PRDM9 gene implicated in recombination and CNV formation in humans. Here we comprehensively assay CNVs using high-density array comparative genomic hybridization in 50 dogs from 17 dog breeds and 3 gray wolves.Copy number variants (CNVs) account for substantial variation between genomes and are a major source of normal and pathogenic phenotypic differences. The dog is an ideal model to investigate mutational mechanisms that generate CNVs as its genome lacks a functional ortholog of the We use a stringent new method to identify a total of 430 high-confidence CNV loci, which range in size from 9 kb to 1.6 Mb and span 26.4 Mb, or 1.08%, of the assayed dog genome, overlapping 413 annotated genes. Of CNVs observed in each breed, 98% are also observed in multiple breeds. CNVs predicted to disrupt gene function are significantly less common than expected by chance. We identify a significant overrepresentation of peaks of GC content, previously shown to be enriched in dog recombination hotspots, in the vicinity of CNV breakpoints.PRDM9. These sequence features may have driven genome instability and chromosomal rearrangements throughout canid evolution.A number of the CNVs identified by this study are candidates for generating breed-specific phenotypes. Purifying selection seems to be a major factor shaping structural variation in the dog genome, suggesting that many CNVs are deleterious. Localized peaks of GC content appear to be novel sites of CNV formation in the dog genome by non-allelic homologous recombination, potentially activated by the loss of  The fraction of genomic variation attributable to copy number variants (CNVs) is larger than single nucleotide polymorphisms (SNPs) and yet the full extent of such structural variation is still relatively unexplored ,2. CNVs A variety of mechanisms are thought to give rise to CNVs . A majorDomestic dogs harbor an astonishing level of phenotypic variation, which is mostly apportioned into distinct breeds. The hundreds of dog breeds recognized today were formed by population bottlenecks accompanied by strong artificial selection, which has led to both their unique collections of characteristics and an increased prevalence of genetic disease. This makes the dog an ideal genetic model for uncovering the genetic basis of normal and pathogenic phenotypic variation . Many trThere is a possibility that the genomic features that promote CNV formation in dogs differ from other mammals. The dog genome differs from the majority of other mammals in that it lacks an active copy of PRDM9, which suggests that formation of meiotic DSBs is controlled differently in dogs . A fine et al. [et al. [Three studies have identified canine CNVs using array comparative genomic hybridization (aCGH) -31. Chenet al.  used a 3 [et al.  focused et al. [Despite extensive efforts to type CNVs in dogs, several questions remain about their mechanisms and effects. There is evidence that meiotic DSBs localize to different sites in dogs than other mammals ; does thet al. , with prWe performed aCGH analysis using a 2.1M probe technology platform spanning the assayable portion of the dog reference genome with a median spacing of 1 kb. We restricted our analysis to identifying high-confidence CNVs containing at least 10 probes, which allows identification of CNVs down to approximately 9 kb in length. We assayed CNVs in 53 samples, comprising purebred dogs from 17 breeds plus 3 wolf samples. Two breeds were represented by ten unrelated individuals each to enable CNVs segregating at lower frequencies in these breeds to be identified, whereas the other breeds were represented by two individuals to maximize coverage of different breeds. A male Boxer was used as the reference sample. CNVs were analyzed in autosomes and × chromosome by comparing the ratio of signal intensities between test samples and the reference.We identified CNVs using a three-stage procedure comprising segmentation, identification of CNV loci, and genotype calling. We first performed a comprehensive comparison of five segmentation algorithms, including NimbleGen, DNAcopy, Ultrasome, pennCNV and cghFLasso, and selected the algorithm most robust to noise . We first identified segments falling above a stringent threshold, designed to exclude false positives, to identify CNV loci in single samples. The final set of CNV loci is the union of individual calls at each locus, which were merged across samples into a single CNV. The breakpoints were defined by the outermost boundaries of all individual CNV calls at each locus. After identification of loci, the genotype of each individual was inferred at each locus using less stringent criteria to determine the most likely state of each sample . This method allowed us to distinguish which samples exhibited deletions and duplications relative to the reference. Each change was then categorized as simple or complex based on variance between samples: changes where the mean value exceeds the threshold were considered simple, whereas changes where the mean value is less than the threshold but the mean deviation from zero exceeds the threshold were considered complex. This happens especially when the segmentation algorithm fails to discern two adjacent CNVs, of which one is a deletion and the other is a duplication . Loci where at least one sample exhibited a complex change from the reference were defined as complex. It is important to note that it is not possible to discern how the copies are distributed between alleles using aCGH on diploid samples, that is, distinguish if a CNV is heterozygous or homozygous.In total, this procedure detected 430 CNV loci distributed along the chromosomes Figure . Out of et al. [On average, 130.9 loci (30%) differ from the reference per sample, and among breeds with two samples an average of 172.4 loci (40%) differ from the reference in one or both of the samples  and analysis on the CanineHD high-density SNP genotyping array. Because of the high overlap (50%) with previously identified and confirmed CNVs (see 'Distribution and genomic effects of CNVs'), we focused our validation on novel CNVs detected only in our study, and performed qPCR on four loci ranging in size from 17 to 80 kb. A total of 53 sample-locus combinations were tested and 3 tests did not match the state predicted from aCGH .All of the samples used for aCGH were also genotyped on the Illumina CanineHD array, which contains >174,000 probes designed for assaying SNPs with an average spacing of 13 kb . The proet al. [P < 0.001). The reason that some CNVs do not overlap between studies is likely to be a combination of differences in breed selection, sample size, array resolution, genotyping algorithms and errors. Furthermore, we generated a canine segmental duplication map using a modified version of the method of Bailey et al. [P < 0.001, random redistribution test) considering <5% of the dog genome is composed of recent SDs. The high proportion of CNVs in SDs together with our non-targeted approach indicates a large involvement of SDs in CNV formation as indicated by previous studies. However, it should also be noted that a small proportion of SDs are likely not fixed in the dog genome and may actually be CNVs.We compared our dataset to CNVs previously identified in dog in multiple studies merged and augmented by Nicholas et al. . We ideny et al.  , to whiRNPC3 and upstream of the collagen genes COL11A1 and COL5A1.The largest CNV locus we identified in the dog genome is located at 48.5 to 50.0 Mb on chromosome 6. The pattern of variation in this region is consistent with a deletion in all samples except both Labradors and one Boxer, which match the reference sequence. Inspection of probe intensities in this region on the CanineHD array shows a pattern consistent with the presence of duplicated sequence in these Labrador and Boxer samples. Considering the high frequency of the deletion allele, it is most likely that the duplication allele is the derived state. The CNV encompasses a gene-sparse region downstream of the RNA-binding region gene VPS13D, RDM1, ARHGAP27 and members of the LRRC37 family. These genes are not co-located in human, so this region must represent synteny to multiple loci. RDM1 is near members of the TBC1D3 family, which shows primate-specific expansion via SDs on chromosome 17, and is present in a majority of human-specific breakpoints of conserved synteny to mouse [LRRC37 and ARHGAP27 are orthologous to regions flanking an inversion in the human genome at 17q21.31, thought to have arisen through NAHR between large blocks of flanking SDs, which are distributed throughout chromosome 17 and contain the LRRC37 gene family [The second largest locus is a complex CNV on chromosome 9 between 20.1 and 21.6 Mb. There are no RefSeq annotated dog genes in this region, although it is orthologous to the human genes to mouse . Both LRe family -31.P < 0.001), are more likely to be complex (P < 0.001), tend to be longer (P < 0.001), and are more likely to overlap genes (P < 0.001) than CNVs not associated with SDs , which confirms previous observations [P < 0.001). Complex CNVs are preferentially observed in SDs, where every third CNV is complex, compared to less than 10% complex CNVs outside SDs. These observations may reflect a higher mutation rate of CNVs in SDs, with recurrent events around the same genomic location leading to both higher frequencies and more complexity, but could also reflect the involvement of SDs in more complex rearrangements. CNVs in SDs are also larger than non-SD CNVs; with a median size of 40 versus 20 kb. The increased frequency and complexity of CNVs in SDs may reflect the dynamic nature of SDs, and that these CNVs have arisen from overlapping but distinct events.CNVs overlapping SDs occur at higher frequencies in the population . A great proportion of the affected genes, 53 (41%), had their entire coding sequence covered by a CNV (Table P < 0.005). They also show a slight tendency towards lower frequencies, although the difference is not significant .We next examined the functional effects of CNVs by identifying genes they overlap. In order to use a high-confidence gene set, 24,232 dog genes annotated under Ensembl ID were filtered with the g:Orth tool from the g:Profiler website to extract only human-dog 1:1 orthologs. Out of 15,258 1:1 orthologous genes, 130 genes (0.85%) are overlapped either completely or partially by 90 CNVs . This number is significantly less than predicted by chance . This pattern is even more pronounced in intragenic CNVs that are predicted to remove a stop codon, where deletions occur at a proportion of 0.2 relative to duplications, which is significantly lower than other intragenic CNVs . This tendency for duplication enrichment among stop codons has previously been detected in humans [In addition to their paucity, CNVs overlapping genes are characterized by a much higher proportion of duplications than deletions Table . In inteP = 7.31 × 10-13), the cellular component 'integral to membrane' (P = 6.07 × 10-5) and the molecular function 'olfactory receptor activity' (P = 6.22 × 10-6). Cell adhesion also appears as a strongly enriched category among human CNVs [P = 5.04 × 10-3). Analyzing simple deletion and simple duplication loci separately reveals a difference in the kind of genes they overlap: no functional categories are inferred to be enriched in both deletions and duplications. For example, the 'homophilic cell adhesion' category is only enriched in deletions, whereas 'olfactory receptor activity' is only enriched in duplications. The enrichment of specific GO categories could reflect changes in patterns of selective constraint in dogs [The set of 130 1:1 orthologous genes overlapping CNVs were scanned for enrichment of gene ontology (GO) categories against a background of all 15,258 1:1 human-dog orthologs , which rapidly decays with increasing distance from the breakpoint, both within and outside of the CNV . These findings provide additional support for an important role of NAHR in dog CNV formation. Surprisingly, the mean length of homology between breakpoints of CNVs overlapping SDs is slightly shorter than those outside of SDs (133.1 bp compared with 116.6 bp). This suggests that NAHR is not restricted to segmental duplications and may be equally or more common outside of them.We also searched for regions of extended perfect homology between pairs of breakpoints flanking CNV loci. Of the 430 CNV loci, 86 have runs of perfect sequence homology greater than 75 bp and 11 have stretches longer than 1 kb. The mean length of runs of perfect homology between breakpoints is 126.8 bp. We tested the significance of these stretches of homology by simulating 1,000 datasets with the same number of loci and distance between breakpoints, located at randomly chosen positions on the same chromosomes. The mean length of homology in the simulated dataset was 35.0 bp, with none of the simulated means exceeding the observed one . Some variation in the number of CNVs between samples was seen both within and between breeds. Notably, the total number of CNVs identified in Boxers was lower than in any other breed, with an average of 64.5 loci different from the reference per sample, largely due to the reference being a Boxer. Of the remaining breeds, the average number of CNVs per sample varied from 116.5 (Swedish Elkhound) to 160 (English Springer Spaniel). On average, a sample differs from the reference at 130.9 CNV loci, of which 2.8 are specific to that breed. Fewer than 6% of CNVs found in any one breed are specific to that breed. These patterns are broadly consistent in the wolf samples, which exhibit a slightly lower than average number of CNVs per sample. On average, 2 dogs from the same breed differ at 83.1 CNV loci whereas 2 dogs from different breeds differ at 103 CNV loci.We next analyzed the distribution of CNVs among breeds Table . The majA more detailed picture of polymorphism in breeds with two samples is given in Table To assess the frequency distribution of CNVs among samples, we used their presence in all samples to build a site frequency spectrum. Figure The two breeds for which ten individuals were analyzed were selected for their large (Golden Retriever) and small (Irish Wolfhound) population sizes, respectively. We first attempted to see how many CNVs were present in all ten dogs of a breed, suggesting fixation Table . For GolDPYD, which encodes the enzyme dihydropyrimidine dehydrogenase (DPD) involved in pyrimidine catabolism. Examples of breed-specific fixations include a 32.7 kb deletion on chromosome 28 in German Shepherds downstream of DUSP10, which is involved in immune responses and mediates various physiological processes, and a 18.1 kb deletion on chromosome 21 in Finnish Spitz immediately downstream of CYP2R1  and overlapping PDE3B . These regions are good candidates for governing breed-specific characteristics, although further investigation is necessary to determine if they are really fixed, or have any phenotypic effect.We scanned our dataset for CNVs that were fixed for one allele in some two-sample breeds and another allele in all other two-sample breeds . There are 24 such CNVs, of which all but one is specific to a single breed. This CNV is a 12.7 kb deletion (located at 55.5 Mb on chromosome 6) shared by Cavalier King Charles Spaniel and English Springer Spaniel, which overlaps the gene et al. [We explored the extent to which patterns of CNV variation can be used to infer population structure between breeds. Based on the proportion of shared CNVs between each pair of samples, a neighbor-joining phylogeny was constructed Figure . With a et al. . One cluPRDM9 gene, which initiates recombination at separate specific sequence motifs in humans. In support of a strong role of NAHR in dog CNV formation, we also identify associations between CNV breakpoints and L1 elements and long stretches of sequence homology. We also show that dog CNVs are affected by the signal of purifying selection and identify candidate CNVs for involvement in breed-specific characteristics. This comprehensive catalogue of CNVs will be useful for future studies to uncover the genetic basis of complex traits in dogs.This study provides insights into the mutational mechanisms and functional effects of CNVs in the dog genome. Our results suggest that many CNVs are generated by NAHR events directed towards peaks of GC content, which is consistent with observations that these sequence features are also enriched in dog, but not human, recombination hotspots. Hence, GC peaks may represent novel sites of elevated recombination and genome instability in dogs. This shift in recombinational activity towards GC peaks in dogs is likely to be due to the lack of a functional copy of the EDTA-blood was collected as part of the LUPA project  from pedet al. [(1+mean log2ratio) and rounded to whole numbers.aCGH was used to detect DNA copy number alterations using NimbleGen's canFam2 Whole Genome CGH oligo array platform with 2.1 million probes on a single slide and a median probe spacing of 1 kb. The array design is based on the annotated CanFam2.0 genome sequence of a female Boxer genome. The isothermal 50-75mer probes were evenly distributed throughout the unique sequence of the genome. The genomic DNA samples were sent to NimbleGen's service facility where the hybridizations were performed in a two-color format according to Selzer et al. . Copy nuCNV calling was conducted in three stages: smoothing, segmentation and selection. Prior to segmentation and selection, triangular smoothing was done on the ratios, which implements an 11-point weighted smoothing function along the chromosomes. This is equivalent to several passes of fewer-point rectangular smoothings (unweighted sliding-average), and is more effective at reducing high-frequency noise in the signal. Segmentation was done with the R package cghFLasso to segment a continuous distribution of intensity ratios into discrete regions of consecutively different ratios. From these putative CNV segments, those with significant deviation are selected as the final set of CNVs. The selection was performed in three stages: first, ascertain CNV locations per sample using a stringent threshold (discovery threshold); second, consolidate the calls from all individuals by mapping them onto one 'master genome' to get merged general CNV locations; and third, decide the state of the general CNVs in each breed using a less stringent threshold (genotyping threshold) to assign individuals to diploid copy number classes. Noisy data were handled by recalculating the ratios into values of standard deviation from a theoretical normal distribution of ratios. These were used to allow samples with little noise to utilize lower thresholds. The two thresholds were then individually chosen on chromosome basis from a fixed log2 ratio and a standard deviation value.The fixed genotyping ratio was set to correspond to a deviation of 0.5 copies from the reference, while the fixed discovery ratio was set to the slightly higher 0.65 copies deviation. The standard deviation genotyping value was chosen to include 5% of the data points, while the standard deviation discovery value was chosen to include 0.1% of the data points, from the theoretical normal distribution (any copy number variants should be clearly distinct from the distribution). These were chosen so that a similar number of samples used the fixed values and the standard deviation values. The discovery threshold was picked as the maximum of these two numbers, while the genotyping threshold was picked as the minimum of the two numbers. This means that no CNVs are identified in the first stage if the deviation from the reference is below 0.65 copies, and in the final stage all CNVs with a deviation above 0.5 copies from the reference are identified as CNVs.T method was used for the quantification of copy numbers in test individuals relative to the same reference Boxer sample used in the aCGH experiments. The CT values for each set of triplicates were averaged and adjusted for PCR efficiency (E) as log2(ECT). The CT values were then normalized against the control primers. The relative copy number for each site was calculated as 2-(t-r), where t = normalized CT for the test sample and r = normalized CT for the reference sample.aCGH results were experimentally validated by qPCR in randomly selected CNV loci. Relative copy numbers of the selected regions were determined in comparison to the reference sample . Regions were selected based on the aCGH profiles across breeds. qPCR experiments were performed on ABI Prism 7500 Fast instrument  using SYBR Green detection chemistry according to the manufacturer's instructions. Primers (available upon request) were designed inside CNVs using Primer3 and NCBI primer design programs. Each assay was performed in triplicate using 20 μl reactions containing 10 μl of qPCR master mix , 10 nM concentration of dNTPs, 250 nM concentration of forward and reverse primer and 10 ng of genomic DNA. Amplification was performed under the following conditions: one cycle at 50°C for 2 minutes, one cycle at 95°C for 10 minutes, 40 cycles at 95°C for 15 seconds and 62°C for 45 seconds. Beta-actin and GAP175 were used as controls. Serial dilutions were performed for each assay to estimate the PCR efficiency (E) prior to analysis. The ddCet al. [Large CNVs, by virtue of their potential functional impact, were validated in an additional validation step. The same set of individuals was genotyped according to manufacturer's instructions on the CanineHD 170K SNP array (Illumina) with a resolution of 1 SNP per 13 kb. The GenomeStudio V2010.3 software package (Illumina) was used to obtain normalized total signal intensity, Log R ratio, and B allele frequencies for all SNPs according to the manual and Peiffer et al. . Exporteet al. . DefaultPrior to analysis, all chromosomes were centered on their mean log2 ratio to remove potential chip biases. The × chromosome was treated differently in this manner, since the pseudo-autosomal regions were centered separate to the rest of the chromosome, which differs in relative copy number to a male reference. To identify the pseudo-autosomal regions, the raw log2 ratios from all female samples were used to determine the average position of the probe where copy number changed from diploidy. Since no CNVs were successively called in that particular region, the position seems to be accurate.The metric used to call CNVs from the aCGH data was the log2 ratio, which is the log (base 2) ratio of the observed normalized R-value for a signal intensity divided by the mean signal of a reference sample. The threshold of log2 ratio was defined as in the discovery and genotyping section to define a true copy number change that presents a pattern consistently different from a diploid region. CNVs were defined as |log2 ratio|>threshold, where deletions are indicated by negative log2 ratios and duplications are indicated by positive log2 ratios. Deletion and duplication variants are special cases of multi-allelic CNVs. To minimize the risk of false positives, we required each locus to be targeted by at least ten consecutive probes to call a CNV, resulting in a final resolution of approximately 9 kb.et al. [Segmental duplications, defined as regions at least 1 kb in length, at least 90% identical at two or more loci, and not consisting entirely of mobile elements, were identified by self-alignment of the genome as described in Zody et al. . In the GO analysis was made with the web-based tool g:GOSt Gene Group Functional Profiling provided by g:Profiler , , with deStatistical significance of overlap between genomic features was assessed by estimating the distribution of sample means. This was done by either random redistributions per chromosome or bootstrapping. Chromosome-wise redistributions were done by repeatedly and randomly redistributing all features on the same chromosome to get a distribution of means from which significance can be assessed. Bootstrapping was done by re-sampling the individual observations with replacement from the population of CNVs to get an empirical bootstrap distribution from which a bootstrap confidence interval could be derived to infer statistical significance.R was used to smooth the aCGH data and the R package cghFLasso was used to assess copy number alterations. For genome annotation the University of Santa Cruz (UCSC) Genome Browser  and EnseA database of genomic copy number variants, DoG_CNV, in the dog genome has been developed to provide the annotation of CNVs discovered in this study and a useful resource to assist with the assessment of CNVs in the contexts of canine variation and disease susceptibility . In addiaCGH: array comparative genomic hybridization; CNV: copy number variant; DSB: double-stranded break; GO: gene ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; LINE: long interspersed nuclear element; NAHR: non-allelic homologous recombination; qPCR: quantitative polymerase chain reaction; SD: segmental duplication; SINE: short interspersed nuclear element; SNP: single-nucleotide polymorphism.The authors declare that they have no competing interests.JB performed the majority of the bioinformatics analyses and drafted the manuscript. EMN performed the array experiments. A-MM analyzed the SNP array data. MP helped to coordinate the array experiments. CA participated in useful discussions. MCZ and TS identified segmental duplications. CH contributed bioinformatics analyses and constructed the website. KL-T and HL conceived of the study and participated in its design and coordination. MTW conceived and coordinated the bioinformatics analyses and wrote the paper. All authors read and approved the final version.Supplementary methods, tables and figures. Click here for file


Spinal intramedullary ependymomas are very rare and occur more commonly in the cervical and upper thoracic regions. These neoplasms tend to manifest in young adulthood, and patients typically present with mild clinical symptoms without objective evidence of neurologic deficits. The mean duration of symptoms is 40 months until the lesion is diagnosed.A 48-year-old male police officer was referred to a chiropractic clinic by a general practitioner for the evaluation of recurrent acute low back pain (LBP). Although the first episode of LBP was resolved, the clinical examination during the second episode revealed subtle changes that warranted referral to magnetic resonance imaging (MRI). The MRI revealed a spinal myxopapillary ependymoma.Because the primary symptoms of spinal intramedullary ependymomas can mimic ordinary LBP presentations, in particular lumbar intervertebral disc herniations, clinicians need to be sensitive to subtle changes in the clinical presentation of LBP patients. Prompt referral to advanced medical imaging such as MRI and early neurosurgical intervention is key to achieve best possible outcomes for patients with spinal intramedullary ependymomas. Primary intraspinal neoplasms are rare, with an age-adjusted incidence rate of 0.5 in females and 0.3 in males per 100,000 population per annum . Spinal Spinal myxopapillary ependymomas tend to manifest in adulthood  and is more commonly seen in male patients . PatientThus it becomes vital to promptly recognise important clues in the clinical presentation and evaluation that will lead to early diagnosis. Here we describe a rare case of an adult male with spinal myxopapillary ependymoma presenting as recurrent acute low back pain with subtle neurologic symptoms. The clinical presentation, chiropractic examination and management, imaging manifestations, and subsequent neurosurgical intervention and outcome are discussed below.A 48-year-old male police officer was referred to a chiropractic clinic by a general practitioner for the evaluation of acute low back pain (LBP) of two weeks duration. The patient reported that the LBP had come on after spending a 12-h night shift driving a patrol car. The LBP was described as diffuse and the patient reported sharp pain radiating down the left lateral thigh to the knee. The average pain intensity was rated as 6/10 on a visual analogue scale (VAS), but had progressed to 8/10 VAS at the time of presentation. The symptoms were aggravated by sitting, sleeping on the left side, and inactivity; while standing provided the most relief. The pain was worse in the morning, and improved only marginally during the day. Self-care with heat packs and over-the-counter non-steroidal anti-inflammatory drugs (6 × 200 mg ibuprofen tablets per day) provided only a 2-point reduction on the VAS. The patient denied the presence of any paraesthesia (e.g. pins or needles), saddle anaesthesia, loss or changes in bladder or bowel control, fever, or nocturnal pain. The patient reported an approximately 20 kg reduction in body weight over the past 10 months, which he attributed to dietary changes and uptake of a substantial exercise regimen.The patient also complained of left-sided headaches in the suboccipital and temporal regions of three weeks duration. The headaches were accompanied by occasional dizziness (clarified to be a feeling of unease) that were aggravated by sustained awkward neck postures. He had a family history of hypertension, type 2 diabetes, and migraines. His grandmother had passed away from bowel cancer. The patient was currently being treated for haemochromotosis, for which he had been managed successfully for 8 years. The rest of the systems review was unremarkable.The physical examination revealed a marked antalgic posture to the right with limited active lumbar range of motion (ROM) in left lateral flexion and flexion (finger-to-floor reach was noted to be 2 cm above the knees). Coughing, sneezing, and Valsalva manoeuvre reproduced the LBP, but not the leg pain. Straight leg raise was positive for leg and back pain at 40° on the left and negative at 70° on the right. Bragard’s test was positive on the left, while slump test and well leg raise were negative. Deep tendon reflexes were normal (2+) in both upper and lower limbs, except for the patella reflex on the left which was slightly diminished (1+). There was no decreased sensation to pin prick or vibration sense in either the upper or lower limbs; however, sensation to light touch was slightly decreased in the left L4 dermatome. Motor and cranial nerve exams were unremarkable. Blood pressure was 152/92 mmHg and the resting heart rate was 75 bpm (measured in the sitting position).The left temporal headache and dizziness (uneasiness) could be reproduced by combined flexion and 25° of rotation of the head to the right. The same manoeuvre on the contralateral side was pain free at 50° of rotation. There was marked tenderness to palpation over the left C2/3 zygapophysial joint and the lumbar spinous processes.On the basis of the clinical presentation, history, and findings of the physical examination, a working diagnosis of posterolateral L3/4 intervertebral disc herniation affecting the left L4 nerve root was made, and a trial of conservative management was suggested to the patient. The patient also received a concurrent working diagnosis of cervicogenic headache. In addition, the patient was recommended to seek further evaluation of his blood pressure.Conservative management consisted of cryotherapy (ice packs), high-velocity, low-amplitude spinal manipulation to the lumbar region, isometric stretching, and neurodynamic mobilisation techniques . This passive care was complemented by ergonomic advice and a prescribed back extension exercise program. The patient received a total of 6 treatments over a 4-week period. After this treatment regimen the patient reported the LBP to be 1-2/10 VAS, with no pain radiating down the left leg, and that he was able to perform all activities of daily living. Neurodynamic tension tests (e.g. straight leg raise and Bragard’s test) were negative. Lumbar spine ROM was improved (finger-to-floor reach 34 cm below the knees during flexion) and pain free.The patient was discharged with a 4-week core muscle strengthening program and asked to return for a follow-up consultation after its completion, or earlier if symptoms returned. The patient returned asymptomatic after 4 weeks, and was able to satisfactorily perform the functional core strength tests.Two months after the last follow-up of the first episode of LBP, the patient presented to the chiropractic clinic with a new episode of acute LBP with pain radiating down the left leg. Similar to the first episode, the patient reported that the LBP had come on after an 8-h shift of driving a patrol car. However, this time the leg pain was radiating down the anterior aspect of the left thigh to the knee. He was taking prescription pain killers as directed by his general practitioner . However, the medication provided minimal relief and the pain was unrelenting, worse at night, and interfering with his sleep. Bladder and bowel function was normal. The physical examination mirrored the initial presentation but for two findings: (1) light touch was decreased in the left L3 and L4 dermatomes, and (2) slump test produced left anterior thigh pain radiating down to the left knee and lateral leg to the foot.Because the patient now presented with indications of the possible involvement of multiple lumbar nerve roots, severe pain waking him up at night, and not responding to strong prescription pain medication, he was referred for magnetic resonance imaging (MRI) for an evaluation of possible compression of neurological structures at the L2 to L4 vertebral levels.The MRI revealed a substantial heterogeneous space-occupying lesion with an intramedullary orientation within the filum terminale at the L2/3 vertebral level see Fig. . The lesThe patient was scheduled for surgery the day after the neurosurgical consultation. A gross total resection was achieved and radiological adjunctive therapy was not pursued. Although the patient reported resolution of the neurological symptoms immediately after the surgery, low-grade LBP (3/10 VAS), described as a dull ache, still persisted for several weeks post-surgery. The patient received standard, post-surgical rehabilitation with a hospital-based physiotherapist for 6 months post-surgery. In long-term follow-ups at 6 and 12 months post-surgery, the patient reported no pain (0/10 VAS), with only a vague sensation of persistent numbness in the left anterior leg.The present case highlights a number of important points: (1) the primary symptoms of intramedullary ependymomas of the caudal spinal cord can mimic ordinary LBP presentations, in particular lumbar intervertebral disc herniations; (2) clinicians need to be sensitive to subtle changes in the clinical presentation of LBP patients; and (3) prompt referral to advanced medical imaging such as MRI and early surgical intervention is key to achieve best possible outcomes for patients like the one described in the present case.LBP is a very common health problem, and one of the leading causes of activity limitation and work absence worldwide , 7. It iAt first glance the second episode appeared to be a typical recurrence of the first episode of LBP, with both the precipitating event and the clinical presentation of the second episode being very similar to the former. Furthermore, successful conservative management of the first episode could potentially lead to confirmation bias, that is, clinicians are likely to believe their working diagnoses are correct when their patients respond satisfactorily to care . In the It is difficult to estimate how often patients with an undiagnosed spinal ependymoma present to chiropractors with LBP because only a couple of cases have been reported in the literature to date. Lensgraf and Young described two cases: (1) a 46-year-old woman with 3–4 year history of intermittent LBP who eventually did not respond to conservative care; and (2) a 38-year-old man who presented with an unusual episode of LBP with leg pain . In bothIn most jurisdictions, chiropractors can request advanced diagnostic imaging such as MRI. However, full government rebates for such referrals are only available in a few jurisdictions (e.g. Norway and Denmark). In Australia (the location of the present case), patients referred to MRI by chiropractors are not eligible for a Medicare rebate. Although the patient in the present case was both willing and able to pay for the MRI, this may of course not be true for all, or even the majority of, patients. It is, therefore, conceivable that such restrictions on advanced medical imaging referrals can contribute to delayed diagnosis of some patients.Early detection and treatment is key to achieve best possible patient outcomes for people with spinal intramedullary ependymoma. These neoplasms are best treated surgically and a complete resection indicates complete resolution for the vast majority of patients , 15. AdjThe primary symptoms of spinal intramedullary ependymomas can mimic ordinary LBP, in particular lumbar intervertebral disc herniations. Clinicians need to be sensitive to subtle changes in the clinical presentation of LBP patients. Prompt referral to advanced medical imaging such as MRI and early surgical intervention is key to achieve best possible outcomes for patients with spinal intramedullary ependymomas.This study was approved by the Human Research Ethics Committee at Central Queensland University . Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Patients with RA (Rheumatoid Arthritis) are more sedentary than the general population. Reduction of Sedentary Behaviour (SB) has been suggested as a mean for improvement of health in patients with chronic diseases and mobility problems. Short-term intervention studies have demonstrated that SB can be reduced by behavioural interventions in healthy populations. However, it remains unexplored whether it is valid for patients with RA also.Therefore, the aim of this trial is to investigate the efficacy of an individually tailored, theory-based motivational counseling intervention on reducing daily sitting time in sedentary patients with RA. Additionally, to explore whether a reduction in daily sitting time is associated with reduced pain and fatigue, self-reported physical function, self-efficacy, improved health-related quality of life (HR-QoL) and cardiovascular biomarker levels, and finally to assess the cost-effectiveness of the intervention.For this parallel group randomized trial, 150 patients with RA and at least 5 hours of sitting time per day, will be recruited from a rheumatology outpatient clinic, and block-randomized to the intervention group or the control group receiving usual care. The intervention includes: 1) individual motivational counseling  on reduction of daily sitting time in combination with 2) individual Short Text Message Service (SMS) reminders over a 16-week intervention period. Primary outcome is change in daily sitting time (minutes) from baseline to 16 weeks measured objectively using an ActivPAL® Activity Monitor. Secondary outcomes include fatigue, pain, physical function, HR-QoL, self-efficacy, costs and cost-effectiveness. Furthermore, anthropometric measures will be included as well as measurement of blood pressure and serum lipids. All outcomes are assessed at baseline and repeated after 16 weeks. Follow-up assessments are made at 6 and 18 months post-intervention.The intervention is simple, non-invasive and may be implemented at low costs. If the study confirms the positive results expected, the intervention might be implemented in clinical practice and potentially transferred to other clinical populations.NCT01969604. Date of registration: 17 October 2013.ClinicalTrial.gov registration number:  Physical inactivity is an established risk factor for chronic disease and premature death ,2. In adet al. found that 71% of waking hours were spent sedentary compared to 62% of waking hours in healthy controls [The everyday life of patients with RA is periodically influenced by increased disease activity (flares) with symptoms such as swelling and stiffness of the joints accompanied by intense pain, which can lead to severe limitations in physical functioning and potential progressive joint destruction ,11. Intecontrols . Objecticontrols -25. A recontrols .A few short-term intervention studies applying objective measures of sitting time in older people , and in The primary objective of this trial is to investigate the efficacy of an individually tailored, theory-based motivational counseling intervention on reducing daily sitting time in sedentary patients with RA. The secondary objectives are to explore whether a reduction in daily sitting time is related to reduction in pain and fatigue, improved health-related quality of life (HR-QoL), self-reported physical function, self-efficacy and improved cardiovascular biomarker levels, and to assess the cost-effectiveness of the intervention.The current study is part of the ‘Joint Resources’ research program devoted to health promotion studies in patients with RA ,31 and iPatients will be recruited from lists of RA patients from the rheumatology outpatient clinic at Copenhagen University Hospital, Glostrup, the Capital Region of Denmark .Patients can be included in the study if they have been diagnosed with RA (defined by the American College of Rheumatology (ACR) 1987 criteria ), are ovPatients will be excluded from the study if they have severe physical disability (HAQ score > 2.5) that would prevent them from reducing daily sitting time ; are pregnant; participate in vigorous physical activity in their leisure time for more than 8 hours a week (measured by PAS 2.1). Excluded patients or eligible ones who do not want to participate will be registered in one of the following three categories: 1) not meeting the inclusion criteria, 2) refused to participate, or 3) other reasons .The patients will be recruited from the Danish National Board of Health biological therapies (DANBIO) database, a nationwide registry that has included > 30,000 patients with inflammatory arthritis in a longitudinal observational cohort . The patt-test of a normal mean difference with a 2-sided significance level of 0.05, assuming a common standard deviation of 102 minutes, a sample size of 67 RA patients per group is required to obtain a power of at least 80% to detect a group mean difference of 50 minutes. Enrolling a sample size of 75 in the intention-to-treat (ITT) population per group has a reasonable power (84.7%) to detect a mean difference of 50 minutes .A feasibility study has been conducted with the aim of testing and evaluating selected methods and instruments for this trial, as well as testing all elements of the planned study (November 2012 to May 2013). The feasibility study included 19 patients with RA with an average daily sitting time at baseline of 10.13 ± 1.7 hours (mean ± SD). The SD for calculation of sample size in the current trial was based on ActivPAL® data from the feasibility study measured as total daily sitting time. We expect a reduction in average daily sitting time of 50 minutes in the intervention group. This estimated reduction in sitting time is based on a US pilot study from 2012 on obese office workers who reduced their daily sitting time by 48 minutes as a result of a behavioral intervention . For a 2Immediately following the baseline measurements, randomization to either (A) the intervention group (N = 75) or (B) the control group (N = 75) will be performed via computer-generated ‘random numbers’ for each of the 2 groups (blocks of 10 patients). Patients will be informed by TT about their group allocation 1 week after baseline measurements. Patients will not be blinded to the intervention; however, all measurements will be conducted by two occupational therapists who will be blinded to group allocation.Individual motivational counselingThe intervention includes: 1) Individual motivational counseling including hand-outs of 4 key messages regarding reduction of daily sitting time  in combiThe intervention consists of 3 individual motivational counseling sessions (60 to 90 minutes), conducted by one of the project staff , who have been trained and calibrated in motivational interviewing (MI) techniques  for thisCounselors will simultaneously be trained and supervised by a psychologist to ensure that the intervention will be delivered in a similar way based on the theoretical understanding of MI. The first counseling will take place immediately after randomization to the intervention group, the second will be 2 weeks after, and the third 10 weeks after the first counseling Individual SMS remindersIn addition to the motivational counseling, four key messages or themes including ideas and suggestions for reduction of sitting time (that are written in booklets) will be handed to the patients at each session. The 4 key messages are: 1) reduce daily TV-viewing, 2) substitute sitting with standing when possible - at work and/or at home, 3) break up prolonged sitting - by standing up frequently and 4) maximum 30 minutes of sitting per episode . All intHello X. Stand up and allow gravity to assist you to digest your lunch. Bonus: you burn more energy when you standHi X. Regard vacuum cleaning as a free fitness hour. Make a playlist, put music in your ears and do not stop until the list and the cleaning are doneHi X. You have some truly privileged colleagues who will be able to see you stand up by your table this afternoon. Show them how to do it, and they might follow your good exampleHey X. Put the remote control next to the TV if you turn on the television today. Every time you change the channel your entire body gets some exerciseBased on the patients’ own individual behavioral goals the patients will decide how many weekly SMS reminders they want to receive during the 16-week intervention period and at what time of the day. Examples of SMSs are shown below:The control group will be encouraged to maintain their usual lifestyle during the 16-week intervention period. When the last follow-up examination is completed they will be offered the opportunity to join an information session (in groups) about the principles of the intervention.Change in total daily sitting time in minutes ,42 from Measurements will be obtained using an ActivPAL® 3 TM Activity Monitor . This is a small (2.0 × 1.4 × 0.3 inches) and light (20.1 grams) uniaxial accelerometer-based device  that is Changes in self-reported daily sitting time at work and during leisure time will be measured by the Physical Activity Scale 2.1 (PAS 2.1) , a modifThe 20-item Multi-dimensional Fatigue Inventory (MFI 20)  will be The Visual Analogue Scale (VAS)  transforHealth Assessment Questionnaire (HAQ)  is an inHR-QoL is assessed with: 1) SF-36   assessesAdditional questions on duration of sitting time and interruptions in sitting time on weekdays and in weekends are included. The questions have been validated against Actigraph  accelerometers in a recent Danish Study .At termination of the objective diurnal measurements, a self-administered questionnaire is applied regarding specific recall of time sitting during the measurement period. Time spent sitting during working hours is obtained by the question: ‘during working hours in the measuring period, in average per day, how long time did you spent sitting?’ -7,11 rep2) and waist-hip ratio are calculated.Height is measured without shoes to the nearest centimeter; weight is measured in light clothing without shoes to the nearest 0.1 kg ; waist circumference is measured midway between the lower rib margin and the iliac crest to the nearest 0.5 centimeter, without any pressure to the skin and with an unstretched tape measure; hip circumference is measured at the point yielding the maximum circumference over the buttocks to the nearest 0.5 centimeter. The tape should be held in a horizontal plane touching the skin but not indenting the soft tissue. Body mass index . Total cholesterol, high-density lipoprotein cholesterol (HDL), and triglycerides will be measured by an enzymatic method on the Vitros 5.1 FS from Ortho Clinical Diagnostics . Low-density lipoprotein cholesterol (LDL) and very low-density lipoprotein cholesterol (VLDL) will be calculated by the formulae:In addition, C-reactive protein (CRP) and glycosylated hemoglobin, type A1c (HbA1c) will be measured on Vitros 5.1 FS  and G8 HPLC Analyzer from TOSOH .Blood pressure will be measured, after 5 to 10 minutes of rest, 3 times at the right upper arm (average of the 3 measurements) with the participant in a sitting/lying position.In addition, information regarding demographics, lifestyle, time of diagnosis, consumption of pain killers and comorbidity , cancer, osteoarthritis, osteoporosis, asthma and depression) will be obtained; all self-reported. Information about DAS 28  they will be stored and analyzed through Statistics Denmark’s Research computer.The trial will last 22 months including a 16-week intervention, and 6 and 18 months follow-up  at baseline; 2) at the end of the intervention (16 weeks after intervention start); 3) 6 months post intervention and finally (4) 18 months post intervention  via a self-report questionnaire (using tablets). Two occupational therapists will attach the ActivPAL® monitor  and instSince all data will be supplied directly by the patients and the assessors through an online interface via a tablet there is no risk of data loss or distortion along the way. Data will be stored encrypted and in unidentifiable form (using participant-numbers) in the server at Glostrup Hospital, University of Copenhagen. Standard missing data analysis will determine whether or not unexpected but missing data due to participant drop-out are random.P-values and 95% confidence intervals (95% CI) will be 2-sided and will not be adjusted for multiple comparisons. Unless stated otherwise, results will be expressed as the difference between the group means and 95% CI with the associated P-values, based on a general linear model (GLM). Data will be analyzed using a one-factor ‘Analysis of Covariance’ (ANCOVA), with a factor for Group, using the baseline value as covariate to reduce the random variation and increase the statistical power.The scoring of the standardized questionnaires will be carried out according to the guidelines from instrument developers. All data analyses will be carried out according to a pre-established analysis plan; all analyses will be done applying SAS software . All descriptive statistics and tests will be reported in accordance with the recommendations of the ‘Enhancing the QUAlity and Transparency Of health Research’ (EQUATOR) network: the CONSORT statement . In ordeProportions will be compared by estimating the risk difference with 95% CIs for each dichotomous outcome; including a Wald-Z-test for the null hypothesis that there is no difference between the proportions.Secondly, to relate the results to compliance, a ‘per protocol’ analysis will also be used. The ‘per protocol’ population is defined as the patients who have ‘completed’ the intervention to which they were allocated according to the principles described in the intervention section above.The cost analysis will report the resource use and cost of the intervention. For the intervention and control group, survey and registry-based data on resource use, including services provided in the health care sector (primary and secondary care) and pharmaceuticals, will be aggregated for each individual in intervals determined by the time of data collection. Cost data will be interpolated to total cost for the whole observation period (18 months). The aggregated cost data will be analyzed as costs attributable to the intervention (that is difference in difference) and reported as mean values with 95% (bootstrap) CIs. Due to the non-normal distribution of cost data generalized linear models will be employed with appropriate choice of link function.The analysis of cost-effectiveness will compare the observed gain in Quality-Adjusted Life Years (QALYs) (HR-QoL over 18 months) with the observed incremental cost (over 18 months) by estimating incremental cost-effectiveness ratios (ICER). To express statistical uncertainty in the ICER estimates acceptability curves will be developed to express the likelihood of the intervention being cost-effective at various QALY threshold values.The trial will be performed in accordance with the Helsinki Declaration. The project has been approved by the Regional Committee on Biomedical Research Ethics September 2012 as registry-based research . The study was approved by the Danish Data Protection Agency (ref number 711-1-08). All formal and safety rules in the process from data collected for publication are observed  issues, and so on). Anyone who has access to the full relevant data sets  is familiar with the formal and safety rules. All information collected during the course of the study will be kept strictly confidential in accordance with Danish Data Protection Agency rules; operationally, this will include consent from the patients.It is planned to publish at least four scientific papers based on the trial in peer-reviewed journals.Papers (peer-reviewed and popular science)Presentations at conferences  or similarCollaboration with the Danish Rheumatism Association - both members and groups of employeesOther relevant patient organizations covering patients with musculoskeletal diseases, and pressIn addition, the following sources are included in the communication plan:RC will analyze data in collaboration with TT, BAE, MA. JS will analyze the cost-effectiveness data. The project group will have access to data as well as to the DANBIO registry ,56. The This trial protocol describes the design of an innovative randomized controlled trial, which aims to test whether sedentary behavior can be reduced in patients with RA through behavioral lifestyle change. To the best of our knowledge, this is the first randomized trial to investigate the effect of an individually tailored motivational counseling intervention on daily sitting time in patients with RA. The intervention is developed and based upon results from studies in population-based samples of sedentary adults and in elderly and obese sedentary populations. The population under study, men and women with RA, represents a group of patients with chronic disease and increased risk of developing diabetes and cardiovascular diseases. In addition, a large proportion of patients with RA struggle with strained joints and immobility in daily life. If effective, the intervention may reduce the risk of cardiovascular disease and type 2 diabetes in patients with RA by reducing their daily sitting time. It should be noted that a possible reduction in sitting time, the primary outcome, does not necessarily imply a reduction in cardiovascular disease (CVD) risk. However, a possible effect of the intervention on secondary outcomes, CVD biomarkers (cholesterol and so on) will be simultaneously registered and studied.We cannot rule out that wearing an ActivPAL® may cause reactivity and as such in itself act as a ‘behavioral intervention’. However, participants in both groups are measured at baseline and follow-up, and the size of the between arm difference is, therefore, not likely to be affected or considered in the sample size calculation. Also ActivPAL® monitors start measuring from midnight following the first day of application in order to minimize reactivity by allowing participants to get used to wearing the device. Finally, the ActivPAL® measurements are not shown to participants until after the study is completed.It should be noted that in behavioral intervention RCTs patients are difficult to blind. We could have offered the control group participants a ‘sham’ counseling intervention with counseling on something else than reduction of sedentary behavior. However, this could potentially induce an effect in the control group which we wanted to avoid.We expect the intervention to result in reduced pain (less use of painkillers), reduced fatigue, increased daily physical functioning, improved self-efficacy and better general health status. The intervention is simple and could relatively easily be implemented in clinical practice along with lifestyle advice and recommendations on physical activity, sports and exercise, diet and smoking habits. In addition, we expect the intervention method to be transferable to other groups of chronically ill individuals with prolonged sitting time and mobility limitations.Recruitment for the trial is planned to start in April 2013 and will end in December 2014.


Differential diagnosis between keratoacanthoma (KA) and squamous cell carcinoma (SCC) is difficult due to their similarities. The mechanisms that drive their distinct biological behavior are poorly understood.To investigate whether the assessment of microvessel density (MVD) could be helpful in KA and SCC differential diagnosis and to gain insight into the pathogenesis of KA-like neoplasms, we compared the density of CD105- and CD34-stained vessels in KAs and SCCs and their relation to the expression of the p53 oncoprotein and proliferation marker Ki67.This is an observational retrospective cohort study. Forty lesions with clinical appearance of KAs (29 KAs and 11 SCCs) entered the study. A biopsy was taken from each lesion at presentation and the natural clinical course was monitored for at least 1 month. Growing or minimally regressing lesions were submitted to complete surgical excision. The diagnoses were established on combined clinical, histological, and follow-up evaluations. The MVD and p53 or Ki67 expression in neoplastic cells were assessed through morphometry.The MVD did not show discriminating power between KAs and SCCs. The Ki67 proliferation rate was significantly higher in SCCs. Although neoangiogenesis (CD105-MVD) in KAs was associated with cell proliferation, in SCCs it was not. There was significant correlation between p53 expression and neoplasia size in SCCs but not in KAs.From our results, we may conclude that KA and SCC have similarities, as CD105- and CD34-MVD. However, the low Ki67 proliferation index and the positive correlation between Ki-67 index and neovascularization in KA suggest a dependence in neovascularization to grow in KA, pointing to involvement of distinct pathogenesis. The real nature of this neoplasma and its relationship to squamous cell carcinoma (SCC) have been disputed for a long time. Some authors consider KA as a benign neoplasia, others as a precursor of SCC,2 others as a well-differentiated variant of cutaneous SCCs 3 or at least an abortive malignancy that only rarely progresses into an invasive SCC.4 Differential diagnosis between KA and SCC is challenging due to their similarities and the lack of reliable diagnostic criteria to distinguish both entities.5Keratoacanthoma (KA) is a cutaneous neoplasia arising preferably from hair follicle cells on sun-exposed skin and characterized by self-limiting growth and involution. The life cycle from origin to spontaneous resolution takes about 4 to 6 months in the majority of cases6 studied the expression of p53 protein in KAs and SCCs and found no clear-cut distinction between these neoplasms. Kerschmann et al7 analyzed the expression of p53 tumor suppressor protein and the proliferation index (Ki67) in KAs and SCCs and found that both neoplasms contain stainable quantities of p53 protein suggesting that these neoplasms are best viewed as a spectrum. These authors also did not find a statistically significant difference in proliferation rate between neoplasms that express p53 and those that do not, for either KA or SCC.Several studies have attempted to find more specific methods to distinguish KAs and SCCs accurately. Among them, Stephenson et al8 studied the expression of p16, a tumor suppressor protein, in KAs and SCCs and found no significant differences that could make this marker useful to this differential.Kaabipour et al9 found that the expression of a peanut lectin receptor is usually uniformly positive in KAs and negative in the majority of SCCs, indicating that this could aid in the histologic differentiation of these neoplasms. Takeda et al10 demonstrated differences in the expression of angiotensin type 1 receptors between SCCs and KAs. However, both of these methods are neither definitive nor reliable to differentiate KAs and SCCs in all cases.Kannon et al13 In fact, previous works have shown that the neoplastic microenvironment plays a major role in several phases of neoplasm development.15 Takahara et al16 studied the microenvironment of epidermal neoplasms and suggested that increased macrophages and decreased Langerhans cells are associated with proliferation and invasion of malignant epidermal tumors. These authors found a significant higher number of dermal macrophages, which are thought to be associated with angiogenesis in SCC, compared with KA and other epidermal tumors.The study of the microenvironment in neoplasia growth, invasion, and metastatic potential is of increasing interest because its stimulatory and restrictive effects upon neoplasm progression have become evident.13 In fact, angiogenesis has been reported to be an important prognostic factor of outcome for several malignancies. Differences in microvessel density (MVD) could also be related to the distinct behavior between KA and SCC and help distinguish between these entities. Weninger et al19, however, could not find a significant difference in factor VIII-determined MVD comparing KAs and SCCs.Induction and remodeling of angiogenesis have been pointed out to be relevant for tumor progression and malignancy.20 The observations that CD105 was strongly up-regulated in the endothelium within various tumors compared with normal tissues supported the important role of CD105 in the investigation of tumor angiogenesis.23 CD105 has been demonstrated to be an endothelial cell proliferation-associated marker and to represent a powerful indicator of neovascularization.20 To the best of our knowledge, CD105-determined MVD has not been studied in KAs.CD105 (endoglin) is an 180-kDa homodimeric transmembrane protein overexpressed in proliferating endothelial cells.To investigate whether the assessment of MVD could be helpful in KA and SCC differential diagnosis and to gain insight into the pathogenesis of KA-like neoplasms, we compared the density of CD105 and CD34 stained vessels in KAs and SCCs and their relation to the expression of the p53 oncoprotein and the proliferation marker Ki67.This is an observational retrospective cohort study.This study has been approved by the Research Ethics Committee of the State University of Campinas (ID number 822/2005).24 in which we analyzed the histopathologic features associated with clinical regression in KA-like lesions.Routinely, formalin-fixed and paraffin embedded specimens of 43 KA-like lesions from 41 patients were selected from the files of the Department of Pathology, State University of Campinas (UNICAMP). These cases corresponded to the cohort of a previous study In brief, during a period of 4 years, when a patient with a cutaneous KA-like lesion  was referred to the dermatological outpatient center, the following routine was taken by the same dermatologist (RFM): cutaneous examination findings were recorded; photos were taken and a partial biopsy was performed so as to include its center and side throughout its depth, to allow the evaluation of the neoplasia architecture and the bottom neoplasia border. A follow-up appointment was scheduled 1 month later for clinical re-evaluation by the same dermatologist as well as lesion measurement and definition of treatment modality. In cases of growing  or minimally regressing lesions, complete surgical excision, as for SCC, was done. In contrast, involuting lesions were left untreated. The patients’ ages ranged from 27 to 93 (mean 71.5) years and the lesions diameter ranged from 5 to 40 (mean 19) mm.After combined clinical, histological, and follow-up evaluations, 32 of 43 lesions were assessed as KA and 11 as SCC . Twelve KAs were partially excised (first biopsy) in the intermediate stage between proliferative and fully developed, and 20 in the intermediate stage between the fully developed and regressing phase. An additional 3 lesions were excluded from the present study because of lack of sufficient material to perform immunostaining.No lesions with obvious clinical signs of involution at presentation nor special morphologic or syndromic types of KAs were included. No recurrence or metastasis developed after a follow-up of 3 months to 10 years.The biopsies of all 40 lesions (29 KAs and 11 SCCs) were formalin-fixed. Sections (4-μm thick) were cut from the retrieved neoplasm blocks, dewaxed, and rehydrated in graded ethanol. The primary antibodies used were either p53  at a dilution of 1:100, Ki67  at a dilution of 1:150, or CD34  at a dilution of 1:100 or CD105  at a dilution of 1:10. For all antibodies with the exception of CD105, a steamer was used for epitope retrieval with either citrate buffer (CD34) or Tris-ethylenediaminetetraacetic acid (EDTA) buffer (p53 and Ki67). For CD105, antigen retrieval was performed using 0.4% pepsin at 37 °C for 30 minutes. Only the CD105 sections were incubated at 37 °C with protein block serum-free (Dako). The EnVision Plus polymer (Dako) was used as a reaction amplifier. Visualization of the antibody complex was achieved using 3,3-diaminobenzidine tetrahydrochloride according to the manufacturer's instructions. Sections were counterstained with hematoxylin. Appropriate controls were included in each assay. For negative controls, primary antibodies were omitted and the specimens were incubated with antibody diluent. Tissue from cutaneous SCC with optimal fixation and blocking was used as positive control.25 For each section, starting at the area assumed as the “hot spot,” 10 images of consecutive high-power fields were obtained using a E200 Nikon microscope equipped with a A630 Canon camera, both for CD34 and CD105. When the size of the biopsy did not allow the capture of 10 different images, all distinct fields were photographed. Areas along the border between neoplasm and surrounding tissue were preferred, whereas areas next to hair follicles were avoided. Any brown-stained single cells or clusters of cells, which were separated from other stained areas, were considered a single countable vessel. Unstained lumina were not taken into account. For each image, the total stromal area was calculated using computer-assisted image analysis , which allows manual segmentation of target areas. The stromal areas measured up to 0.04 mm2 in each digital image. MVD was defined as the number of manually counted stained vessels per mm2 (v/mm2). The MVD of the most vascularized area of each section and the MVD corresponding to the sum of all vessels and stromal areas analyzed per section were calculated .The sections were blindly scanned at low magnification (×100) by a single pathologist for areas of highest microvessel density), so-called “hot spots.”As for the vascular markers, the sections stained with p53 and Ki67 were blindly scanned for “hot spots” and ≤10 images of consecutive high-power fields were obtained according to the size of the biopsy. A quantitative analysis of the expression of each marker was performed using computer-assisted image analysis . All positive and negative nuclei of epithelial neoplasm cells were counted in each image. The neoplasm expression of these markers was calculated as the percentage of positive cells in the most positive area as well as the percentage of positivity corresponding to the total amount of nuclei counted per section.t test was used to evaluate differences between both groups. Correlations among the variables were assessed by Pearson correlation coefficient analysis. In addition, multiple liner regressions were calculated, which enables us to determine whether correlations are independent. Moreover, we calculated the coefficients of determination R2. The significance level was 0.05.The data obtained from the incisional biopsies of the 40 KA-like lesions were analyzed (KM and ICW). The CD34 and CD105-determined MVD and the p53 and Ki67 expression percentage were calculated with Winstat 3.1 software. Normal distribution of the variables was tested according to Kolmogorov–Smirnov. Student The study comprised 29 KAs and 11 SCCs, which had been classified primarily according to clinical follow-up.CD34 and CD105 antibodies provided a uniform and moderate (CD105) to intense (CD34) membrane staining of endothelial cells with no or very low background Figure A-D. In tP > 0.2) or CD105 staining (P > 0.2), as well as the density of p53 positive nuclei (P > 0.2). The Ki67 staining index was significantly higher in SCCs (P = 0.0058)  between the groups. The lowest values were found in the fully developed/regressing KAs, followed by the proliferative/fully developed KAs, and the highest values were found in SCCs. The post-hoc test showed only significant differences between the regressing KAs and SCCs. There was, however, considerable overlap between the values in the 3 groups.There were no statistically significant differences between KAs and SCCs in microvessel density revealed by CD34 . We found a positive correlation in KAs between the CD105 density and the Ki67 index , whereas in multiple linear correlation for SCCs, this result was not found.In SCCs, there were no significant correlations between the density of CD105-positive vessels and age, neoplasm size, Ki67, or p53 index . However, in SCCs, there were significant positive correlations between the neoplasm size and p53 index  and the neoplasm size and age , which demonstrated independence in a multiple linear regression , SCCs, and KAs and found that the vascular density was significantly lower in BCC than in both KA and SCC, but no significant difference in MVD was demonstrated between the latter neoplasms. Our results are in accordance with their findings because the assessment of MVD using both a pan-endothelial marker (CD34) and a neovascularization marker (CD105) did not reveal statistical differences between them. We share the opinion of these authors that neoplasm MVD probably does not reflect the neoplasm progression differences between them.Angiogenesis is considered to play a major role in neoplasm progression, as neoplasm growth is dependent on a sufficient supply of nutrients and oxygen.Our quantitative data from the proliferation rate (Ki67) did show discriminating power between KAs and SCCs, with a rate significantly higher in SCCs. However, there was a large overlap between both groups, making it difficult to apply in routine practice because pathologists most commonly use semiquantitative methods of evaluation. Still, is of interest that the SCCs, neoplasm with a higher malignant potential than KAs, had the higher proliferation rate. With respect to the time of neoplasm growth, it is known that KAs generally grow faster than most SCCs, which seems to conflict with the proliferation rate found by our study. This can be explained by the fact that part of our KAs was in the intermediate stage between the fully-developed and regressing phases. Indeed, when SCCs were also compared with each KA phase by the post hoc test, significant differences were only found between the regressing KAs and SCCs. Our other quantitative data did not show discriminating power between KAs and SCCs, adding to the hall of similarities previously described for these 2 neoplasms.27 found rarity of loss of heterozygosity in KAs, in contrast with what was described for SCCs. In Clausen et al’28 study, KA showed a surprisingly high degree of genetic imbalance. Many of the most frequently found aberrations in KAs were not seen in any of the SCCs analyzed by the authors and could be linked to their peculiar rapid growth followed by regression.The immunostains assessed in our study demonstrated important differences regarding neoplasm pathogenesis. Although neoangiogenesis (CD105-MVD) in KAs was associated with neoplasm proliferation, in SCCs, it was not. It is probable that KA needs neovascularization to grow and SCCs have independent growth, probably due to genetic differences. Waring et alIn addition, the correlations observed between age and neoplasm size or p53 expression and neoplasm size in SCCs but not in KAs also indicate further different mechanisms of progression for each neoplasm.We consider that the strengths of this study are as follows: the diagnoses of SCC and KA were established on clinical/pathological/follow-up basis; the lesions were relatively uniform in size and the patients’ ages relatively uniform, in each group. To the best of our knowledge, this is the first systematic study on angiogenesis of lesions with clinical appearance of KA in which, to improve the histopathologic criteria that discriminate the 2 neoplasms, clinical evolution was used as gold standard. However, the small sample size for KA-like SCCs may have restricted the test power for this group and is a limitation of our study.In summary, from our results, we may conclude that KA and SCC have similarities, as CD105- and CD34-MVD. However, the low Ki67 proliferation index and the positive correlation between Ki-67 index and neovascularization on KA suggest dependence in neovascularization to grow in KA, pointing to involvement of distinct pathogenesis.


Learning audiovisual associations is mediated by the primary cortical areas; however, recent animal studies suggest that such learning can take place even in the absence of the primary visual cortex. Other studies have demonstrated the involvement of extra-geniculate pathways and especially the superior colliculus (SC) in audiovisual association learning. Here, we investigated such learning in a rare human patient with complete loss of the bilateral striate cortex. We carried out an implicit audiovisual association learning task with two different colors of red and purple . Interestingly, the patient learned the association between an auditory cue and a visual stimulus only when the unseen visual stimulus was red, but not when it was purple. The current study presents the first evidence showing the possibility of audiovisual association learning in humans with lesioned striate cortex. Furthermore, in line with animal studies, it supports an important role for the SC in audiovisual associative learning. There is strong evidence from neurophysiological animal experiments for the confluence of visual and auditory cues in superior colliculus (SC) . This suA rare patient (TN) with a complete bilateral lesion of striate cortex gave us the opportunity to study potential contributions of the SC to human audiovisual learning in the absence of the retino-geniculo-cortical pathway. Establishing audiovisual learning in hemianopic patients and understanding the underlying mechanisms is important in view of the potential for audiovisual learning in such patients which can improve self-reliance.The indication that there is sufficient visual information being processed that could be associated with normally perceived auditory information led to the idea to study audiovisual learning in an individual with a striate cortex lesion. Although damage to striate cortex severally disrupts normal visual processes causing blindness , some patients show remarkable residual visual abilities as evidenced by above-chance detection or discrimination of movement , wavelenFigure 1A). Previous studies documented his inability to perceive color, movement, and object shape presented in the visual modality alone, yet, he demonstrated above-chance level performance in a facial expression discrimination task (The participant was a 56 year-old right-handed patient (TN) who became cortically blind bilaterally following two strokes within 6 weeks . In a subset of trials a brief square-wave modulation in volume was given superimposed on the overall increasing intensity of the sounds. This modulation represented the target to which the patient had to respond as quickly as possible in the target trials. The target was clearly audible during the trial, and occurred in the middle of the target trials . In other trials the auditory target was absent . Hence, auditory target and non-target trials were identical except for the brief square-wave modulation in the middle of target-trials. One third of the trials consisted of target trials, and two thirds were non-target trials. A single experiment comprised 270 1-s-stimulus presentations, followed by a 500 ms response window, and an inter-trial interval lasting between 400 and 700 ms.TN was presented with an auditory looming stimulus of 1 s, during which the stimulus increased in volume , increasing in size (looming) or decreasing (receding). To ensure visual stimulation, we used a small viewing distance (∼25 cm) and gaze was monitored by two of the experimenters. The patient was not given information about the presence of any systematic associations between auditory and visual stimuli, but was informed that visual stimuli were being presented. The audiovisual pairings were designed as follows: In non-target trials (n = 180), half of the trials were associated with simultaneously receding visual stimuli, and the other half were associated with looming visual stimuli. However, among auditory target trials (n = 90), two-thirds of the trials (n = 60) paired the auditory stimulus with a receding visual signal, and only one-third (n = 30) paired the auditory stimulus with a looming visual signal. Therefore, in the overall distribution of audio-visual pairings, the presence of a receding visual stimulus is predictive of an auditory target (cued target). Thus, the auditory target is cued by the receding visual stimulus, because the auditory target is paired more frequently with the receding visual stimulus than with the looming visual stimulus. To maintain the parallelism of visual and auditory stimuli, we included a visual target analog in the visually looming or receding stimuli, paired with auditory targets. The visual target consisted of a maximally sized disk that briefly interrupted the smoothly varying size of the disk . Experiment 2 consisted of a repetition of Experiment 1 with purple visual stimuli instead of red.In Experiment 1, we created an audio-visual association which was predictive of the presence of the auditory target. Two red visual disk stimuli were used  without a possible confounding advantage due to congruency. If we had chosen a congruent pairing, levels of performance may have reflected contributions from both a congruency effect and a learning effect. The chosen cueing strategy therefore enabled us to study a more pure effect of learning.t = 500 ms, duration = 10 ms, loudness = 80 dB).At the patient’s position, the background noise was 35 dB, and the auditory looming stimulus was added on top of the noise, increasing from 35 dB to a maximum of 80 dB. The noise (measured in the patient’s position) was due to ambient noise in the room due to computers and other equipment. The patient was seated in a dark room at a distance of approximately 25 cm from the monitor (0.2 cd/m2) and a chin rest supported his head. The audiovisual stimuli were prepared as follows: first, an auditory wave of 500 Hz looming sound of 1 s was generated with and without target sounds, using GoldWave v.5.68 . The target sound was generated by inserting the maximum amplitude in the middle of the looming wave time course (in the middle of the stimulus at www.adobe.com) and synchronized with the auditory waves resulting in four different video files (60 Frame/s) for each of the red and purple colors used . At the viewing distance used, the visual looming stimulus varied from 4.8° to 67° diameter. The visual receding stimulus was exactly the same as the looming stimulus, only with opposite order of frames in time. Target analogs in looming and receding visual stimuli corresponded to a replacement of the middle frame (31st frame) by a frame containing the largest disk (67°).Next, looming and receding videos were produced in Adobe Flash CS6  shows the data for audiovisual learning in Experiment 1, with the darker red trend line corresponding to RTs to auditory targets that were associated with the visual cue, and the lighter red trend line corresponding to the RTs to auditory targets that were not associated with the visual cue. The trend lines were generated by a moving average including a window of six trials . The first 18 trials were excluded from analysis as the patient didn’t respond within the 500 ms response window. Of the 90 auditory target trials administered in Experiment 1, we therefore kept 44 trials with visually cued auditory targets, and 24 trials with non-cued targets. RTs above 500 were excluded, resulting in the first 20 trials of Experiment 1 not being included. The data clearly show a systematic tendency of the RTs on cued trials (dark red) to be lower than those on non-cued trials (light red).Figure 2A, combined for the two experiments , show an overall reduction in RT that fit well in a power law curve. This suggests an aspecific learning effect, with the patient becoming more skilled in responding to the auditory target.The data in D = 0.14, p = 0.20; purple: D = 0.16, p = 0.16). This allowed us to apply a paired t-test. Cued and non-cued trials were paired based on trial-cycle, with each consecutive cycle of nine trials containing all trial types, including the three cued and non-cued trials. As shown in Figure 2B, we found a significant RT advantage for the cued over the non-cued condition in Experiment 1 , but no significant difference between the two conditions in Experiment 2 .Prior to testing the differences between cued and non-cued conditions, we verified whether the distribution of RT differences was Gaussian. A Kolmogorov–Smirnov test showed for both datasets that normality was not rejected  = 3.1, p < 0.05; purple: t(6) = 0.99, p = 0.36], suggesting that the purple color used in our experiment would indeed lead to a reduction in the contribution of SC to audiovisual association learning.To verify that the colors used in our experiment would show the properties expected for contribution (red) or non-contribution (purple) from the SC, we performed a control experiment, based on the idea that RTs in a speeded RT test to two targets shown bilaterally are faster than to a single target shown unilaterally. It has been shown that this RT advantage disappears when stimuli are made purple , and henThe result from Experiment 1 shows that the patient with complete striate damage can implicitly learn the association between the auditory and the visual stimuli when the stimuli are colored red. We demonstrated in Experiment 2 that the patient can no longer make use of the learned association when the stimuli are rendered purple.The present study is the first demonstrating the fast development of implicit audiovisual associations  between consciously perceived auditory stimuli and visual stimuli that cannot be perceived because of complete bilateral striate lesion. The finding that the implicit audiovisual association did not take place for the purple visual stimuli strongly supports the notion that SC plays a critical role in this form of learning. A control experiment in seven normal participants  showed a RT advantage when responding to two red, compared to one red target in the visual field; an advantage that disappeared with purple stimuli. As the SC is thought to contribute to this RT advantage, these findings support the idea that the purple color used in our visual stimuli indeed could not be processed by the SC .Our data are in line with demonstrations of automatic neural audiovisual integration in the anesthetized cat  and withThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lack of CBG does not modify the progression of inflammation associated to pancreatitis but resulted in the loss of gender differences in corticosterone serum levels. In the lung, CBG expression and protein level were detected, and it is noteworthy that these showed a sexual dimorphism opposite to the liver, i.e. with higher levels in males. Reduced expression of 11β-HSD2, the enzyme involved in the deactivation of corticosterone, was also observed. Our results indicate that, in addition to glucocorticoids transporter, CBG is involved in the gender differences observed in corticosteroids circulating levels and plays a role in the local regulation of corticosteroids availability in organs like lung.Corticosteroid-binding globulin (CBG) is the specific plasma transport glycoprotein for glucocorticoids. Circulating CBG is mainly synthesized in liver but, its synthesis has been located also in other organs as placenta, kidney and adipose tissue with unknown role. Using an experimental model of acute pancreatitis in  Acute pancreatitis is a serious inflammatory process with significant morbidity and mortality. The most relevant complication during acute pancreatitis is the systemic inflammation that, in the severe forms of the disease, may lead to an acute respiratory distress syndrome . This puAs occurs with other critical illnesses, such as sepsis, trauma and septic shock, the hypothalamic–pituitary–adrenal axis plays an important modulatory role in the control of the inflammatory process. Several reports suggested a link between an impaired adrenal secretion and the progression of systemic inflammation in acute pancreatitis . In addiIn addition to glucocorticoids, changes in the levels of corticosteroid-binding globulin (CBG) have also been reported in patients with acute pancreatitis 8]. CBG . CBG 8].It is accepted that CBG actively deliver glucocorticoid to inflamed tissue due to the action of elastase released by activated neutrophils. This protease cleaves CBG and disrupts the glucocorticoid-binding site, thus resulting in the release of the corresponding glucocorticoid in the areas of inflammation . Howevercbg+/+ and in cbg-/- animals. Moreover, considering the remarkable differences in the metabolism of glucocorticoids between males and females, we did the experiments comparing the effects between the two genders.Herein, our study was designed to investigate, in an experimental model of acute pancreatitis in mice, whether changes in CBG could affect the progression of the disease as well as the metabolism of glucocorticoids in the lung. To do this, we compared the effects of pancreatitis in cbg+/+ and cbg-/- were used in all experiments. The colony of cbg-/- was established through crossing heterozygous breeding kindly provided by Dr. Willnow. The procedure followed for disruption of the CBG gene was described by Petersen et al.  and incubated at room temperature for 30 minutes with occasional shaking. Samples were then centrifuged and the clear supernatants were further diluted 1/5 with PBSG buffer. These hormone-stripped samples were used for corticosterone binding assays. The final concentration of labelled corticosterone in the tubes was 15 nM in a total volume of 200μl. Then, 200 μl of a constant stirring charcoal-dextran suspension were added to each tube. These were shaken, left to stand for 10 min in an ice bath, and centrifuged for 10 min. Aliquots of 200 μl of the clear supernatants were added to 4 ml of a water-miscible scintillation cocktail  and counted in a standard scintillation counter. In all samples, individual unspecific binding (measured in a 500-fold excess of non-radioactive corticosterone) was measured and subtracted from the total radioactivity bound to diluted stripped serum.The data were analyzed using the GraphPad software program version 5.0 and were expressed as the mean ± SEM. Statistical comparisons were made by two-way ANOVA analysis, evaluating the variables "treatment"  and "sex" . Bonferroni was used as a post-test. The P values < 0.05 were considered significant.cbg+/+ mice, this increase was higher in females than in males. By contrast, there no gender differences were observed in the cbg−/− mice. In males the increase of lipase circulating levels promoted by pancreatitis was higher in cbg−/− than cbg+/+ mice, but in female the inverse effect was observed.  and type 2 (11β-HSD2). No differences were observed in 11β-HSD1 expression in any condition, nor due to genotype, cerulein treatment or gender .cbg+/+ nor in cbg-/- mice. Pancreatitis, in males and females cbg+/+ mice, significantly reduced the expression of 11β-HSD2. In the cbg-/- mice, control expression was lower than in cbg+/+ and pancreatitis also resulted in a decreased expression although in this case only in females achieved significantly values.With respect to 11β-HSD2 expression , gender cbg-/- mice.Since 11β-HSD1 catalyzes the conversion of 11-dehydrocorticosterone to the active corticosterone, whereas 11β-HSD2 catalyzes the opposite reaction, the ratio 11β-HSD1/11β-HSD2 indicates the changes in the local glucocorticoid metabolism . InductiThe progression of the inflammatory response results from the balance of several pro- and anti-inflammatory mediators. Cytokines, free radicals, lipid mediators and activated enzymes promote the activation of inflammatory pathways, not only locally, but also in circulating cells and in distant organs . These eIt is known that the glucocorticoids ability to regulate the inflammatory response is strongly related with their availability on the site of inflammation. Tissue availability of glucocorticoids, namely the proportion of glucocorticoid able to bind to the receptor and carry out a response, depends of its synthesis at adrenal gland , its bincbg+/+ animals. This difference was not observed in cbg-/- mice and the levels achieved were similar to that observed in male cbg+/+ mice  indicating a major activity of the gland. These features suggest that the HPA axis is irresponsive to the negative feed-back exerted by free corticosterone in cbg-/- pointing to a role of CBG in the HPA axis control and regulating corticosterone synthesis.The low levels observed in circulating corticosterone in en et al . It has en et al . The totactivity . Furthercbg-/- mice, the gender difference in corticosterone levels was not observed in controls neither in pancreatitis induced animals. The role of CBG in the gender differences on circulating glucocorticoids have been observed recently in a model of emotional stress [cbg-/- mice showed no differences between males and females in stress reactivity and neither in the absence of estrogens. Nevertheless, there are some differences in the response of cbg-/- animals to stress or to acute pancreatitis inflammation. While under the stress, cbg-/- animals failed to increase corticosterone levels [In addition to the higher levels of total serum corticosterone in female  there wel stress  where wae levels , in our It is well known that CBG regulates the amount of free corticosterone released during inflammation  but our Although the circulating levels of corticosterone reflect the general systemic anti-inflammatory response, the action of corticosterone also depends on the local activity of the enzymes 11β-HSD1 and 11β-HSD2. In general, it is accepted that in response to inflammation the levels of 11β-HSD1 are increased in order to potentiate the anti-inflammatory effects of glucocorticoids. Less information is available on 11β-HSD2. In vitro it is known that pro-inflammatory cytokines, as TNFα, upregulate 11β-HSD1  and downcbg-/- mice lung expression of 11β-HSD2 showed lower levels than cbg+/+. This fact could be a mechanism to increase corticosterone levels into the tissue to counteract the impaired availability of tissue corticosterone observed in cbg-/- [Interestingly, in control n cbg-/- .In conclusion, our results indicate that the role of CBG is not only restricted to act as a carrier of corticosteroids and a modulator of its tissue availability. It is also an important factor involved in the gender differences observed in corticosteroids levels. CBG is generated in the lung showing a pattern of expression opposite to the liver either in the response to the acute pancreatitis as in the sexual dimorphism. These differences between liver and lung suggest that CBG has a specific, and yet unexplored, role in the lung.