mediated cell-cell adhesion that are known to occur during gastrulation ,39,40. MIn embryos Wnt-mediated signals have been shown to activate Cdc42, a process that is required for normal gastrulation movements ,12. In fThe known functional properties of the CEP class of effector proteins, and the characteristics of CRIB domain effector proteins in general, suggest some interesting possibilities relating to the control of cell adhesion during gastrulation. Consistent with the observed functions of the XCEP2 homologs in cultured cells, XCEP2 in embryos may impinge on regulatory circuits downstream of Cdc42 that control actin filament assembly, which in turn may affect diverse cellular processes, including assembly of adherens junctions. Alternatively, XCEP2 may more directly impinge upon cadherin functional activity, perhaps by influencing the association of IQGAP or other molecules with cadherin complexes. In future studies, it will also be important to establish whether embryonic activation of whether there are links between Wnt-mediated activation of Cdc42 and functional activation of XCEP2 and to characterize the mechanism(s) by which XCEP2 contributes to cell adhesion between cells of gastrulating embryos.Xenopus gastrulation. For this reason it will be interesting and important to discern further the role of XCEP2, with regard both to its relationship to intracellular signalling pathways and its effects on cellular behavior during gastrulation.It has become clear in recent years that an integrated network of signals involving Rho GTPase proteins and their effector proteins help to control and regulate the diverse and intricate morphogenetic processes that occur during embryonic development. Less clear are the specific modes of functional interaction between the multiple Rho GTPases and the diversity of potential effector proteins. We have shown that XCEP2 is one component in the complex regulatory puzzle contributing to morphogenetic processes during Adaptor 1-5'-ATGAGTCCTGAC-3' (upper strand)4-CGGTCAGGACTCAT-3'(lower strand).5'-POAdaptor 2 (the 3' phosphate group of the lower strand inhibits DNA polymerase extension)-5'-ACTGGTCTCGTAGACTGCGTACC-3'(upper strand)4-CGGGTACGCAGTC-PO43' (lower strand).5'-PO"Universal" RFDD-PCR primer (complementary to Adaptor 2)-5'-ACTGGTCTCGTAGACTGC-3'"Selection" Primer 4 AAG-3' (3-nucleotide extension is underlined)5'-ATGAGTCCTGACCGAXCEP2 cDNA forward-ATGTCCGCCAAG-3'5'-ATTGCAAAGXCEP2* cDNA forward-CGGGATCCTAGATGTCGGCGAAAGCGCCGATATACCTAAAGAG AAG-3'5'-XCEP2 cDNA reverse-5'-AACGTATCCCCTTCCCCA-3'XCEP2A forward (complementary to 5' untranslated region of the cDNA sequence)5'-AACGTATCCCCTTCCCCA-3'XCEP2A reverse (complementary to 5' untranslated region of the cDNA sequence)5'-AAAGAGAAGTAGCCGTAAAGGA-3'XCEP2B forward5'-GCCAAGGCCCCGATATAC-3'XCEP2B reverse5'-CCAATAGCAGGTAGGGAA-3'Brachyury forward5'-GGATCGTTATCACCTCTG-3'Brachyury reverse5'-GTGTAGTCTGTAGCAGCA-3'Goosecoid forward5'-ACAACTGGAAGCACTGGA-3'Goosecoid reverse5'-TCTTATTCCAGAGGAACC-3'ODC forward-5'-GTCAATGATGGAGTGTATG-3'ODC reverse5'-TCCATTCCGCTCTCCTGA-3'Antisense morpholino oligonucleotides, which specifically block translation of targeted mRNAs  were synAntisense XCEP2 MO1 -CATCTTTGCA-3'5'-GGGCCTTGGCGGAXenopus Genbank sequence accession # BC045241)-Antisense XCEP2 MO2  are ligated to restriction digested, double stranded cDNA. Amplification of this cDNA is conducted with the "universal primer", complementary to one of the adaptor sequences, and one of 64 "selection" primers that bind largely to the to the second adaptor sequence but have a 3-nucleotide 3' extension that is complementary only to a subset of cDNA inserts. An extension-inhibiting chemical modification of the lower strand Adaptor 1, consisting of a 3' phosphate group in our modified procedure, and the 3-nucleotide extension of the selection primer largely restricts amplification to cDNA fragments that have different adaptor sequences ligated on opposite ends. Furthermore, any particular selection primer will selectively amplify only a subset of cDNA fragments .Restriction Fragment Differential Display PCR (RFDD-PCR) procedures were patterned closely after the commercially available DisplayProfile kit from QBiogene , with minor modifications. In this differential display procedure, Adaptors 1 and 2 . RNA from Xenopus laevis embryonic stages 7, 8.5, 9.5, 10.5 and 12.5 were prepared. First strand cDNA was synthesized using oligo dT primer and Superscript III reverse transcriptase (Invitrogen), followed by second strand synthesis catalyzed by DNA polymerase I . Double stranded cDNA was purified using GeneClean silica resin , and then digested with TaqI restriction enzyme . An annealed mix of RFDD-PCR of Adaptor 1/Adaptor 2 oligonucleotides was added, and ligated to the TaqI digested cDNA ends using T4 DNA Ligase (Roche). Touchdown PCR using the universal RFDD-PCR primer, 35S-labelled dCTP, and "Selection" Primer 4 was conducted using Accuprime PCR mix (Invitrogen), with the following cycling parameters:Total RNA from staged, duplicate batches of Pre-dwell: 94°C 4 minutes10 cycle touchdown PCR, with a 0.5 temperature decrement each cycle:94°C 30 seconds60°C→ 55°C 30 seconds72°C 1 minute30 cycle standard amplification:94°C 30 seconds55°C 30 seconds72°C 30 secondsPost dwell: 72°C, 5 minutesXenopus homologue of the Cdc42 Effector Protein 2, which we refer to as "XCEP2". The full length cDNA was amplified using gene-specific primers and poly-dT primers using standard 3' RACE procedures [Samples were run on a standard 6% denaturing DNA sequencing gel, and the gel was dried and subjected to autoradiography. Bands showing differential expression were excised from the gel and eluted by heating at 95°C for 15 minutes. Eluted fragments were re-amplified with 30 cycles of standard PCR, using the original primers (above). Direct cycle sequencing of isolated product  revealed that one of the fragments corresponded to the 5' end  of a The RACE amplified full-length fragment isolated from the RFDD-PCR procedure (see above) was cloned into the pCR2.1 vector, using the pCR2.1 TA cloning kit (Invitrogen), and commercially sequenced (MWG Biotech). To construct a vector encoding XCEP2A fused to a C-terminal 6xmyc tag, PCR was conducted using primers directed to the 5' and 3' end of the XCEP2A cDNA sequence (XCEP2A cDNA forward and XCEP2A cDNA reverse) with the cloned, full length XCEP2A as template. The primers used in this PCR mutated the normal stop codon and introduced a BamHI and ClaI site at the 5' and 3' ends of the amplified product, respectively. BamHI/ClaI digested full length cDNA was directionally cloned in the sense orientation into the corresponding into BamHI/ClaI digested pCS2-myc vector, producing the plasmid pCS2-XCEP-myc. In this context, the full length X-CEP2A cDNA encodes a fusion protein with a C-terminal 6x myc tag. A second construct, containing introduced mutations in the XCEP2A antisense morpholino target region, was also derived by PCR. To construct this vector, PCR was conducted using XCEP2* forward and XCEP2 reverse primers with the full-length XCEP2 DNA as template. XCEP2* forward introduced multiple changes in the sequence of the cDNA encoding the translational start site region , producing the plasmid pCS2-XCEP2-anti. In a similar fashion, an RNA expression vector containing the XCEP2 insert in the sense orientation in pCS2+ was also constructed.For transcription of antisense probe for in vitro using the mMessage mMachine kit (SP6) from Ambion , and were dissolved in distilled water prior to injection.Capped mRNAs were synthesized RT-PCR Primers, above). 25 cycles of PCR were carried out using Accuprime PCR mix (Invitrogen) in 25 ul reactions containing 2 ul of 200 ug/ml reverse transcribed cDNA, 0.3μM of each primer, and a trace of radiolabelled nucleotide to allow for autoradiographic visualization. Parallel reactions containing primers specific to the metabolic gene, ornithine decarboxylase (ODC), were used as a loading control in all semi-quantitative RT-PCR experiments. RT-PCR products were separated electrophoretically on 5% acrylamide gels, which were dried and subsequently subjected to autoradiography. RT-PCR of the XCEP2A allele resulted in stronger amplification than with the XCEP2B primer set. XCEP2B bands required correspondingly longer exposure times (2–4 fold) for detection.Total RNA was isolated from embryos using Trizol (Invitrogen) according to manufacturer's instructions. Reverse transcription was carried out using SuperscriptIII (Invitrogen). Assessment of relative levels of gene expression over developmental time, or under different experimental conditions, was carried out using standard semi-quantitative RT-PCR methods , with thXenopus laevis were purchased from Nasco . Eggs were obtained and fertilized according to standard methods. Staging was determined according to Nieuwkoop and Faber [in vitro transcribed mRNA's were injected into two-cell stage embryos, with injected volumes of approximately 13 nl in doses as described. All injections were carried out using the Nanoject positive displacement microinjector .Adult nd Faber . AntisenMeasurement of blastopore to embryo diameter ratios was accomplished by dividing the measured blastopore diameter  by the embryo diameter. Diameter measurements were obtained using digital images of individual embryos, and the "Ruler" tool of the Adobe Photoshop software.To assess the effects of morpholino antisense oligonucleotides on animal cap explants, morpholino injected and control embryos were allowed to develop until stage 8.5. At stage 8.5 animal caps were dissected, and then cultured in 1xMMR for 24 hours. Non-dissected sibling embryos were allowed to develop until post-neurula stages in order to asses the effectiveness of the morpholino treatment.Proteins from embryos were extracted in 10 volumes of isotonic buffered saline containing 1% NP40. Extracts were cleared by a brief centrifugation, and supernatants were denatured by a 5-minute incubation at 95°C after diluting samples with SDS-PAGE sample buffer. Proteins were separated on 8–16% gradient polyacrylamide gels, and transferred to nitrocellulose. After blocking, blots were probed with monoclonal antibody 9E10 , which rin vitro from linearized pCS2-XCEP2anti plasmid using a digoxygenin nucleotide labeling mix (Roche) and the mMessage mMachine in vitro transcription kit (SP6) from Ambion. In situ hybridization was carried out essentially as described [Digoxygenin-labelled antisense RNA probe was transcribed escribed , using nin situ hybridization methodology in the laboratory, and conducted in situ hybridizations. RWN conceived of the study, designed and conducted molecular biological, embryological and biochemical experiments, and wrote the manuscript. Both authors read and approved of the final manuscript.KKN played a critical role in establishing


Our obstetrics and gynaecology undergraduate teaching module allocates 40–50 final year medical students to eight teaching hospital sites in the West Midlands region. Based on student feedback and concerns relating to the impact of new curriculum changes, we wished to objectively assess whether the educational environment perceived by students varied at different teaching hospital centres, and whether the environment was at an acceptable standard.A Dundee Ready Education Environment (DREEM) Questionnaire, a measure of educational environment, was administered to 206 students immediately following completion of the teaching module.The overall mean DREEM score was 139/200 (70%). There were no differences in the education climate between the teaching centres.Further research on the use of DREEM inventory, with follow up surveys, may be useful for educators to ensure and maintain high quality educational environments despite students being placed at different teaching centres. The undergraduate curriculum at our medical school was redesigned in 1998/99 to bring it in line with recommendations suggested by the General Medical Council (GMC) in Tomorrow's Doctors [Based on previous student feedback reporting differences in educational experiences, together with our concerns relating to the impact of new curriculum changes, we wished to objectively assess whether the educational environment perceived by students varied at different teaching hospital centres, and whether the environment was at an acceptable standard. In particular, was there any potential loss of teaching experience when students were placed away from the principal Teaching Hospital. Thus, the null hypothesis we wished to test was that there was no difference in the learning environment between centres. Several questionnaire-based educational tools are available that set out to 'quantify' the educational environment -4. HowevThe DREEM questionnaire, based on a Likert scale, was administered to the full class of 206 final-year Birmingham University medical students undertaking the exam module in Obstetrics and Gynaecology in 2000. All questionnaires were distributed and returned the same day of the exam, which allowed us to achieve a 100% response rate. Students were told to only comment on their recent 8-weeks experience of Obstetrics and Gynaecology. Statistical analysis was performed using Microsoft Excel and Arcus Quickstat Biomedical Statistical software, and utilised single-sample T test and One-way analysis of variance (ANOVA).The year group comprised 42% male and 58% female. The overall mean DREEM score for the study group was 139/200 (95% CL 136.1 to 141.9), or expressed as percentage of the maximal score, 70% (95% CL 68% to 71%). There was no statistically significant difference between the mean scores for the contributory DREEM domains, which were as follows: perception of learning, 34.52/48 (72%); perception of teaching, 32.05/44 (73%); academic self-perception, 19.46/32 (61%); perception of atmosphere, 34.07/48 (71%), and for social self perceptions, 18.90/28 (68%). The DREEM scores for each hospital, with comparison of all contributory elements of the DREEM inventory, are depicted in Table When converting the raw DREEM score to percentages, two-sided P-value single-sample Student's T test showed no statistically significant difference between hospitals by each DREEM domain, or between each DREEM domain within the same hospital. Greatest variation between hospitals occurred in the Students' Perception of Atmosphere domain, where there were four hospitals beyond the 95% Confidence Limits; this compared to three hospitals beyond 95% Confidence Limits in all other DREEM domains Table s. One-WayWe have used the Dundee Ready Education Environment Measure (DREEM) in 'diagnosing' the educational environment of eight different teaching centres and making comparative analysis between these centres. The overall mean DREEM score was 139/200, or expressed as a percentage, 70% (95% CL 68–71%). The educational learning environment did not vary between centres. The two lowest scoring contributory domains, academic self-perception (61%) and social self-perceptions (68%), were not statistically significantly different from the other three DREEM domains or overall mean DREEM score.This study has benefited by using an established educational measure and obtaining a 100% response rate. No students had been previously taught at the principal teaching hospital as this was solely used for Obstetrics and Gynaecology teaching. However, some of the students  had previously attended the other seven teaching hospital centres due to prior clinical teaching attachments. Thus, previous experiences may have biased the teaching assessment completed by some students. Furthermore, the DREEM questions are of such a nature that it is likely that the environment of the entire curriculum was being assessed. However, by performing the DREEM survey immediately at the end of the obstetrics and gynaecology module, and emphasising reporting only the last eight weeks experience, we believe this maximised the chance that the DREEM measure assessed only the recent hospital teaching site and minimised any recall bias. Other groups  have higThe DREEM domains are unlikely to be independent variables, and may be less of an environment test but more of a measure of the overall motivation and learning attitude of the individual. The Course Valuing Inventory (CVI) score is made up of five domains: worthiness of learning experience, emotional awareness, personal development, cognitive enhancement and task drive. A recent study of first year medical students showed a correlation between higher Course Valuing Inventory (CVI) scores, female gender, stronger self-confidence as a learner, greater motivation to learn and higher DREEM scores .There is no accepted agreement on what is an acceptable DREEM inventory score from published literature. Nevertheless, our DREEM score of 139/200 was higher than other reports. A study of final year medical students in Trinidad reported an overall mean DREEM of 109.9/200 . A largeThe non-significant differences between the DREEM domains and between hospitals were significant findings. This was conveyed to our tutors based at the various teaching centres as a positive and encouraging result. In practical terms, this meant that regardless of hospital capacity or student group size, their education delivery and environment was no different to other centres in the student's curriculum. The DREEM inventory may thus be a useful tool for educators to ensure and maintain high quality educational environments and uniformity in educational delivery despite students being placed at different teaching centres.The author(s) declare they have no competing interests.RV and ET carried out the statisitcal analysis, data interpretation, and drafted the manuscript. JKG conceived and coordinated the study, acquired the results, and made revisions of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


In clinical practice, visual gait observation is often used to determine gait disorders and to evaluate treatment. Several reliability studies on observational gait analysis have been described in the literature and generally showed moderate reliability. However, patients with orthopedic disorders have received little attention. The objective of this study is to determine the reliability levels of visual observation of gait in patients with orthopedic disorders.The gait of thirty patients referred to a physical therapist for gait treatment was videotaped. Ten raters, 4 experienced, 4 inexperienced and 2 experts, individually evaluated these videotaped gait patterns of the patients twice, by using a structured gait analysis form. Reliability levels were established by calculating the Intraclass Correlation Coefficient (ICC), using a two-way random design and based on absolute agreement.The inter-rater reliability among experienced raters  was comparable to that of the inexperienced raters . The expert raters reached a higher inter-rater reliability level . The average intra-rater reliability of the experienced raters was 0.63 (ICCs ranging from 0.57 to 0.70). The inexperienced raters reached an average intra-rater reliability of 0.57 (ICCs ranging from 0.52 to 0.62). The two expert raters attained ICC values of 0.70 and 0.74 respectively.Structured visual gait observation by use of a gait analysis form as described in this study was found to be moderately reliable. Clinical experience appears to increase the reliability of visual gait analysis. Patients exhibiting gait deviations caused by orthopedic impairments are often referred to a physical therapist for treatment. In order to determine treatment goals or to evaluate the effect of a therapeutic intervention, physical therapists visually observe the patient's gait -3. This et al. [et al. [Several reliability studies on observational gait analysis have been described in the literature. These studies included patients with hemiplegia -7, amputet al. , found oIn the Netherlands a gait analysis form has been developed which focuses mainly on orthopedic disorders . Visual The purpose of this present study is to determine the inter- and intra-rater reliability of videotaped observational gait analysis with use of an orthopedic gait analysis form when applied to a cohort of patients suffering from orthopedic impairments. In addition, this study determines how well the raters perform observational gait analysis by comparing their assessments with a criterion, based on the experts' opinion. In order to gain insight into how the results may give guidance to physical therapy treatment, this study also investigates which items on the gait analysis form, that have been considered to be disturbed by visual observation, receive high priority in the physical therapy treatment program according to the physical therapist who performs the visual gait analysis.Thirty videotapes of patients' gait were selected from the archives of the department of Physical Therapy of the University Medical Center Nijmegen, the Netherlands. These videotapes involved patients who had been referred to a physical therapist for gait treatment. It is common practice at this department that prior to gait training therapy the gait of each patient is videotaped according to a standardized protocol.The criteria for inclusion of the videotapes were: (1) the presence of mild to severe gait deviations due to an orthopedic impairment; (2) patient was wearing shorts or underwear to allow for a more accurate observation of the joint movement; (3) ability of a patient to walk 15 meters at least four times, twice in a semi-circle and twice in a straight line on a gymnasium floor; (4) and patient's written informed consent. The first thirty patients who complied with these criteria were included.The group consisted of 15 male and 15 female patients with a mean age of 37.8 years (range: 15 to 62 years). The type of orthopedic impairments varied from status post hip, knee, ankle surgery (n = 8), status post hip or knee prosthesis (n = 6), status post femur, tibia or ankle fracture (n = 3) and traumatic or non-traumatic non-specific hip, knee or ankle pain (n = 13) , anterior and posterior view at each of the three sub-phases of stance and the two sub-phases of swing. All patients were recorded from a lateral view (both sides) while walking 15 meters in a semi-circle (radius approximately 10 m) at a comfortable self-selected walking speed. We used a semi-circle in order to be able to observe the patient's gait in the sagittal plane from one position. The anterior and posterior views were videotaped while the patient walked five meters toward and away from the camera.®). Manufactured videos were reduced into a one-minute film-clip in which the patient's gait could be viewed in the lateral and frontal plane. Subsequently, these videos were converted to analog format again, so that they could be played by a regular video player. Sampling frequency was 24 Hz.The collected videos were edited with use of the computer program adobe premiere 6.0  to the items they scored as disturbed, with respect to a physical therapy treatment program. In other words, which items would receive important attention in the physical therapy intervention if the rater was going to treat this patient for his or her gait disorder.In order to determine the level of performance of observational gait analysis of all experienced and inexperienced raters, we compared their ratings with a criterion. This gives us an indication about how well the raters were capable in performing visual gait analysis. The criterion was attained during a consensus session of the two expert raters: After individually assessing the 30 patients for the second time, the two expert raters jointly observed the videotaped gait of all 30 patients for the third time.Inter- and intra-rater reliability levels were assessed by using Intraclass Correlation Coefficients (ICCs), validated for use with multiple raters and calculated in a two-way random model based on absolute agreement. We used ICCs because it has been shown that with data that are rated as a dichotomy, the ICC is equivalent to measures of nominal agreement, simplifying computation in cases where more than two raters are involved . In addiThe inter-rater reliability among experienced raters was 0.42 (95%CI: 0.38–0.46). This level of reliability is comparable to the inter-rater reliability of in-experienced raters, which reached an ICC value of 0.40 (95%CI: 0.36–0.44). The expert raters reached the highest inter-rater reliability (ICC: 0.54 (95%CI: 0.48–0.60)).There were no differences in inter-rater reliability between the first and second rating session of all three groups separately, based on the overlap of 95% confidence intervals.The average intra-rater reliability of the experienced raters was 0.63 (ranging from 0.57 to 0.70). The inexperienced raters reached an average intra-rater reliability of 0.57(ranging from 0.52 to 0.62). The two expert raters attained ICC values of 0.70 and 0.74 respectively.The agreement between the outcome of the joint assessment of the expert raters (criterion) and those of the individual experienced raters ranged from 0.43 to 0.55 with an average ICC value of 0.48. The inexperienced raterrs attained agreement levels ranging from 0.41 to 0.55, with an average of 0.49. There is no difference in the level of performance of visual gait assessments of experienced or inexperienced raters, when compared to the experts' opinion.The inter-rater reliability per item on the gait analysis form between the two experts is generally moderate to substantial . Only one item, extension movement of the knee during mid stance, had an ICC value for intra-rater reliability of less than 0.6. The experienced raters were able to attain good intra-rater reliability for item 2, posture of the trunk during walking (ICC = 0.81). Three items reached substantial intra-rater reliability . Two items of the gait analysis form, pelvis rotation and ankle movement during late stance, were not intra-rater reliable (ICC < 0.40). The inexperienced raters reached the highest intra-rater reliability for the assessment of arm swing during walking (ICC = 0.81). Three items had inadequate intra-rater reliability levels; flexion of the knee in early stance (ICC = 0.36), extension of the knee in mid stance (ICC = 0.22), and ankle movement during the late stance phase (ICC = 0.37).No reliability score was obtained from item 3, which describes a trunk position behind the hips, because this item was observed only once.On average, with respect to all items, in about a quarter of the cases items were judged to be disturbed by the expert and experienced raters . Expert raters showed the least variability between the first and second session; five items showed to have a mean intra-rater reliability level that is considered good (ICC > 0.80).The results of this study suggest that a brief introduction in normal gait kinematics in inexperienced raters gives comparable reliability levels of observational gait analysis in patients with orthopedic impairments compared to experienced physical therapists, who have worked for several years with patients with gait disorders. However, expert raters – those that work significantly more intensive with patients with gait disorders – accomplish higher reliability levels.As mentioned in the methods section, the gait analysis form used in this study is also used in daily practice to guide the treatment of the patient's gait disorder. In the physical therapist's treatment program, some items on the form will obviously receive higher priority than others. The results of this study show that physical therapists mainly focus their intervention on movement disorders of the lower extremity. However, the expert raters also report to give priority to asymmetry of the stance phase and excessive lateral flexion of the trunk during gait. Of the three items in this study that achieved the highest reliability levels, only the movement of the knee received generally a high priority in the treatment program of experienced raters. This implies that experienced raters will mainly focus their treatment on items that have generally a low inter- and intrarater reliability.Structured visual observation of a patient's gait by use of a gait analysis form as described in this study is found to be only moderately reliable, but may be a useful guide to the physical therapist in setting up a gait training or exercise therapy program. Intra-rater levels have shown that visual gait analysis will supply the observer with a fair indication of changes in a person's gait. However, to evaluate the effect of an intervention on a patient's gait we recommend more objective instrumentation which has been proven reliable and valid.The author(s) declare that they have no competing interests.JB carried out the data analysis, participated in the design of the study and drafted the manuscript. CvU participated in the design and coordination of the study, assisted with statistical analysis, and helped to draft the manuscript. SvM participated in the design of the study and supplied the videotapes. JK participated in the design and coordination of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Salmonella enterica serovar Typhimurium aroA strain in two Raf dependent lung tumor mouse models.Serine-threonine kinases of the Raf family  are central players in cellular signal transduction, and thus often causally involved in the development of cancer when mutated or over-expressed. Therefore these proteins are potential targets for immunotherapy and a possible basis for vaccine development against tumors. In this study we analyzed the functionality of a new live C-Raf vaccine based on an attenuated Escherichia coli α-hemolysin and expressed in secreted form by an attenuated aroA Salmonella enterica serovar Typhimurium strain via the α-hemolysin secretion pathway. The effect of the immunization with this recombinant C-Raf strain on wild-type C57BL/6 or lung tumor bearing transgenic BxB mice was analyzed using western blot and FACS analysis as well as specific tumor growth assays.The antigen C-Raf has been fused to the C-terminal secretion signal of Salmonella enterica serovar Typhimurium aroA strain using the E. coli hemolysin secretion system. Immunization of wild-type C57BL/6 or tumor bearing mice provoked specific C-Raf antibody and T-cell responses. Most importantly, the vaccine strain significantly reduced tumor growth in two transgenic mouse models of Raf oncogene-induced lung adenomas.C-Raf antigen was successfully expressed in secreted form by an attenuated Salmonella enterica serovar Typhimurium could form the basis for a new generation of live bacterial vaccines for the treatment of Raf dependent human malignancies.The combination of the C-Raf antigen, hemolysin secretion system and  The Raf proteins  are located upstream of MEK and downstream of Ras and represent an essential part of the mitogenic cascade -3. InterSalmonella enterica serovar Typhimurium aroA strain. Such recombinant live vaccines have been shown to efficiently elicit both, humoral and cellular immune responses against a variety of heterologous antigens [E. coli α-hemolysin (HlyA) secretion system which is fully active in Salmonella [s), which is recognized by the HlyB/HlyD/TolC-translocator, leading to direct secretion of the entire protein into the extracellular medium without the formation of periplasmic intermediates. In addition, fused to the C-terminus of heterologous proteins, the HlyAs leads to efficient secretion of such proteins by the recombinant bacteria. In our case, the whole C-Raf antigen fused to the C-terminal secretion signal of hemolysin was efficiently expressed and secreted by an attenuated Salmonella enterica serovar Typhimurium aroA strain. The effects of this recombinant vaccine were assessed in wild-type C57BL/6 or tumor bearing transgenic BxB mice.Here we describe the development of a C-Raf vaccine on the basis of an attenuated antigens ,8. In orlmonella . This trThe bacterial strains, plasmids, cell lines and mice used in this study are listed in Table Salmonella enterica serovar Typhimurium LB5000  at 2.5 kV, 25 microfarads (μF), and 200 Ohm in a 0.1 cm electroporation cuvette.The plasmids pMOhly1 and pMOhly-Raf were first transformed in competent  E. coli ,11. SubsATGCATCTTGGAAG-3' and antisense primer 3'Raf: 5'-CAACTAGAATGCATGCAGCCTCGGGGA-3' were used to amplify by PCR a 1950 bp DNA fragment representing the entire craf gene from plasmid pUC13-c-raf-1 [NsiI restriction enzyme, the DNA fragment carrying the craf gene was inserted into the single NsiI site of the export vector pMOhly1 [E. coli DH5α (Invitrogen), analyzed and transformed in S. typhimurium SL7207  were centrifuged at 5000 × g at 4°C for 5 min. The supernatant proteins were precipitated with 10% (V/V) trichloroacetic acid (TCA) for 1 h on ice, collected by centrifugation and resuspended in SDS sample buffer. The supernatant proteins were separated on 10% gels by SDS-PAGE [SDS-PAGE . For imm9 bacteria/100 μl phosphate-bufferd saline (PBS) at 5-day intervals. At day 45 after the start of the vaccination, these mice were boosted intravenously (i.v.) with a single-dose of 5 × 105 bacteria/100 μl PBS. The BxB23 mice received a second i.v. boost of 5 × 105 bacteria at day 90 after the start of the vaccination. Induction of Raf-specific immune responses were analyzed at day 50 for C57BL/6 or at day 95 for BxB23 respectively.In order to achieve a broad immune response encompassing both, the mucosal and systemic immunity against C-Raf, we combined oral immunization (p.o.) with an intravenous (i.v.) boost. In these experiments, seven weeks old C57BL/6 or BxB23 mice were immunized, first p.o. three times with 5 × 107bacteria/30 μl phosphate-buffered saline (PBS) at 14-day intervals. The vaccine was applied using a micropipette onto the nares of mice under anesthesia.Seven weeks old BxB23 mice were immunized i.n. four times with 1 × 10Induction of Raf-specific immune responses were analyzed at day 70.8salmonellae in 10 μl PBS at 14-day intervals. The vaccine was applied using a micropipette into the nares of mice without anesthesia.Four months old BXB11 mice were immunized i.n. three times with 1 × 10b haplotype . The EL4 tumor cell line is a murine thymoma cell line of the H-2ype Tab. . 10 μg pAnimals were sacrificed and a single cell suspension of splenocytes was prepared by passage of the spleen through a sieve into RP 10 medium [RPMI medium (Life Technologies) supplemented with glutamine (1%), 50 μM β-mercaptoethanol , penicillin , streptomycin  and 10% fetal calf serum .4Cl, pH 7.3) for the lysis of erythrocytes. After 2 minutes, 10 ml RP 10 medium was added to stop lysis. After centrifugation, cells were resuspended in 2 ml RP 10 medium and counted.The cell suspension was centrifuged and resuspended in 3 ml lysis buffer  or 5 × 105 EL-4 cells or with the addition of 10 ng/ml of phorbol myristyl acetate  and 500 ng/ml of ionomycin (Sigma) or with medium alone.In 6 ml FACS tubes and a total volume of 100 μl RP 10 medium in the presence of 30 U/ml recombinant IL-2 and 10 μg/ml Brefeldin A at 37°C, 5% COAfter restimulation, cells were washed with PBS-0.1% bovine serum albumin  and incubated with 1 μl of anti-CD8-CyChrome (Pharmingen Nr. 01082A) in a volume of 100 μl PBS-0.1% BSA for 20 min on ice. Subsequently, cells were washed and fixed for 20 min at room temperature with PBS-2% paraformaldehyde (Sigma). After washing with PBS-0.1% (BSA), cells were permeabilized with PBS-0.1% BSA-0.5% saponin  buffer. After incubation for 5 min at room temperature, cells were washed with P-B-S buffer and incubated in a volume of 100 μl at room temperature with polyclonal rat immunoglobulin G (IgG) antibodies (JacksonImmunoResearch) to block nonspecific binding and 1 μl anti-IFN-γ-FITC  for 20 min. After incubation, cells were washed twice with P-B-S buffer and another time with P-B buffer. Cells were resuspended in 400 μl PBS-PFA 0,5% and kept at 4°C until analysis.+ cells.Cells were analyzed by flow cytometry in a FACS Calibur flow cytometer (Becton Dickinson) using CellQuest 3.0 software (Becton Dickinson). Lymphocytes were chosen according to their size and granularity in a forward/side – scatter diagram. Numbers are expressed as percent IFN-γ positive CD8t test. Findings were regarded as significant, if P values were <0.05. Survival curves were compared using a log rank test.The statistical significance of differential findings between experimental groups was determined by Student's Salmonella enterica serovar Typhimurium aroA strain SL7207 secreting the C-Raf antigen was achieved by cloning the human craf cDNA from pUC13-c-raf-1 [craf-hlyAs fused gene and the functional hlyB and hlyD genes required for its secretion  and with Salmonella enterica serovar Typhimurium SL7207 as control in order to test the induction of Raf-specific immune responses. The data showed that 20% of the sera of BxB23 mice immunized with SL7207/pMOhly-Raf contained Raf-specific antibodies .To assess the induction of Raf-specific T-cell responses, mice were sacrificed and Raf specific T-cell responses were assessed using intracellular IFN-γ staining followed by FACS analysis. For this purpose, T-cells were restimulated with C-Raf overexpressing EL-4 cells. Using this technique, we could detect Raf specific CD8Salmonella strains, groups of 6 to 10 heterozygous BxB23 mice at the age of 7 weeks were immunized p.o./i.v. or i.n. with recombinant Salmonella enterica serovar Typhimurium strain (SL7207/pMOhly-Raf) or with SL7207 as control. BxB23 mice normally show an induction of lung adenomas with short latency and at 100% incidence [To test the protective capacity of the immune responses induced by the recombinant ncidence ,16. The craf transgenic strain, which develop lung adenomas after a shorter latency period compared to BxB23 mice [In order to confirm these data we repeated the protection experiments using BxB11, another B23 mice . In thisThese results suggest that the immunization with the vaccine strain SL7207/pMOhly-Raf can achieve a partial protection against tumor growth in the BxB mouse model.The major problems for vaccine development against cancer are the heterogeneity of the tumor cells and the fact that all tumor antigens are self-antigens. Therefore specific T-cells might be anergic or tolerant ,18.However, despite these problems, several cancer vaccines have already reached clinical trials . The sucSalmonella enterica serovar Typhimurium aroA strain as a "live vaccine". In general, recombinant aroA salmonellae are efficient live bacterial vectors that stimulate strong mucosal immunity, humoral and cell-mediated responses with a great potential as live vaccine carriers in both humans and animals [aroA Salmonella vaccines include their safety and easy administration [aroA Salmonella enterica serovar Typhimurium strains were already successfully used as carriers for DNA vaccines against cancer in mice [Here we describe a new strategy for achieving an anti-tumor immune response with a C-Raf vaccine on the basis of an attenuated  animals ,20. Advastration ,22. In a in mice .Salmonella enterica serovar Typhimurium aroA strain using the E. coli hemolysin secretion system. This system allows an efficient antigen secretion and presentation, which are necessary for an optimal immune response against the given heterologous antigen [Salmonella enterica serovar Typhimurium aroA strain SL7207 secreting C-Raf resulted in the induction of a humoral immune response, manifested by the presence of Raf-specific antibodies in some of the vaccinated mice. In addition, C57BL/6 mice immunized with Salmonella enterica serovar Typhimurium SL7207/pMOhly-Raf developed a Raf-specific CD8+ T-cell response. The C-Raf vaccine thus is able to break the peripheral tolerance of the immune system towards C-Raf and to induce a specific immune response. Although the transgenic model is closer to the human setting in comparison to challenge models with tumor cell lines, we can not formally exclude that lung specific expression of the transgene in our BxB model is not sufficient for the induction of peripheral tolerance. Furthermore, side effects due to autoimmunity might occur in humans who have a more generalized expression pattern. However, the lung tissue does not belong to immunoprivileged sites and we have never observed any signs of lung pathology in immunized animals which strongly suggests that the data could, in principle, be translated into the human setting.In this study the C-Raf antigen was delivered in secreted form by attenuated  antigen . In factSalmonella enterica serovar Typhimurium SL7207/pMOhly-Raf. However, we were not able to assess Raf specific CTL responses in these mice due to the high variability and background and therefore we cannot conclude whether protection is really due to cytotoxic T-cells or might be the result of other effects, e.g. an increased intratumoral level of IFN-γ produced by Raf specific CD4+ T-cells. Interestingly, the observed variation occurred only in treated BxB23 mice, not in naïve BxB23 mice. Therefore, the observed effect might be due to a spontaneous induction of an immune response mediated by tumor infiltrating bacteria. It is interesting to note that spontaneous Raf specific immune responses also occur in the human context, which we have recently demonstrated B-Raf V599E specific CTL and B-Raf /B-Raf V599E specific humoral responses in melanoma patients [We demonstrated also a partial tumor protection both in BxB23 and in BxB11 mice after immunization with recombinant patients ,25.+ T-cells are effective in eliminating antigen positive tumor cells. The combined data thus provide proof of concept for the development of Raf-based anti-cancer vaccines.Most importantly, the lack of B-Raf V599E mutations in metastases of melanoma patients with strong B-Raf V599E CD8 response  supportsE. coli hemolysin secretion system is a versatile tool for the delivery of cancer antigens in Salmonella enterica serovar Typhimurium. Moreover, we have recently shown that this secretion system is also fully active in Salmonella enterica serovar Typhi Ty21a, the only Salmonella vaccine strain registered for human use [S. typhi Ty21a could form the basis for a new generation of live bacterial vaccines against cancer.This is the first example demonstrating that the uman use . TherefoSalmonella enterica serovar Typhimurium aroA SL7207 strain via the E. coli hemolysin secretion system. In addition, the immunization of wild-type C57BL/6 or tumor bearing transgenic mice with this C-Raf secreting Salmonella strain provoked specific C-Raf antibody and T cell responses. Most importantly, the vaccine strain induced partial protection against lung cancer in two transgenic mouse models of Raf oncogene-induced lung adenomas.Taken together we have demonstrated that the C-Raf antigen can be successfully expressed and secreted by the attenuated The approach may provide a new strategy for the rational design of cancer therapies.The author(s) declare that they have no competing interests.IG, JF and JT designed the study. IG drafted the manuscript. JF was also involved in writing the report. AS and JF did the FACS analyses. JT constructed the EL4Raf cell line. TP, AS, JF and IG carried out the immunization of mice and the Western blot analyses. WG and URR were involved in providing the conceptual framework for this study. All authors approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:Supplementary figure Click here for file


EMO syndrome, defined as a triad including exophthalmus, pretibial myxedema and osteoarthropathia, is a rare condition in patients suffering from hyperthyreosis.We here describe an interesting case of EMO syndrome associated with unilateral fibromatosis of the hand and an initial stage of generalized myxedema of the skin. To our knowledge a similar case has not yet been described in literature though reports about associated fibromatosis, e.g. located retroperitoneally, already exist. Familiar explanations include its initiation by autoimmune processes or aberrant T-cell cytokine stimulation leading to an overwhelming production of glycosaminoglycans.Interpreting our case in context with previous reports we conclude that associated fibromatosis induced by autoimmune processes may affect a variety of different localizations and therefore requires careful monitoring. A therapeutical attempt by using UVA1 irridation for pretibial myxedema remained without a satisfying regression. EMO syndrome is a rare condition seen in patients suffering from hyperthyreosis. It is defined as a triad of exophthalmus, pretibial myxedema and osteoarthropathia occurring in less than 1% of patients suffering from Graves' disease [In October 2003, a 64-year-old male Caucasian patient was admitted with aggravating pretibial myxedema. Five years ago, he was diagnosed with hyperthyreosis caused by Plummer's disease. Laboratory findings revealed a repressed TSH of 0.01 mU/L (0.4–4.0 mU/L). The patient was treated with radioiodine therapy. Today he suffers from hypothyreosis and a daily substitution of 100 μg L-Thyroxin is performed. Within the months following the radioiodine therapy, an erythema progressing into a manifest pretibial myxedema developed, followed by exophthalmus and fibromatosis of the right hand within the next three years. Surgical orbita decompression due to massive exophthalmus was performed, followed by a subsequent correction of the ocular muscles. Additionally, the fibromatosis of the right hand was excised. Retrospective histological findings revealed a regressive sclerotic fibrosis of firm kollagenous fibers embedded in soft and fat tissue were consistent with a generalized fibrosing process as known in myxedema. Within the last months, decent mucous plaques of the upper limps decreased. The patient now reported progressing congestion of lymph and decreasing flexibility of the lower legs caused by the pretibial myxedema. Clinical examination confirmed massive pretibial myxedema, lymphatic congestion of the lower legs, generalized myxedema accentuating the upper limps, a residual postsurgical nodular tumor of the ulnar aspect of the right hand, exophthalmus, and severe hypertrophic osteopathy of the distal phalanges with clubbed fingers and hippocratic nails . While the congestion of lymph clearly improved, pretibial myxedema remained without any signs of regression.Grave's disease is known to eventually develop after radioiodine therapy  and thisA massive recurrence after excision of thyroid dermopathy, as described in literature, could not be observed in our patient during a follow-up of 2.5 years.Generalized fibrosing processes are thought to be based on an accumulation of glycosaminogylcans. Common explanations comprise its initiation by autoimmune processes, e.g. thyreotropin receptors on fibroblasts, or aberrant T-cell cytokine stimulation, leading to overwhelming glycosaminoglycan production  Fibrosin2 UVA1 (seven treatment sessions). However, we suggest that phototherapy might also be considered as an adjunct therapeutic alternative in persistent EMO syndrome.Therefore, we conclude that concomitant fibromatosis might appear in various localizations requiring elaborated diagnostic procedures and monitoring in all patients affected. We started low-dose UVA1 irradiation of the pretibial myxedema known to be able to degrade pathologic collagenous architecture by inducing dermal matrix-metalloproteinases as well as by decreasing abnormal cytokine liberation following T-cell apoptosis . NeverthThe author(s) declare that they have no competing interests.C.A. attended to the patient, participated in the design of the case report, and drafted the manuscript. A.K. conceived the study. F.B., A.B. and P.A. participated in its design and coordination.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: