Estrogens are known to regulate the proliferation of breast cancer cells and to modify their phenotypic properties. Identification of estrogen-regulated genes in human breast tumors is an essential step toward understanding the molecular mechanisms of estrogen action in cancer. To this end we generated and compared the Serial Analysis of Gene Expression (SAGE) profiles of 26 human breast carcinomas based on their estrogen receptor α (ER) status. Thus, producing a breast cancer SAGE database of almost 2.5 million tags, representing over 50,000 transcripts.Estrogen Responsive Elements (EREs) distributed on the promoter regions of 163 out of the 473 up-modulated genes in ERα (+) breast tumors. In brief, we observed predominantly up-regulation of cell growth related genes, DNA binding and transcription factor activity related genes based on Gene Ontology (GO) biological functional annotation. GO terms over-representation analysis showed a statistically significant enrichment of various transcript families including: metal ion binding related transcripts (p = 0.011), calcium ion binding related transcripts (p = 0.033) and steroid hormone receptor activity related transcripts (p = 0.031). SAGE data associated with ERα status was compared with reported information from breast cancer DNA microarrays studies. A significant proportion of ERα associated gene expression changes was validated by this cross-platform comparison. However, our SAGE study also identified novel sets of genes as highly expressed in ERα (+) invasive breast tumors not previously reported. These observations were further validated in an independent set of human breast tumors by means of real time RT-PCR.We identified 520 transcripts differentially expressed between ERα-positive (+) and ERα-negative (-) primary breast tumors . Furthermore, we identified 220 high-affinity The integration of the breast cancer comparative transcriptome analysis based on ERα status coupled to the genome-wide identification of high-affinity EREs and GO over-representation analysis, provide useful information for validation and discovery of signaling networks related to estrogen response in this malignancy. Estrogen plays essential roles in the development, growth control and differentiation of the normal mammary gland. However, it is well documented that endogenous estrogens are powerful mitogens critical for the initiation and progression of human breast and gynecological cancers . This ce2) is mediated by two main mechanisms. In one, the E2-ER complex binds to a specific DNA sequence called the estrogen response element (ERE), this receptor-ligand DNA bounded complex interacts with co-regulatory proteins, promoting chromatin remodeling and bridging with the general gene transcription machinery thus resulting in transcription initiation [2 also exerts rapid, non-genomic effects attributed to cell membrane-initiated signaling [Transcriptional regulation of target genes in response to 17β-estradiol  at the time of diagnosis and expression of this receptor is determinant of a tumor phenotype that is associated with hormone-responsiveness. Patients with tumors that express ERα have a longer disease-free interval and overall survival than patients with tumors that lack ERα expression . However2 induced proliferative response predominantly via the regulation of estradiol-responsive genes. Nevertheless, the expression of additional subsets of genes not necessarily directly regulated by estrogen may also be fundamental in defining the breast cancer hormone-responsive phenotype.Several studies have been carried out using cDNA and oligonucleotide microarrays identifying breast cancer subclasses possessing distinct biological and clinical properties -13. AmonTo further elucidate the molecular basis of estrogen-dependent breast carcinogenesis, we here report a comparative transcriptome profiling of invasive breast tumors based on ERα status obtained by SAGE. The SAGE method provides a statistical description of the mRNA population present in a cell without prior selection of the genes to be studied, and this constitutes a major advantage . The brei.e. ERα (+) vs. ERα (-) breast tumors.The primary goal of our study was to identify the most commonly deregulated genes in invasive breast carcinomas related to ERα status. To this end SAGE data was obtained from a set of primary breast carcinomas. Thus, a breast cancer SAGE database of almost 2.5 million tags was analyzed, representing over 50,000 tag species. We performed a comprehensive evaluation and comparison of gene expression profiles using a recently developed supervised method , to idenThis statistical analysis revealed 520 genes differentially expressed  between ERα (+) and ERα (-) primary breast carcinomas see . Among ttrefoil factor 1 (TFF1/pS2), synaptotagmin-like 4 (SYTL4), regulating synaptic membrane exocytosis 4 (RIMS4), dual specificity phosphatase 4 (DUSP4), chromosome 1 open reading frame 34 (C1orf34), necdin homolog (NDN), n-acetyltransferase 1 (NAT1) and caspase recruitment domain family 10 (CARD10) (Table The most commonly over-expressed transcripts in ERα (+) tumors were: 0) Table .signal peptide CUB domain EGF-like 2 (SCUBE2) (p = 0.0001), SYTL4 (p = 0.0005), KIAA0882 protein (p = 0.0005), tetraspan 1 (TSPAN1) (p = 0.001), myeloblastosis viral oncogene homolog (C-MYB) (p = 0.002), epidermal growth factor-like 2 (CELSR2) (p = 0.011), nuclear receptor subfamily 4 (NR4A1) (p = 0.029), and enolase 2 (ENO2) (p = 0.033)  (p = 0.079) transcript  breast tumors including: p = 0.000, SYTL4  encodes a secreted and cell-surface protein containing EGF and CUB domains that defines a novel gene family [SCUBE2 has been detected in vascular endothelium and may play important roles in development, inflammation and perhaps carcinogenesis [e family . The epie family . Expressogenesis .CELSR2 gene  encodes a protein member of the nonclassic-type cadherins (flamingo subfamily). These 7-pass transmembrane proteins have nine cadherin domains, seven-epidermal growth factor-like repeats and two laminin A G-type repeats [The  repeats . It is p repeats  playing  repeats ,23.SYTL4  contains an N-terminal Slp homology domain (SHD) than can specifically and directly bind the GTP-bound form of Rab27A, a small GTP-binding protein involved in granule exocytosis in cytotoxic T lymphocytes [SYTL4 is associated with ERα (+) tumors  encodes one of three enolase isoenzymes found in mammals. This isoenzyme was described to be expressed in cells of neuronal origin. Interestingly, in a recent report Hao et al. (2004) showed high expression of ENO2 transcripts in breast cancer lymph node metastases when compared with primary breast tumors [t tumors .TSPAN1 gene  encodes a cell-surface protein member of the transmembrane 4 superfamily (TM4SF), involved in the regulation of cell development, activation, growth and motility. A number of tetraspanins were described as tumor-specific antigens, and it was suggested that the function of some TM4SF proteins may be particularly relevant to tumor cell metastasis [TSPAN1 protein complexes may control the invasive migration of tumor cells and contribute to ECM-induced production of MMP2 in breast cancer cell line [The tastasis . Sugiuraell line .NR4A1, a nuclear receptor subfamily 4, group A gene  encodes an orphan member of the steroid-thyroid hormone-retinoid receptor superfamily whose members mainly act as transcriptional factors to positively or negatively regulate gene expression and play roles in regulating growth and apoptosis [NR4A1 in cell proliferation has been previously reported. It was shown that its expression is rapidly induced by various mitogenic stimuli such as: serum growth factor, epidermal growth factor and fibroblast growth factor [poptosis ,29. A roh factor .Taken together, the genes that we identified and validated appear to be involved in signaling pathways related to cell proliferation, invasion and metastatic processes, but their exact role in breast carcinogenesis remains to be elucidated.Expression Analysis Systematic Explorer software (EASE) [Classification of genes based on Gene Ontology (GO) terms is a powerful bioinformatics tool suited for the analysis of DNA microarray and SAGE data. Analysis of GO annotation allows one to identify families of genes that may play significant roles related to specific molecular or biological processes in expression profiles . We usede (EASE)  to annote (EASE) . The GO cell growth and/or maintenance, 21% are related to cell communication, and 16% are related to regulation of transcription. Approximately 16% of these deregulated genes are related to molecular functions associated with DNA binding and more specifically with transcription factor activity (10%) , calcium ion binding proteins  and steroid hormone receptor activity related proteins  , androgen receptor (AR), hydroxysteroid 17-β dehydrogenase 4 (HSD17β4), glucocorticoid receptor (NR3C1), oxysterol binding protein (OSBP), and retinoic acid receptor α (RARA). The observation of functionally related groups of genes identified in the SAGE dataset via GO over representation analysis allows the identification of distinct biological pathways directly or indirectly associated to estrogen response related processes and provides the basis for future mechanistic studies.We observed that 31% of ERα associated transcripts are involved in biological processes related to = 0.031) . The GO et al. [We used a recently reported genome-wide high-affinity ERE database  to identet al. .The observed frequency of these elements in our study was 220 EREs in 3260 kb (considering a DNA window of 20 kb for each one of the 163 up-modulated genes with EREs). Compared with the expected frequency from random distribution of high-affinity EREs found in the genome   [et al. [2-ER binding sites related to transcriptional activation.Fifty percent (110 out of 220) of the detected EREs mapped within a 10 kb region 5' of the TSS, while the rest mapped to 3' regions Figure . Approxiet al. . HoweverSYTL4 (-8384 bp from the TSS: tggacatcatgacct), TSPAN1 (+974 bp and +9384 bp from the TSS: tggtctgaatgaccc and aggtcatttccacct respectively), CELSR2 (+173 bp and +3607 bp from the TSS: tgctcagggtgaccc and aggtcaccatgaccg respectively), and NR4A1 (-3478 bp and +4217 bp from the TSS: tgttcactctgacct).Of the validated transcripts previously discussed Figure , we deteovalbumin  and c-fos ; two transcripts detected as over-expressed in ERα (+) breast tumors by SAGE (retinoic acid receptor α (RARA) , vascular endothelial growth factor (VEGFC) , insulin-like growth factor binding protein-4 (IGFBP4)  and heat shock protein 27 (HSPB1) ; four transcripts detected as over-expressed in ERα (+) tumors in our study (It is interesting to note that we were unable to identify high-affinity EREs on the majority of deregulated genes 65%) associated with a positive ER α status. The possibility exists that many of these genes are transcriptionally regulated by non-ERE mediated mechanisms such as those involving ER binding to the AP1 or SP1 transcription factors . The AP15% associ by SAGE . Similarur study .nuclear orphan receptors with significant homology to ERs, which do not bind estrogen and have unknown physiological ligands. ERRs are known to bind to the steroidogenic factor 1 response element (SFRE) and also bind to classic EREs, by means of which they exert constitutive transcriptional activity [NR4A1 by SAGE and subsequently validated this observation by real time RT-PCR , activity . We deteR Figure . Interes by ERRs .As described, ERα can mediate estrogenic response through multiple genomic and non-genomic mechanisms, many of which affect proteins and pathways not necessarily directly or exclusively associated with ERα. Thus it is worth stressing that it will the totality of deregulated proteins the ones that ultimately define the phenotype of ERα (+) breast carcinomas regardless of whether a "direct association" with ER transcriptional regulation exists or not.2-responsive genes in MCF-7 cell line, previously reported by us [et al, we observed a general up-regulation cell cycle progression-related genes including: CCT2, CCND1, PES1, RAN/TC4, CALM1, CALM2; and tumor-associated genes such as: RFP, D52L1, TFF1/PS2, CAV1, and NDKA among others [2-ER transcriptional regulation.The SAGE profiles for Eed by us , was comg others . These tin vitro  and in vivo  gene expression profiles, to our surprise we detect that only few transcripts: TFF1, CCND1, H19, SREBF1 and WWP1 behaved similarly (i.e. up-regulation) in both studies. This is similar to observations made previously by Meltzer and co-workers whom showed that the majority of genes regulated in cell culture do not predict ER status in breast carcinomas [in vitro response of a single cell line to E2, in this particular case the widely used MCF-7 cells, may not faithfully reproduce the physiological effects of ER signaling in vivo.By comparing the rcinomas ,37. Thiset al. [et al. [In order to identify and validate the most reliable set of genes able to discriminate breast carcinomas based on their ERα status, we performed a cross-platform comparison between the described SAGE dataset with two previously reported breast cancer studies based on DNA microarray methods ,13. van'et al.  reportedestrogen receptor 1 (ESR1), GATA-binding protein 3 (GATA3), mucin 1 (MUC1), v-myb-myeloblastosis viral oncogene homolog (C-MYB) , X-box-binding protein 1 (XBP1), hydroxysteroid 17-β dehydrogenase 4 (HSD17B4), BTG family member 2 (BTG2), transforming growth factor β-3 (TGFB3), member RAS oncogene family (RAB31), START domain containing 10 (STARD10), and KIAA0089  breast tumors. One hundred and eighty-three genes were identified by more than one method Figure . Eleven One hundred and fourteen genes were identified as over-expressed by oligonucleotide microarrays  and SAGEInterestingly, we found a higher number of overlapping genes between the oligonucleotide microarray and SAGE platforms 114 genes), while only 66 genes were observed overlapping when comparing both microarray platforms. It is worth noting that 96% of the 470 genes  breast tumors. More importantly this analysis identifies the most promising biomarkers for further evaluation as ERα associated genes in breast cancer. Furthermore, the identified proteins may be of value as breast cancer prognostic indicators analyzed either as a group or individually. It is also likely that groups of co-regulated genes in ERα (+) breast cancers may be associated to the hormonal control of mammary epithelial cells growth and differentiation. Finally, a better understanding of the signaling networks controlled or associated with the estrogen response may lead to the identification of novel breast cancer therapeutic targets.. For the generation of our SAGE libraries, snap frozen samples were obtained from the M.D. Anderson breast cancer tumor bank, and SAGE analysis was performed as previously described [To perform the comparative breast cancer SAGE analysis based on ERα status, we analyzed 26 Stage I – Stage II invasive breast carcinomas (8 ERα-negative tumors and 18 ERα-positive tumors). To this end, we generated and sequenced 24 breast cancer SAGE libraries at an approximate resolution of 100,000 tags per library, combined with 2 additional breast cancer libraries (ERα-negative tumors) downloaded from the Cancer Genome Anatomy Project – SAGE Genie database (SAGE_Breast_Carcinoma_B_95-259 and B_IDC_4) escribed ,38.. In our analyses we only considered tags with single tag-to-gene reliable matches. To compare these SAGE libraries, we utilized a modified t-test recently developed by us [SAGE tag extraction from sequencing files was performed by using the SAGE2000 software version 4.0 . SAGE data management, tag to gene matching as well as additional gene annotations and links to publicly available resources such as GO, UniGene, LocusLink, were performed using a suite of web-based SAGE library tools developed by us ed by us . This teed by us ..All raw SAGE data reported as Supplementary tables in this manuscript is publicly available at ® 7700 Sequence Detection System . Experiments were performed in duplicate for each data point and 18s rRNA was used as control. Results were expressed as mean ± 2 Standard Error based on Log2 transformation of normalized real time RT-PCR values of the assayed genes. We used t-test to compare the gene expression levels of validated genes between ERα (+) and ERα (-) breast tumors (p < 0.05).Template cDNAs were synthesized on mRNAs isolated from an independent set of 36 Stage I – Stage II human breast carcinomas (13 ERα-negative tumors and 23 ERα-positive tumors) obtained from our tumor bank. Primers and probes were obtained from the TaqMan Assays-on-Demand™ Gene Expression Products . All the PCR reactions were performed using the TaqMan PCR Core Reagents kit and the ABI Prism2O2 in methanol for 10 min. After pretreatment with Tris-EDTA buffer, in order to block non-specific antibody binding, the slides were incubated with 10% goat serum in PBS for 30 min. Primary monoclonal ERα antibody  was used at 1:50 dilution and detected following standard immunohistochemical techniques. DAB was used as chromogen and Mayers hematoxylin is used as counterstain. Scoring was performed by breast pathologist (AS). Cuttoff for positivity was determined at 5% of tumor cells staining positively for ER (i.e. < 5% of cells the tumor was considered negative for ERα).IHC staining and ER status determination was performed by the Pathology Department, MDACC following routine immunohistochemical procedures. Briefly, five micrometer sections of invasive breast carcinomas paraffin embedded tissues were used. Endogenous peroxidase activity was blocked with 3% HEASE [Database for Annotation, Visualization and Integrated Discovery (DAVID) at [EASE software calculates over-representation of specific GO terms with respect to the total number of genes assayed and annotated. Statistical measures of specific enrichment of GO terms are determined by means of an EASE score (p < 0.05). The EASE score is a conservative adjustment of the Fisher exact probability that weights significance in favor of biological themes supported by more genes and is calculated using the Gaussian hypergeometric probability distribution that describes sampling without replacement from a finite population [EASE analyzed genes.For automated functional annotation and classification of genes of interest based on GO terms, we used the EASE  availablAVID) at . The EASpulation . This al[11 = 732 high-affinity EREs) [To identify the occurrence of EREs within the promoter regions of up-modulated genes in ERα (+) breast tumors, we used a human genome-wide high-affinity ERE database . This puERα status associated genes identified in previous breast cancer studies ,13 using and ) [All over-expressed genes in ERα (+) breast tumors obtained from these studies were downloaded from the corresponding web sites ( and ) ,13.EASE software [e.g. SAGE study vs. DNA microarray) was determined. We used the normal approximation to the binomial distribution as previously described [These datasets were annotated by LocusLink ID using the software , and theescribed  to calcuM.C.A. conceived the study idea and carried out the real time RT-PCR validations, the biostatistical/ bioinformatics analysis and writing the manuscript. Y.H., H.S. and J.A.D. carried out the breast cancer SAGE libraries and provided practical feedback on aspects of the manuscript. K.B. and S.G. developed the biostatistical and web-page base methodology. A.S. provides the tissue samples and clinical information. C.M.A. is the principal investigator and was involved in the conceptualization, design and writing of the manuscript. All authors read and approved the final manuscript.The author(s) declare that they have no competing interests.Differentially expressed genes between ERα (+) vs. ER α (-) breast carcinomas .Click here for fileGene Ontology overrepresentation analysis.Click here for fileHigh-affinity EREs identified in ERα (+) up-modulated genes.Click here for fileCross-platform comparison of the up-modulated transcripts in ERα (+) breast carcinomas.Click here for file


This review article is aimed at comparing the results of histopathological and clinical imaging studies to assess coronary collateral circulation in humans. The role of collaterals, as emerging from morphological studies in both normal and atherosclerotic coronary vessels, is described; in addition, present role and future perpectives of echocardiographic techniques in assessing collateral circulation are briefly summarized. In the past 25 years, the concept of a compensatory function of the coronary collaterals (or anastomoses) – i.e. vessels that join different coronary arteries or branches – has been practically cancelled from the mind of cardiologists since cineangiography shows that the onset of coronary heart disease (CHD) occurs independently of their presence. The assumption, therefore, was and is that they have no compensatory meaning  and corohomocoronary (between branches of the same coronary artery) and intercoronary (between different coronary arteries) anastomoses were present everywhere joining at any level the intramural branches. Only in two of more than 600 hearts, superficial collaterals between extramural coronary arteries were seen and sampled for histology. The diameter of the innumerable normal collaterals ranged from 20  to 350 microns, frequently assuming a corkscrew aspect, possible adaptation to the contraction cycle of the myocardium ; when more than one severe stenoses exist each one had its own satellite collaterals. However, an identical obstruction located at the same level of an artery might show relatively few highly enlarged collaterals (the only ones visible by cineangiography), or numerous relatively small collaterals  in apparently healthy people acting their normal life, 89% with a fatal AMI had one or more (47%) severe atherosclerotic stenosis greater than 70% ;65% of sudden and unexpected death (SUD) showed the same finding in one or more (35%) vessels; 66% of non cardiac patients dead from other diseases and 39% of normal subjects dying from accident had the same severe atherosclerotic stenosis in one or more (40% and 16% respectively) coronary arteries  does not correspond to that observed in pathological studies in which the mean figure is 50% for AMI and 29% for SUD patients. Nevertheless, different selection of material, divergent definition and an absence of a correct correlation of all pertinent variables give reason of dissimilar conclusions. In 200 selected AMIs and 208 SUD cases the unique cause of occlusion was a thrombus found in 41% and 29% respectively. In AMI group it correlated significantly with a lumen reduction greater than 70% (93%), length of plaque more than 6 millimeters (95%), its concentric shape (100%), prevailing atheroma (84%), medial neuritis (92%) infarct size greater than 50% (86%). SUD cases showed a similar behavior.In reality, both clinicians and pathologists observe a phenomenon which started before, missing its onset and sequence of events to distinguish whether primary or secondary. In only one case reported in literature , this seOne case is only one case but when for the first time shows how the events developed, it becomes a precious mile stone for our knowledge demonstrating that the cineangio occlusion was a pseudocclusion namely a blood flow stasis in LAD secondary to an increased intramyocardial resistance. The first main question is how many of the 87% cineangio occlusion are pseudocclusion and whether the "layered" thrombus recovered at bypass surgery was a true thrombus or a coagulum which frequently show a layering of blood elements not seen in thrombus formation."Red" thrombus, namely a coagulum, is frequently and erroneously considered as thrombus. The second question concerns the nature of increased intramyocardial resistance: spasm of intramural arterial vessels or their extravascular compression by an asynergic myocardium? The first sign of CHD is hypokinesis of a myocardial zone which particularly in systole may compress vessels. Any time there is an increase of the intraventricular pressure with bulging of hypokinetic myocardium such a compression may abolish blood flow with subsequent infarction. In the reported patient location and infarct size corresponded to the hypokinetic area observed before the infarct onset.A last comment deserves the supposition that small atherosclerotic plaques undetectable at cineangio, may rupture causing an infarct. A supposition based on the cineangio finding of a non critical stenosis observed in a vessel tributary of a territory in which an infarct will develop. Since, when the latter occurred, stenoses in other non supplying vessels did not show a further lumen reduction, the conclusion was that even the plaque related to infarction had a non critical lumen reduction . A conclIn the past years, clinical methods available to measure collateral flow have been too crude and showed major limitations, thus contributing to debate and confusion about the functional relevance of collateral circulation in the human myocardium. Coronary angiography allows visualization of collateral vessels having a diameter ≥100 μm, that actually prevents the majority of them from being detectable with this technique ,17. On tFinally, intravenous MCE has been recently reported to provide qualitative and quantitative evaluation of collateral blood flow in the presence of an occluded infarct-related artery, and to emerge as the only predictor of true collateral blood flow among other markers .caveat, however, have to be taken into account. First, although the peak contrast pixel intensity has been reported as the most accurate of the variables obtained to measure collateral flow, there is a remarkable scatter in the correlation between peak pixel intensity and true collateral flow [All these reports as a whole support the concept that MCE provides important information on collateral flow and represents a promising mean for evaluating the status of coronary collateral circulation in clinical practice. Some important ral flow . Second,ral flow . FinallyBesides the attempt to obtain direct echocardiographic assessment, coronary collateral circulation can indirectly affect the result of diagnostic stress testing with the use of echocardiographic technique. Increased vulnerability to myocardial ischemia induced by pharmacological coronary vasodilation was reported consistently with the hypothesis of a facilitated steal phenomenon in the presence of good collateral circulation . On the The application of low-frequency ultrasound to intravascular microbubble contrast agents has been receiving attention in the last few years due to its potential therapeutic application, primarily as targeted gene delivery systems . FurtherAny hypothesis on the pathogenic role of a plaque and its activity and vulnerability should consider all interrelated variables for a correct interpretation of findings. When only one or few variables are investigated erroneous conclusions can be reached. An atherosclerotic plaque is always an active structure since its progression depends on a sequence of events due to a variety of correlated phenomena; while vulnerability is just an hypothesis which believe that some findings indicate a risk of plaque rupture.The known variables are: degree of lumen reduction, shape, length, satellite collaterals, tunica media changes, inflammatory reaction per se and associated with media nerves , survival Table  macrophaMost studies analized few variables mainly observed in animals after hypercholesterol diet or in familial hypercholesterolemia. A pattern ,47 totalThe presence of functioning collaterals induces a particular hemodynamic condition within the residual lumen at the plaque level with proximal flow reduction counterbalanced by distal collateral flow. Any time there is a regional asynergy Figure  with incProf. Giorgio Baroldi contributed to the conception and organization of this review and to the final comments. Dr. Riccardo Bigi and Dr. Lauro Cortigiani summarized the use of ultrasound techniques in atherosclerotic plaque imaging.


The aim of this study was to describe practice activity trends among oral and maxillofacial surgeons in Australia over time.All registered oral and maxillofacial surgeons in Australia were surveyed in 1990 and 2000 using mailed self-complete questionnaires.Data were available from 79 surgeons from 1990 (response rate = 73.8%) and 116 surgeons from 2000 (response rate = 65.1%). The rate of provision of services per visit changed over time with increased rates observed overall (from 1.43 ± 0.05 services per visit in 1990 to 1.66 ± 0.06 services per visit in 2000), reflecting increases in pathology and reconstructive surgery. No change over time was observed in the provision of services per year . Time devoted to work showed no significant change over time , while the number of visits per week declined (70 ± 4 visits per week in 1990 to 58 ± 4 visits per week in 2000).The apparent stability in the volume of services provided per year reflected a counterbalancing of increased services provided per visit and a decrease in the number of visits supplied. In Australia the majority of dentists work in private general practice . RelativIdentifying trends over time in the practice activity of oral and maxillofacial surgeons is a key element in planning by informing debate on issues relevant to the speciality such as the future supply of services and training needs. Previous Australian data have shown the distribution of oral and maxillofacial surgery was dominated by dentoalveolar services . HoweverConsidering the trend towards oral and maxillofacial surgeons gaining medical qualifications and the potential impact that this may have on practice activity, the aims of this study are to describe practice activity trends among oral and maxillofacial surgeons in Australia in 1990 and 2000 in terms of time worked, visits supplied and services provided.All registered oral and maxillofacial surgeons in Australia were surveyed in both 1990 and 2000. A surgeon must be registered with a dental board in the state/territory in which they practice. For the purposes of this analysis trainees were excluded. Although some of the same surgeons may have responded to the survey at both points in time for this study the design and analysis is treated as two sequential cross-sectional surveys.Data were collected using mailed self-complete questionnaires with a primary approach letter sent initially to each surgeon, followed a week later by the survey materials, with a reminder card two weeks later, and up to four follow-up mailings of survey materials to surgeons who had not yet responded in order to ensure higher response rates . SurgeonThe questionnaire was designed to provide comprehensive coverage of a range of workforce issues and the analysis presented here is limited to a subset of the total number of variables that were collected. The questionnaire included surgeon demographic and background variables , practice details , and a log of services provided in a typical week. Surgeons recorded details of the patients they treated over a one-week period. Main areas of service were classified as dentoalveolar, trauma, pathology, orthognathic, reconstructive surgery and other/major medical compromise. An outline of the key variables collected and how measures of time worked, services provided and visits supplied were derived is presented in Figure Data were available from 79 surgeons from 1990 (response rate = 73.8%) and 116 from 2000 (response rate = 65.1%). Service provision data were available for 4,847 patients from 1990 and 3,292 patients from 2000. Table The rate of provision of services is presented in Table The number of hours per year devoted to work by surgeons did not change significantly between 1990 and 2000, reflecting stable levels of hours per day, days per week and weeks per year worked. However, the number of visits per week supplied by surgeons decreased between 1990 and 2000. The number of visits per year supplied by surgeons also showed a trend towards a decreased number of visits over time, but the change (P = 0.0508) was not significant at the P < 0.05 level. The relationship between the service and visit measures is presented in Figure While the findings of this study are based on a small sample size this primarily reflects the size of the population studied. Oral and maxillofacial surgeons comprise a small percentage of the dental workforce , hence aThe practice patterns of oral and maxillofacial surgeons have been reported to be largely stable, showing no change between 1990 and 2000 in their age and sex distribution, place of birth, practice activity level, referral sources, mix of cases and perceptions of work . The disThe decline in patient visits supplied by oral and maxillofacial surgeons, while statistically significant for visits supplied per week, was not statistically significant at the conventional level of P < 0.05 for visits supplied per year. This partly reflects the stability in weeks per year worked, one of the component measures used to derive visits per year. However, the number of visits per year was at the borderline of statistical significance (P = 0.0508) and considerations other than a reliance on P values is recommended in the literature , with moThe trends observed in practice activity among oral and maxillofacial surgeons show parallels with private general practice dentists in Australia. Trends in private general practice have also shown a counterbalancing effect of declining numbers of visits supplied with increased rates of services per visit resulting in a stable level of provision of services per year. Medical general practitioners have shown a trend towards longer consultations , consistPossible explanations for the lower levels of patient visits per hour among private general practice dentists include increased numbers of older patients , who mayHistorical records have indicated that average length of dental appointments changed little over the period 1960–61 to 1974–75, but there was an increase since 1974–75 that was quite marked across the 1977–78 to 1982–83 period -29, and Estimates of the capacity to supply services and projections of labour force requirements need to consider that while the rate of services per visit has increased this has been counterbalanced by decreases in the number of visits supplied. This has resulted in a stable volume of services provided by oral and maxillofacial surgeons per year over the study period.The author(s) declare that they have no competing interests.DSB performed data analyses and drafting of the manuscript. AJS provided overall supervision of the project. KAS performed data processing and preliminary analyses. DNT was involved in data collection. ANG provided specialist advice on oral and maxillofacial surgery. All authors contributed to and approved the manuscript.The pre-publication history for this paper can be accessed here:


Studies in animal models of AD, in which brain complement activation can be experimentally altered, should be of value for clarifying this issue. However, surprisingly little is known about complement activation in the transgenic animal models that are popular for studying this disorder. An optimal animal model for studying the significance of complement activation on Alzheimer's – related neuropathology should have complete complement activation associated with senile plaques, neurofibrillary tangles (if present), and dystrophic neurites. Other desirable features include both classical and alternative pathway activation, increased neuronal synthesis of native complement proteins, and evidence for an increase in complement activation prior to the development of extensive pathology. In order to determine the suitability of different animal models for studying the role of complement activation in AD, the extent of complement activation and its association with neuropathology in these models must be understood.Complement inflammation is a major inflammatory mechanism whose function is to promote the removal of microorganisms and the processing of immune complexes. Numerous studies have provided evidence for an increase in this process in areas of pathology in the Alzheimer's disease (AD) brain. Because complement activation proteins have been demonstrated  A variety of inflammatory processes are increased in regions of pathology in the Alzheimer's disease (AD) brain -4. Therein vitro protection against excitotoxicity [The significance of complement activation, a major inflammatory mechanism, in AD is particularly problematic. The complement system is composed of more than 30 plasma and membrane-associated proteins which function as an inflammatory cascade. Complement activation promotes the removal of microorganisms and the processing of immune complexes. The liver is the main source of these proteins in peripheral blood, but they are also synthesized in other organs including the brain . Proteintoxicity ,52 and Atoxicity , as welltoxicity ,55. Furttoxicity , althougtoxicity . Understtoxicity , and Mortoxicity ). Condittoxicity , organ ttoxicity , glomerutoxicity , ischemitoxicity , and hertoxicity . ModulatWhile animal models of human disease generally have similar pathological findings to the human disorders, distinct differences remain. These models may be appropriate for studying some aspects of a disease process, while less suitable for others. To determine the significance of complement activation in the development of AD-type pathology, for example, some animal models may be of value primarily for investigating the relationship between early complement activation and SP and NFT formation, whereas others may be more relevant for studying the role of the MAC in neuronal loss.Investigators at the Academic Hospital Free University in Amsterdam first reported the presence of early activation proteins in the classical complement cascade in the AD brain -34,36,37in vitro studies indicating that Aβ and tau protein, the major components in SPs and NFTs, can fully activate human complement [Complement proteins are detectable on or closely associated with SPs, NFTs, and dystrophic neurites in the AD brain. These findings are in agreement with mplement ,68-71. Amplement . In contmplement ,73,74. Cmplement . The aboHow the increased complement activation in AD relates to the development of SPs and NFTs, and to neuronal loss, is unclear. Immunocytochemical staining for complement activation proteins in the aged normal human brain is generally faint, and may be below the level of detection ,69,73; oBoth mRNA expression and protein synthesis of native complement proteins are increased in the AD brain -80.  in transgenic mice expressing mutations in the human amyloid precursor protein (hAPP) gene. The APP mutations expressed in these mice have been associated with early-onset, familial AD . The TGF for C3a , and the for C3a  reported for C3a  found on for C3a  and many for C3a , and (b) for C3a .In addition to APP, mutations in the gene encoding for presenilin-1 (PS-1) have also been associated with familial AD . The PS/- and APP/PS1/C1q- animals, lack of C1q did not alter either plaque density or the time course of plaque deposition. Neuronal cell numbers (NeuN+ cells), assessed only in the Tg2576 (APP) mouse, were not changed by the absence of C1q; however, immunoreactivity to MAP-2  and synaptophysin  in the hippocampus (region CA3) was increased 2-fold in the APP/C1q- animals, compared with APP mice. Microglial and astrocytic activation was significantly reduced in the APP/C1q- animals. These results were interpreted to suggest that in these animal models of AD, (1) early complement activation (as indicated by C1q deposition) in response to fibrillar Aβ deposition might be responsible for the chemotactic attraction of activated glial cells, and (2) the activated microglia, while unable to clear fibrillar Aβ, may have contributed to the loss of neuronal integrity indicated by reduced MAP-2 and synaptophysin staining in the APP mice. By recruiting activated microglia, complement activation could potentially contribute to neuronal injury even if full activation (MAC formation) does not occur.Fonseca et al.  investigCoimbra and colleagues  describeAlterations in native complement mRNA and protein levels have been evaluated in the rat hippocampus following experimental induction of acute neuronal injury. These surgical and pharmacological procedures result in neuronal loss in the entorhinal cortex, and deafferentation of hippocampal neurons, similar to that which occurs in AD . Selectiin vitro study of Webster et al. [Frautschy et al.  examinedr et al. , in whicr et al.  in whichswe mice (Tg2576) with mice expressing the transgene for a human tau mutation (JNPL3 mice) to generate a double mutant tau/APP mouse (the "TAPP mouse"). These mice develop SPs similar to APP mice , and older TAPP mice have NFTs, in association with increased astrocyte proliferation, in limbic areas. The plaques contain both Aβ40 and Aβ42. Oddo et al. [42) detected in the frontal cortex by 6 months of age, and in other cortical regions and hippocampus by 12 months. Many of the extracellular Aβ deposits are thioflavin-S-positive and are associated with reactive astrocytes. Phosphorylated tau initially appears in the hippocampus and subsequently in cortical regions; it is detected within neurons by 12–15 months and within dystrophic neurites at 18 months. Though Aβ immunoreactivity precedes that of tau, these proteins co-localize to the same neurons. The presence of NFTs as well as SPs suggests that the 3xTg-AD and TAPP models may be more relevant than APP or APP/PS-1 mice for studying the significance of complement activation in the development of AD-type pathology. Potential drawbacks for using these models for complement-related studies include, as discussed earlier, functional deficiencies in activation of mouse complement [Mutations in the gene encoding for human tau protein have been linked to the development of frontotemporal dementia with parkinsonism . By combo et al.  injectedmplement , decreasmplement , and themplement . It is nRuberti et al.  developeC. pneumoniae is an intracellular, gram-negative or gram-variable bacterium long identified as a respiratory pathogen. It has more recently been demonstrated to be a causative agent in reactive arthritis [C. pneumoniae infection in BALB/c mice could produce AD-like pathology. Intranasal inoculation with C. pneumoniae resulted in deposition of Aβ1–42 in the hippocampus, amygdala, entorhinal cortex, perirhinal cortex, and thalamus by 3 months post-inoculation. The majority of these Aβ deposits appeared similar to diffuse plaques, though a small number of them were thioflavin-S-reactive. NFTs were not detected. The authors suggested that soluble factors such as lipopolysaccharides, which are present in the cell wall of all Chlamydiae [C. pneumoniae-infected mouse may offer a novel infectious model for studying the relationship of complement activation to the development of Aβ-containing plaques.rthritis  and to brthritis  and atherthritis . Some larthritis -127, altrthritis -131. A rrthritis  examinedlamydiae , may havlamydiae ,135, the42 is the predominant type of Aβ deposited in plaques [Old dogs, in particular the beagle, have been extensively investigated as a model for CNS Aβ deposition and associated age-related cognitive dysfunction. Aβ deposits are detectable in the brains of most older dogs . The reg plaques . Canine  plaques ,139. Age plaques ,141. The40 [Age-related formation of SPs has been reported in a variety of non-human primates including the cynomolgus monkey , rhesus 40 . NFTs ap40 ,148, and40 -151. In 42-containing plaques, and NFTs are not detected [Scattered reports of AD-type pathology in other species have also appeared. Adding trace amounts of copper to the water supply of cholesterol-fed rabbits results in Aβ deposition within SP-like structures in the hippocampus and temporal cortex, with associated learning deficits . The neudetected . A repordetected  describedetected . While t1. Complement activation has been extensively studied in the AD brain. There is convincing evidence for activation of both the classical and alternative pathways, resulting in full activation as indicated by the presence of the MAC. Both aggregated Aβ (in SPs) and phosphorylated tau (in NFTs) are likely to be responsible for this activation.2. Because complement activation generates both both neuroprotective and neurotoxic effects, the significance of increased complement activation in the development and progression of AD is unclear.3. An optimal animal model for studying the significance of complement activation in the development of AD-type pathology would have complete activation of this process, with co-localization of complement activation proteins with SPs and with NFTs (if present). Other desirable features include early complement activation prior to the development of extensive neuropathology, increased CNS production of native complement proteins, and both classical and alternative pathway activation.4. Surprisingly little is known about the extent of complement activation in animal models of AD. The postischemic hyperthermic rat  is the o5. The extent of complement activation and its association with neuropathology must be determined in animal models of AD to clarify the relevance of these models for investigating the significance of complement activation in the development of AD-type pathology.Aβ, amyloid beta; AD, Alzheimer's disease; APP, amyloid precursor protein; CNS, central nervous system; MAC, membrane attack complex; mRNA, messenger ribonucleic acid; NFTs, neurofibrillary tangles; NGF, nerve growth factor; PS-1, presenilin-1; sCrry, soluble complement receptor-related protein y; SPs, senile plaque; TGF-β1, transforming growth factor beta1.The author declares that he has no competing interests.


The aim of this study was firstly, to improve and validate a cerebrospinal fluid (CSF) prefractionation method followed by two-dimensional electrophoresis (2-DE) and secondly, using this strategy to investigate differences between the CSF proteome of frontotemporal dementia (FTD) patients and controls. From each subject three ml of CSF was prefractionated using liquid phase isoelectric focusing prior to 2-DE.With respect to protein recovery and purification potential, ethanol precipitation of the prefractionated CSF sample was found superior, after testing several sample preparation methods.The reproducibility of prefractionated CSF analyzed on 2-D gels was comparable to direct 2-DE analysis of CSF. The protein spots on the prefractionated 2-D gels had an increased intensity, indicating a higher protein concentration, compared to direct 2-D gels. Prefractionated 2-DE analysis of FTD and control CSF showed that 26 protein spots were changed at least two fold. Using mass spectrometry, 13 of these protein spots were identified, including retinol-binding protein, Zn-α-2-glycoprotein, proapolipoproteinA1, β-2-microglobulin, transthyretin, albumin and alloalbumin.The results suggest that the prefractionated 2-DE method can be useful for enrichment of CSF proteins and may provide a new tool to investigate the pathology of neurodegenerative diseases. This study confirmed reduced levels of retinol-binding protein and revealed some new biomarker candidates for FTD. Frontotemporal dementia (FTD) accounts for up to 20% of presenil dementia cases  and is, The diagnosis of FTD is often difficult and would be greatly enhanced by the use of disease specific neurochemical markers . SeveralSome abundant proteins, for example albumin and immunoglobulins, limit the total amount of CSF proteins that can be loaded on the 2-D gel, resulting in difficulties detecting low abundant proteins of CSF. By using liquid phase isoelectric focusing (LP-IEF) as a prefractionation step prior to 2-DE we have previously shown that less abundant CSF proteins can be enriched, thus making them more easily detected and identified by mass spectrometry (MS) . The advThe aim of the present study was to improve the prefractionation procedure for individual CSF samples and to determine its reproducibility. Moreover, the second aim of this study was to apply the method and further explore disease-influenced proteins in CSF from FTD patients compared to controls.Several strategies to reduce impurities, i.e. salt and ampholytes, of the prefractionated CSF samples were performed including, trichloroacetic acid (TCA)-acetone precipitation, ethanol precipitation, chloroform/methanol/water precipitation and, micro Bio-Spin desalting column. The protein pattern on the 2-D gels and protein recovery from the different sample preparation methods were compared to that of acetone precipitation, previously used prior to prefractionated 2-DE of CSF . We founBoth TCA-acetone and chloroform/methanol/water precipitation substantially reduced the number of spots and resulted in streaky 2-D gels (data not shown). The use of biospin columns gave well-focused 2-D gels but several protein spots were reduced or lost (data not shown).Four identical CSF samples were individually prefractionated by LP-IEF. Fractions 6–9 (having a pH of 4.5–6.0) were pooled and analyzed on pH 4–7 strips in four replicates. Coefficients of variation (CVs) were calculated for the protein spots quantities, determined by the PD-Quest software, and a selection of 20 spots had CVs ranging from 1–33%  Figure . The spoComparison of direct and prefractionated 2-DE showed that the prefractionated gels contained more spots with a higher protein concentration Figure . The spoThe prefractionated 2-DE method was used to screen for changes in the CSF proteome of five FTD patients compared to that of five non-dementia controls in the pH intervals 3–6, 4–7, and 5–8 Figure .Comparing the protein densities of the gels, 10 spots were up regulated and 16 spots down regulated, at least two fold, in FTD patients compared to non-dementia controls. Increased proteins are marked with a square and decreased with a circle Figure . ThirteeIn this study we present an improved method for increased detection of CSF proteins by a combination of LP-IEF and 2-DE, followed by SYPRO Ruby protein staining and protein identification by mass spectrometry, for investigation of protein differences in CSF of FTD patients compared to controls. To our knowledge no other prefractionation method combined with 2-DE has so far been developed and evaluated for CSF proteins.The study showed that the reproducibility of prefractionated 2-D gels could be compared to that of direct 2-D gels, indicating that the extra prefractionation step did not introduce additional variation and could be reproduced from sample to sample. The protein detection and quantitative reproducibility of Coomassie Brilliant Blue , silver,In addition to the high salt concentration of CSF (> 150 mmol/L), ampholytes are also introduced into the sample in the prefractionation step, LP-IEF. We previously reported that the focusing time in the first dimension of prefractionated 2-DE had to be increased  probablyWhen comparing direct and prefractionated 2-DE it is evident that the prefractionated gels contain more spots, with higher protein quantities. Thus, the CSF proteins are enriched in the prefractionation step, simplifying their identification by MS, as shown in our previous study . CSF anaIn order to widen our search for protein differences in the CSF of FTD patients the improved prefractionated 2-DE procedure was applied to CSF from five FTD patients and five control subjects. 26 protein spots were changed at least two fold and 13 of these protein spots, representing seven different proteins, were identified as ZAG, ProapoA1, β-2-m, TTR, RBP, serum albumin and alloalbumin. Our previous direct 2-DE study  of the FA reduced level of RBP was consistent between our two studies, and in this case RBP was found reduced in both the pH 4–7 and the pH 5–8 gels. In contrast, CSF analysis of AD showed increased levels of one isoform of RBP , indicatBrain TTR is exclusively produced, secreted and regulated by the choroid plexus -34. TTR One isoform of β-2-m was found increased in this study. Other studies have shown that CSF β-2-m is elevated in patients with various neurological diseases including AD , infectiThe function of ZAG is unknown but studies have shown that it is present in several body fluids, including CSF, sweat, saliva, seminal fluid, plasma, milk, amniotic fluid and urine, suggesting a fairly widespread exocrine function of the protein . In thisThe level of ProapoA1 was also increased in this study. Our previous study of the FTD proteome  did not The reason that several protein changes were inconsistent between this and our previous study of the FTD proteome may be explained by the fact that a smaller sample size and a different population of FTD patients, which is a rather heterogeneous disease, were used in this study. Due to the small sample size it must also be emphasized that the protein changes found in this study are preliminary. Moreover, direct and prefractionated 2-DE are still two different proteomic approaches and a somewhat different analytical window was not unexpected. Indeed, direct and prefractionated 2-D gels show different protein patterns, for example, the apolipoprotein E and apolipoprotein J isoforms seem to be missing in the prefractionated 2-D gels, which may be explained by the fact that lipoproteins tend to adhere to plastics  and coulAs shown in the present study, the levels of specific isoforms are altered and these are unlikely to be detected using methods measuring the total concentration of a protein. Therefore, determination of posttranslational modifications is of importance for understanding the neuropathology in FTD, and 2-DE is a useful method for sensitive detection of different protein isoforms.We have shown that the prefractionated 2-DE method is reproducible to the same extent as traditional 2-DE and can enrich CSF proteins in the gel. This approach may offer new perspectives on the pathology of neurodegenerative diseases. Prefractionated 2-DE analysis of FTD CSF proteins confirmed some of the proteins previously detected by direct 2-DE and revealed some new biomarker candidates. The protein changes should be further validated on a larger patient material, preferably also with complementary methods, in order to assess any of the proteins potential as biomarkers for FTD.CSF samples were obtained from the Clinical Neurochemical Laboratory . All CSF samples had a normal white-cell count, normal blood-brain barrier function and absence of intrathecal IgG and IgM production. The CSF samples for the proteomic study, described in table The study was approved by the Ethical Committee of Göteborg University. All participants or their relatives gave their informed consent to participation in the study, which was performed in accordance with the Declaration of Helsinki.To find a method for effective reduction of impurities and maximal protein recovery, 300 μL of prefractionated pooled CSF samples  were purified using each of the following methods:Ice cold acetone precipitation; acetone: sample  precipitated at -20°C for 2 hours.1. Ice cold acetone-TCA precipitation; 2a) acetone: TCA: sample  at -20°C for 45 min. 2b) acetone: TCA: sample  at -20°C for 45 min. The protein pellet was washed 2 times with acetone after centrifugation.2. Chloroform/methanol/water precipitation, chloroform: methanol: sample  at room temperature for 2 hours.3. Ice cold ethanol precipitation; final concentrations of 60%, 70% and 80% ethanol was added to the sample and precipitated for 2 hours at -20°C.4. Purification using micro Bio-Spin column  with a MW cut off of 6 kDa. The purification procedure w two-dimensional electrophoresis (2-DE) as performed according to the manufacturer's instructions.5. After precipitation all samples were centrifuged and the protein pellet analyzed on 2-D gels, described below.The protein recovery of acetone and ethanol treated samples was measured using the RC DC protein assay (Bio-Rad) according to the manufacturer's instructions.The CSF samples from individual patients were prefractionated using LP-IEF in the Rotofor cell (Bio-Rad). Three mL CSF sample was mixed with 9 mL millipore water, 1% ampholytes , 20 mM dithiothreitol (DTT) and 1 × Complete antiprotease solution . The focusing was performed at 4°C and at 12 W constant power for 2.5 hours. Then the 20 Rotofor fractions were harvested and fraction 2–5 corresponding to pH 1.5–4.5, fraction 6–9 corresponding to pH 4.5–6.0 and fraction 10–14 corresponding to pH 6.0–7.5 were pooled and concentrated to 300 μL in a vacuum centrifuge prior to 2-DE.In the FTD-study, the prefractionated pooled protein fractions were precipitated using 900 μL 95 % ice-cold ethanol  for two hours at -20°C. The mixture was centrifuged at 10,000 × g for 10 min at 4°C. The protein pellets were air-dried and then resolved in a buffer containing 9 M urea, 35 mM tris, 42 mM DTT, 2% 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS), 0.66% sodium dodecyl sulfate (SDS), 2% IPG buffer and bromophenol blue.The first dimension was carried out using immobilized pH gradient (IPG) strips (Bio-Rad), 7 cm, pH 3–6 for Rotofor fractions 2–5, pH 4–7 for Rotofor fractions 6–9 or pH 5–8 for Rotofor fractions 10–14. The IPG-strips were actively rehydrated in the CSF-protein sample for 12 h at 50 V followed by protein focusing for 20,000 Vh using the Protean IEF Cell (Bio-Rad). The IPG strips were placed in 5 ml equilibration solution  containing 1% DTT, and 2.5% iodoacetamide in the second equilibrium step for 2 × 15 min.The second dimension was performed using the Nu-PAGE gel system  with (2-(N-morpholino) ethane sulfonic acid (MES) buffer: 50 mM MES, 50 mM tris, 3.5 mM SDS, 1 mM EDTA), for 35 min at 200 V.In the direct 2-DE procedure, 300 μL CSF proteins were precipitated using 900 μL ice-cold acetone and stored for two hours at -20°C. The mixture was then centrifuged at 10,000 × g for 10 min at 4°C. The 2-DE procedure was performed as described above for prefractionated 2-DE.The gels were stained using SYPRO Ruby Protein Stain  according to the manufacturer's instructions. Image acquisition and analysis were performed on a Fluor-S MultiImager (Bio-Rad). The protein spots were detected, quantified and matched with the PD-Quest 2-D gel analysis software, v.7.0 (Bio-Rad). The gels were normalized according to the total quantity in valid spots . Protein levels increased or decreased two fold were taken into account.The protein digestion method has been previously described in detail . BrieflyMatrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis was performed using an upgraded Reflex II MALDI-TOF MS  equipped with a two-stage electrostatic reflectron, a delayed extraction (time-lag-focusing) ion source, a high resolution reflector detector and a 2 GHz digitizer. The spectra were acquired in the reflection mode at an accelerating voltage of 20 kV. The mass spectra, acquired and analyzed using Bruker software, were initially calibrated by external calibration using a mixture of known peptides and later recalibrated using two auto digestion products of porcine trypsin as internal calibrants. The protein database search tool "MASCOT Peptide Mass Fingerprint" on the Matrix Science web site  was usedCoefficient of variation (CV) was calculated (standard deviation (SD)/ Mean × 100) of the normalized protein spot densities from four replicate 2-D gels.In the proteomic study a 2-fold increase or decrease of normalized protein quantities was taken into account.The authors declare that they have no competing interests.SFH carried out all the 2D gel experiments, mass spectrometry analysis, participated in results evaluation and drafted the manuscript.MP participated in the 2D gel experiments, results evaluation and participated to the manuscript writing.KB contributed with material and critical reading of the manuscript.MJ contributed with material and participated in the design of the study.PD conceived the study, participated in its design and coordination, results analysis and supervised the manuscript writing.


In recent years, outcome prediction models using artificial neural network and multivariable logistic regression analysis have been developed in many areas of health care research. Both these methods have advantages and disadvantages. In this study we have compared the performance of artificial neural network and multivariable logistic regression models, in prediction of outcomes in head trauma and studied the reproducibility of the findings.1000 Logistic regression and ANN models based on initial clinical data related to the GCS, tracheal intubation status, age, systolic blood pressure, respiratory rate, pulse rate, injury severity score and the outcome of 1271 mainly head injured patients were compared in this study. For each of one thousand pairs of ANN and logistic models, the area under the receiver operating characteristic (ROC) curves, Hosmer-Lemeshow (HL) statistics and accuracy rate were calculated and compared using paired T-tests.ANN significantly outperformed logistic models in both fields of discrimination and calibration but under performed in accuracy. In 77.8% of cases the area under the ROC curves and in 56.4% of cases the HL statistics for the neural network model were superior to that for the logistic model. In 68% of cases the accuracy of the logistic model was superior to the neural network model.ANN significantly outperformed the logistic models in both fields of discrimination and calibration but lagged behind in accuracy. This study clearly showed that any single comparison between these two models might not reliably represent the true end results. External validation of the designed models, using larger databases with different rates of outcomes is necessary to get an accurate measure of performance outside the development population. In recent years, outcome prediction studies have become the avante garde in many areas of health care research, especially in critical care and trauma. However acceptable models for outcome prediction have been difficult to develop . AccordiArtificial neural networks (ANNs) are mathematical constructs modeled on interconnection of nodes (neurons) giving a loose association with the animal nervous system. ANNs employ nonlinear mathematical models to mimic the human brain's own problem-solving process. Just as humans apply knowledge gained from past experience to new problems or situations, a neural network takes previously solved examples to build a system of "neurons" that makes new decisions, classifications, and forecasts. ANNs are complex and flexible nonlinear systems with properties not found in other modeling systems. These properties include robust performance in dealing with noisy or incomplete input patterns, high fault tolerance, and the ability to generalize from the input data . Neural Lang EW et al have compared ANN with Logistic Regression in prediction of outcome after severe head injury and concluded that the differences in the results obtained with the two models were negligible .Almost all of the published articles indicate that the performance of ANN models and logistic regression models have been compared only once in a dataset and the essential issue of internal validity (reproducibility) of the models has not been addressed.The objective of this study was to compare the performance of ANN and multivariate logistic regression models for prediction of mortality in head trauma based on initial clinical data and whether these models are reproducible. We used different variables even if they were interdependent.th revision (ICD-10) as well as the outcome of the patients. Data collection was conducted by a group of trained physicians who had completed special training courses to become familiar with the process of extracting Abbreviated Injury Score (AIS-90) codes and filling out the relevant questionnaires. For quality control (QC) purposes each hospital had a physician, who was responsible for overseeing the data gathering process. In the intubated patients GCS were calculated according to the other portions of the scale by this physician. Finally, a medical practitioner examined all the checklists in order to evaluate and amend them if deemed necessary based on pre-arranged and fixed protocols.Among 8452 trauma patients' records admitted to the emergency departments of six major university hospitals in Tehran from 23 August 1999 to 22 September 2000, the records of 1271 patients whose main trauma was head injury, were selected for this study. The selection of head trauma as the main trauma was based on the definition of principal diagnosis in the Uniform Hospital Discharge Data Set (UHDDS). It defines the principal diagnosis as "that condition established after study to be chiefly responsible for occasioning the admission of the patient to the hospital for care". For making determination of the main trauma more practical in the case of ambiguity, hospitalization in the neurosurgical ward was used as an additional guideline. The database was based upon the trauma data registry program began in 1996 in Trauma Research Center, Sina Hospital, a hospital affiliated with the Tehran University of Medical Sciences ), clinicSince we were trying to build and compare models for prediction of outcome mainly based on the initial clinical data, only data related to the GCS, tracheal intubation status, age, systolic blood pressure (SBP), respiratory rate(RR), pulse rate(PR), injury severity score (ISS)(upon admission) and outcome were used in our study. In order to prepare the data for the Neural Network software and to enhance the reliability of the data, three variables of systolic blood pressure, respiratory rate and pulse rate were transformed to dichotomous variables. Low systolic blood pressure was defined according to the following cutoff points: up to 5 years of age, less than 80 mmHg; and 5 years of age or older, less than 90 mmHg. Respiratory rate of 35 per min and pulse rate of 90 per min were selected as limits for definition of tachypnea and tachycardia. Other variables including GCS, age, systolic blood pressure (SBP) and injury severity score (ISS) variables were also converted from decimal (Base 10) to binary (Base 2). This conversion was carried out in order to render the input data suitable for processing by our ANN software with its default settings. The data and the data format were similar for both ANN and logistic regression models.The dataset was divided randomly into two sets, one set of 839 cases (66% of the whole dataset) for training and 432 cases for testing the model. A model was built using a training set with logistic regression. GCS, tracheal intubation status(dichotomous), age, SBP(dichotomous), RR(dichotomous), PR(dichotomous) and ISS were the independent variables and the outcome  was the dependent variable. The logistic regression analyses were performed using Intercooled STATA for windows, Version 6  "logistic" default options.The built logistic model was tested using the testing dataset (432 cases). These steps  were repeated 1000 times. This resulted in 1000 pairs of training and testing datasets  which were saved for further processing by the neural networking.The ANN used in this study was a standard feed-forward, back-propagation neural network with three layers: an input layer, a hidden layer and an output layer. The input layer consisted of 23 input neurons, the hidden layer consisted of fifteen hidden neurons, and the output layer consisted of one output neuron Fig. . The leaThe training and testing datasets were the same as those were used with regression models, thus there was an ANN and a logistic model for each training and testing dataset..PDP++ version 3.1 was used for the artificial neural network analysis as it has a powerful built-in scripting language and is freely available. This software can be downloaded from Discrimination and calibration (goodness of fit) were both measured. Discrimination refers to the ability of a model to distinguish those who die from those who survive. A perfectly discriminating model would assign a higher probability of death to all cases that died than to any case that survived. The discriminatory power of the models was analyzed using the area under the receiver operating characteristic curves (ROC-A(z)). ROC curves were constructed by plotting true positives (patients who died and whom the model predicted as dying [i.e. sensitivity]) versus the false positive fraction (fraction of the patients who lived and were incorrectly classified as dying [i.e. (1 – specificity)]).A ROC-A(z) value of one corresponds to a test that perfectly separates two populations, whereas a ROC-A(z) value of 0.5 corresponds to a perfectly useless test that performs no better than chance.The relative calibration of the models, that is, how accurately the models predicted over the entire range of severity, was compared using Hosmer-Lemeshow (HL) statistics. The HL statistics is a single summary measure of the calibration and is based on comparing the observed and estimated mortality for patients grouped by estimated mortality. The resulting statistic follows a chi-squared distribution, with degrees of freedom equal to two less than the number of groups (10 in this study). The smaller the HL statistics, the better the fit, with a perfectly calibrated model having a value of zero.A probability cut point of 0.5 was used to classify observations as events or nonevents. The overall accuracy  of the final model was determined by comparing the predicted values with the actual events.For each of one thousand pairs of ANN and logistic models (trained and tested on the same datasets), these indices  were calculated and compared using paired T-tests (P < 0.05).All the statistical analyses were performed using Intercooled STATA for windows, version 6 STATA Corp., College Station, TX) and its downloadable add-on ado files.. Further details are available from the corresponding author.Some of the scripts used both for the STATA and PDP++ and the designed neural networks can be downloaded from the site of the Tehran University of Medical Sciences Table As is seen in Table The confidence intervals of the all the measures show higher variation in the ANN models results.Prognostic evaluation of the patients without CT scan findings may have limited applicability considering the availability of CT scans in the majority of the trauma centers. Nevertheless, the idea of prognostic models based on initial clinical data at admission is still worth trying and seems to be of practical value in some situations. Although omission of CT data weakens our armamentarium significantly, patients without paraclinical and imaging data make the real trauma scenes at which medical staff should have an evaluation. Actually as neurosurgeons we have initial clinical judgments based on our growing experiences and in many situations they prove to be true. We can expect computers do similar things and help us. In fact, vague situations like what we see in primary evaluation of head trauma patients are where ANN may prove to be superior to traditional linear modeling. This is one of the reasons that although many paraclinical and imaging factors are known to be of significant predictive value in the outcome of the head trauma patients, this study only used clinical measures which are simply available to a physician in the emergency department.Calculating ISS and AIS are not simple tasks and needs training. This can reduce the practicability of the results. The pupillary size and reactivity are one of the clinical signs with prognostic value that were not considered in the study due to defect of the main database in this regard.As is seen the mortality rate and the percentage of patients with GCS < 8 are the same (7.5%). This is a coincidence, but emphasizes the need for further studies in the populations with different rates of outcome.The mean GCS was 13.5 ± 3. The standard deviation is over the top score of GCS (15) and means that the GCSs were skewed towards higher levels of consciousness.Not considering the exact time interval between head trauma and admission which is simply available for the medical staff is one of the weak points of this study.) somewhat difficult in other situations where pre-hospital care services are more advanced.The lower intubation rate in this population study related to the distribution of the GCS may show the lower quality of the pre-hospital care prevailing in the hospitals selected for this study. This may render the reproduction of the results using the same network , and seem to promise "prediction" without the difficulties associated with use of mathematics.ANNs are rich and flexible nonlinear systems that show robust performance in dealing with noisy or incomplete data and have the ability to generalize from the input data. They may be better suited than other modeling systems to predict outcomes when the relationships between the variables are complex, multidimensional, and nonlinear as found in complex biological systems.The difficulty in developing models using artificial neural networks is that there are no set methods for constructing the architecture of the network. The most common type of artificial neural networks is the feed-forward back propagation multiperceptron (used in this study).Another limitation of neural network models is that standardized coefficients and odds ratios corresponding to each variable cannot be easily calculated and presented as they are in regression models. Neural network analysis generates weights, which are difficult to interpret as they are affected by the program used to generate them . This laSeveral early applications of neural networks in medicine reported an excellent fit of the ANN model to a given set of data. The impressive results usually were derived from over fitted models, where too many free parameters were allowed. Linear and logistic regression models have less potential for overfitting primarily because the range of functions they can model is limited.Neural network models require sophisticated software. The complexity and unfamiliarity of ANN has been a major drawback of this technique so far. However, as palmtop computing becomes increasingly powerful and popular, the complexity of ANNs may become less onerous in real-time clinical settings. Furthermore, there are some theoretical advantages comparing a predictive ANN model over conventional models such as logistic regression.One such advantage is that ANN model allows the inclusion of a large number of variables . AnotherAlthough, one of the strengths of ANNs is their ability to still find patterns despite missing data, in this study a dataset with no related missing values was used.Recently the task of comparison between these two models has been addressed from different points of view. Considering the publication bias, several published works in the medical literature have demonstrated the success of the ANN approaches. In a review carried out by Sargent on 28 major studies, ANN outperformed regression in 10 cases (36%), was outperformed by regression in 4 cases (14%) and the 2 methods had similar performance in the remaining cases. Sargent concluded that both methods should continue to be used and explored in a complementary manner. Gaudart et al. using simulated data, have compared the performance of ANN and linear regression models for epidemiological data and concluded that both had comparable performance and robustness and despite the flexibility of connectionist models (like ANN), their predictions were stable. This study was primarily designed to compare the performance of an ANN and a multivariable logistic regression analysis with the goal of developing a model for predicting the outcome in head injury and for studying their internal validity (reproducibility). Also setting up a standalone practical model for prediction of mortality in the head trauma patients was a secondary goal of this study.Using freely downloadable software (PDP++) and making the networks and scripts accessible by the researchers can be perceived as an advantage of this study.This study showed that ANN models significantly outperformed logistic models in both senses of discrimination and calibration, although lagged behind in accuracy. It is pointed out that the calibration values should be treated with some caution in this study, since according to Hosmer and Lemeshow,  in descrThis study clearly shows that in a single comparison of these models based on the same data there is 22.2% chance of getting discrimination results contrary to our findings in majority of comparisons. This ratio is 43.6% for calibration and 32% for the accuracy results. These figures are practically important and imply that any single comparison between these two models cannot reliably represent their final performance.Although considerable efforts, through many trial-and-errors, were made to optimize the design of the network, the designed ANN models could, and should, be further improved. In line with any other predictive models, likewise the findings of this study need to be externally validated. The networks are downloadable and the results can be studied in other study populations with divergent data and different survival ratios.So far, there is no single algorithm that performs better than all other algorithms for any set of given head injury data. To this end, there is room for much more work to be done before a definite conclusion can be reached.Should the results be reproducible in other populations, using a simple preprogrammed calculator (or other programmable computing devices) and minimal training of the personnel, this model and similar ones may emerge to be of considerable practical value in triage of the patients. At that time a dedicated instead of general purpose ANN software should be designed for this purpose.The authors concur with the conclusion arrived by Tu  that logIn conclusion, this study compared models for the prediction of outcome in head injury using trauma data from hospital registries in Tehran, the data was applied to artificial neural network and multivariable logistic regression analysis. The predictive ability of the artificial neural network model was found to be comparable to that of the logistic regression model. Specifically, the ANN models significantly outperformed logistic models in both senses of discrimination and calibration but lagged behind in accuracy. Although the performance of the models were studied when the models were applied to the different samples of the original population study, external validation is necessary to get an accurate measure of performance outside the development population. Studies using larger databases with different rates of outcomes may further clarify the differences between artificial neural network and logistic regression models in head injury outcome prediction and their clinical implications.The author(s) declare that they have no competing interests.BE carried out the data extraction, performed the analysis and drafted the manuscript. KM supervised the analysis and critically reviewed the statistical viewpoints. HEA, MG and EK supervised the study and participated in its coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


There is limited population-based data on diet and physical activity behaviors and weight loss among users of prescription weight loss medications. Most findings are from clinical settings or from research that includes organized behavioral programs.We analyzed data from the 1998 Behavioral Risk Factor Surveillance System, an annual telephone survey conducted in all fifty states, the District of Columbia and Puerto Rico. The sample consisted of 135,435 noninstitutionalized adults aged 18 years old and older. We determined the prevalence and odds of prescription weight loss medication use, odds of 10% weight loss, and among current weight loss medication users, the prevalence and odds for diet and physical activity behaviors.10.2% of obese women and 3.1% of obese men reported using prescription weight loss medications in the past 2 years. Of users, 28.2% had lost at least 10% of their pretreatment body weight. The odds of losing at least this much weight were higher among women, those who usually consumed ≥ 5 fruits and vegetables daily and those who met physical activity recommendations. Among current prescription weight loss medication users, 26.7% reported both eating fewer calories and meeting recommended leisure-time physical activity levels (<40% of any group met both). Of those meeting both recommendations, almost half (47.2%) had lost 10% of their pretreatment body weight. Of current users, 9% reported using the medications for weight maintenance.Only 26.7% of prescription weight loss medication users reported following recommended diet and physical activity behaviors. Further research is needed to assess whether behavioral changes are associated with greater weight loss and maintenance among prescription weight loss medication users. Lifestyle modifications, including behavior therapy, diet, and physical activity, are the cornerstone of weight management . CurrentThe initial goal of weight loss therapy is to reduce body weight by approximately 10% over a 6-month period ,4. WeighUsing population-based data we estimated that 4.6 million American adults used prescription weight loss medications between 1996 and 1998 . During We examined data from the 1998 Behavioral Risk Factor Surveillance System (BRFSS). The BRFSS is a telephone survey of health practices that is conducted each year by all state health departments. Each state, the District of Columbia, and Puerto Rico select an independent probability sample of noninstitutionalized residents aged 18 years and older. In 1998, 149,806 persons responded to the BRFSS; several investigators have published detailed descriptions of survey methods and quality control indices . The aveTo determine prescription weight loss medication use, respondents were asked, "In the past two years, have you taken any weight loss pills prescribed by a doctor? Do not include water pills or thyroid medications." Responses were coded as: 1) "Yes, I am currently taking them", 2) "Yes, I have taken them but I am not currently taking them", or 3) "No, I have not taken them".Respondents with a positive response were asked about their prepill weight, "How much did you weigh just before you started taking prescription weight loss pills for the first time?"At the end of the interview, respondents were asked to report their current height and weight without shoes. We used current weight and pretreatment weight to calculate current and pretreatment body mass index (BMI), respectively. BMI was calculated as weight (either current or pretreatment) in kilograms divided by current height in meters squared. BMI was categorized into 3 groups: normal weight (<25.0), overweight (25.0–29.9), and obese (≥ 30.0) . PercentWe used the following questions to determine weight control strategy, "Are you now trying to lose weight?" Those who responded "no" were then asked, "Are you now trying to maintain your current weight, that is to keep from gaining weight?" Additional questions on dietary practices and physical activity were asked of the subset of respondents  who responded "yes" to trying to lose or maintain their body weight. These respondents were asked "Are you eating either fewer calories or less fat to lose/maintain weight?" Their responses were either: 1) "Yes, fewer calories", 2) "Yes, less fat", 3) "Yes, fewer calories and less fat", or 4) "No". They were also asked "Are you using physical activity or exercise to lose/maintain weight? Their responses were either "yes" or "no".All individuals were also asked "In the past 12 months, has a doctor, nurse, or other health professional given you advice about your weight?" Responses were "Yes, lose weight", "Yes, gain weight", "Yes, maintain current weight", "No". In an earlier section individuals were asked, "About how long has it been since you last visited a doctor for a routine checkup? The latter question was used to distinguish those who had not received advice due to not seeing a doctor for a routine checkup in the past 12 months.For respondents who were currently using prescription weight loss medication and were trying to lose weight or maintain their current weight, we attempted to determine if they were meeting the minimal lifestyle recommendations. Respondents who stated they were eating fewer calories – either as fewer calories or as both fewer calories and less fat – were classified as meeting minimal dietary recommendations. Respondents were asked about the two physical activities or exercises they engage in most often and about the frequency, duration, and distance (as appropriate) of each activity . ResponsWe also requested demographic and socioeconomic information such as age in years, sex, race/ethnicity, marital status, educational level, and household income. Of the 149,806 respondents, we excluded those who did not report information on prescription weight loss medication use n = 1561), had a missing current weight (n = 5229) or height (n = 1369), were pregnant (n = 1780), were missing current weight goal (trying to lose weight or trying to maintain weight) (n = 749), or were missing information on sociodemographic variables, fruit and vegetable intake, or physical activity (n = 3595). We excluded an additional 88 respondents that had a questionable reported weight, height or BMI that was outside the sex-specific reference values from the Third National Health and Nutrition Examination Survey, 1989–1994 [61, had aWe used SAS and SUDAAN for the statistical analysis to account for the complex sampling design ,17. StudThe results of our multivariate analyses show that the reported use of prescription weight loss medications in the past 2 years was higher among women (4.0%) than men (0.9%). For women, the odds of use was 19% lower among those aged 35–54 years and 75% lower among those 55 years old than among those aged <35 years. The odds of prescription weight loss medication use was 34% lower among non-Hispanic black women than among non-Hispanic white women; and 53% and 39% lower among those women residing in the Northeast and Midwest regions of the United States, respectively, than among women residing in the West region , one tenth lost no weight , one-tenth lost between 1% and 5% , and one fifth lost between 5% and 9% of body weight , data not shown.Additionally among users, one-third of women (35.0%) and one quarter of men 25.0%) reported a 10% weight loss, Table 5.0% repoWe also assessed professional advice, diet, and physical activity among current prescription weight loss medication users by current weight intention Table . AlthougOnly one-quarter (26.7%) of current prescription weight loss medication users met both lifestyle recommendations – eating fewer calories and attaining recommended physical activity levels. Our results indicate that among current users, the odds of meeting both recommendations was 62% lower among men than among women and 56% lower among other race/ethnic groups than among non-Hispanic whites Table . We alsoIn a secondary analyses we assessed prevalence of a 10% weight loss among current prescription weight loss medication users who were trying to lose weight (not maintain) by whether or not recommendations were met for eating fewer calories and physical activity (n = 237). 39.6% (SE 5.3) of individuals who reported both eating fewer calories and who met physical activity recommendations had lost at least 10% of their pretreatment weight; 36.5% (SE 6.4) who reported eating fewer calories but who did not meet the physical activity recommendations had lost at least 10% of their pretreatment weight; 54.3% (SE 9.8) who reported not eating fewer calories but met physical activity recommendations had lost at least 10% of their pretreatment weight; and 25.5% (SE 5.9) who did not meet either lifestyle recommendation had lost at least 10% of their pretreatment weight, (chi-square p = 0.03).Although in 1998 clinical guidelines recommended that prescription medication for obesity be used in conjunction with lifestyle modifications [Although patterns of prescription medication use differed by sex, for both men and women the odds of use was higher among those who were obese compared to those who were overweight although at different magnitudes. Our data show than more overweight women used prescription weight loss medications (4.88%) than did obese men (3.11%). It is possible that pharmacotherapy prescribing patterns differ by sex since more women than men attempt weight loss  or that Eating fruits and vegetables and taking part in recommended levels of physical activity through leisure-time activities were more frequent behaviors among prescription weight loss medications users who had lost at least 10% of their body weight. Although we had no direct measurements of caloric intake, fruits and vegetables in their natural state are low in energy density. Thus, adding or substituting fruits and vegetables for energy-dense snacks and sweets can impact satiety and weight gain ,18,19. OAbout 40% of current prescription weight loss medications users who both reduced calories and met leisure-time physical activity recommendations had lost 10% of their body weight. Our data also show that respondents who consumed usually less than 5 fruits and vegetables per day were less likely to meet both diet and physical activity recommendations than did those who consumed 5 or more fruits and vegetables a day. Because fewer than half of physicians counsel obese patients about weight control ,22 we beThe current study had several limitations. Although the participants self-reported pre-medication body weight and current weight, we did not ask for the date prescription weight loss medication use began and it could not be established whether weight loss was solely attributable to pharmacotherapy usage. We were also unable to determine if respondents had lost a substantial amount of weight but then regained the weight (weight history). Self-reported weights and weight history may also be a concern because individuals, especially those who are overweight, may underreport their weight . We lackAlthough we found that about one fifth of participants had lost between 5% and 9% of their pretreatment body weight and one third of participants had lost at least 10% of their pretreatment body weight, the pill dosage and length of medication use were not collected. Thus, some users may have used pills for a short time, such as less than 6 months, and would not be expected to have lost 5% to 10% of their body weight.In current practice, physicians treating obesity can prescribe phentermine, sibutramine, and orlistat (available April 1999). Analysis of national data on patient visits found that phentermine was the most common antiobesity medication in 2001 and in early 2002 (based on January through March figures) , suggestMost nonsurgical obesity treatments lead to weight loss for the first 6 months followed by regain . Thus, sWe found that one in ten obese women and one in 33 obese men reported using a prescription weight loss medication in the past two years. Among medication users, one-third of women and one quarter of men reported a 10% weight loss. Only one quarter of current medication users reported the minimal diet and physical activity behaviors recommended for weight loss. Ongoing population-based surveillance is needed to assess the prevalence of prescription weight loss medication use and whether pharmacotherapy users are making appropriate lifestyle changes in order to lose or maintain weight.BRFSS: Behavioral Risk Factor Surveillance SystemBMI: body mass indexOR: odds ratio95% C.I.: 95% confidence intervalThe author(s) declare that they have no competing interests.The questions that were asked of all authors:Have you received reimbursements, fees, funding, or salary from an organization that may in any way gain or lose financially from the publication of this paper in the past five years? No.Have you held any stocks or shares in an organization that may in any way gain or lose financially from the publication of this paper? No.Do you have any other financial competing interests? No.Are there any non-financial competing interests you would like to declare in relation to this paper? No.HB performed the statistical analysis and drafted the manuscript.LKK conceived the study questions, provided statistical input, and edited the manuscript.MKS conceived the study questions, provided statistical input, and edited the manuscript.All authors read and approved the final manuscript.