The use of biomarkers in medicine lies in their ability to detect disease and support diagnostic and therapeutic decisions. New research and novel understanding of the molecular basis of the disease reveals an abundance of exciting new biomarkers who present a promise for use in the everyday clinical practice. The past fifteen years have seen the emergence of numerous clinical applications of several new molecules as biologic markers in the research field relevant to interstitial lung diseases . The scope of this review is to summarize the current state of knowledge about serum biomarkers in interstitial lung diseases and their potential value as prognostic and diagnostic tools and present some of the future perspectives and challenges. The use of biomarkers in medicine lies in their ability to detect disease and support diagnostic and therapeutic decisions. New research and novel understanding of the molecular basis of the disease reveals an abundance of exciting new biomarkers who present a promise for use in the everyday clinical practice. The initial evaluation of a serum biomarker concerns its expression in patients with the disease and in normal individuals in order to define sensitivity and specificity. The sensitivity of a test is defined as the proportion of patients with disease having a positive test whereas the specificity is the proportion of patients without the disease who have a negative or normal test Table . ConsequVery few markers present a threshold at which the risk suddenly rises. The interplay between sensitivity and specificity and the nature of the disease under prediction assigns suitable cut-off points. Sensitivity and specificity calculated at various cut-off points give rise to a receiver-operating-characteristic (ROC) curve . A cliniIn diffuse lung disease, there has been confusion for decades because of the term "disease activity" that variably means a) inflammation and responsiveness to therapy; and b) progressiveness. In diffuse lung disease, there are two separate therapeutic goals: a) a short term response as judged by improvements in pulmonary function test (PFT) and regression of imaging abnormalities as judged mainly by high resolution computed tomography (HRCT); and b) slowing or prevention of decline. It follows logically that a useful biomarker might conceivably predict either responsiveness (i.e. the inflammatory component) or the risk of progression of fibrotic disease. Therefore, the clinical utility of a biomarker is critically dependant upon whether clinicians have tools in diffuse lung disease that accurately predict: a) responsiveness and b) progression of disease. There is much less need to predict responsiveness than progressiveness. HRCT is reasonably accurate in the separation between a group of patients in which disease is clearly irreversible and a group of patients in whom responsiveness is reasonably likely . HunningAnother key benefit a biomarker might provide is more accurate prognostic information from change in that biomarker, especially if the information can be obtained rapidly (i.e. short term change with treatment). The current serial tests including serial HRCT and PFT provide scattered statements regarding the prediction of long-term outcome. Flaherty et al.  stated tOn the contrary to the listed theoretical requirements reflecting the profile of an ideal marker, a useful biomarker in the real world of clinical practice, simply needs to fulfil an unmet need. If current non-invasive and semi-invasive tools predict outcome very accurately, a biomarker will add little extra information of value. Therefore, it simply needs to provide more accurate information about progressiveness than current modalities, not independent information. Is it mandatory for a biological predictor to add independent information about prognosis? Is it necessary for a clinician to score the scan or the factor in the lung function severity mathematically, every time a biomarker is to be measured? Additionally, this information should be transferable, since the definition of interstitial lung diseases (ILDs) is not yet constant and therefore test accuracy may vary considerably from one setting to another .Regarding serial monitoring, whether or not a biomarker is highly reproducible is less crucial than clear quantification of the reproducibility, in order to distinguish between true change and measurement "noise". A marker might be poorly reproducible and yet highly accurate when it does clearly change. Furthermore, a poorly reproducible biomarker may be highly accurate when the range is categorized into for example a five – point semi quantitative scale, in order to address reproducibility issues.Moreover, there is confusion between diagnostic and prognostic requirements. A diagnostic marker needs to be sensitive and specific . HoweverIn conclusion, the clinical need for a biomarker in diffuse lung disease regarding the presence or absence of the disease, the histospecific diagnosis and the prediction of responsiveness has probably little value. On the other hand, clinical necessity becomes invaluable regarding the baseline prediction of rate of progression and the early prediction of progression based on serial change with treatment. This will allow anti-inflammatory or other treatments to be evaluated or eventually modified before they have failed.The scope of this review is based on the fact that although there are numerous published papers investigating the utility of biomarkers in the clinical research field, the number of review articles summarizing the current state of knowledge about the clinical applications of these molecules as diagnostic and prognostic tools in the research field relevant to common ILDs such as idiopathic pulmonary fibrosis (IPF), scleroderma, sarcoidosis, as well as other ILDs including radiation and drug- induced pneumonitis, pediatric ILDs and occupational and environmental diseases, still remains inadequately small.Beyond other important functions, the lung epithelium produces complex secretions, including mucus blanket, surfactant proteins, as well as several proteins important for host defense .Sampling the epithelial lining fluid by bronchoalveolar lavage (BAL) represents the common means of studying the proteins secreted by the lung epithelium and investigating their alterations in lung disorders . However• Leakage from the lung into the bloodstream resulting from the increased permeability of lung vessels and the destruction of the barrier between alveolar epithelium and endothelium caused by injury to the basement membrane• Increased production by the alveolar type II cells coupled with an increase in total type II cells per lung due to diffuse hyperplasia and• Diminished clearance rates from the circulation.In the present review article we have focused on recent publications concerning the most studied and interesting lung peripheral biologic markers in ILDs, namely: lung epithelium specific proteins , circulating cytokines estimating various types of inflammatory activity and finally enzymes and metabolites, products of epithelioid cells and derivatives of activated macrophages. Table Pulmonary surfactant is a complex and highly surface active material covering the alveolar space of the lung. Biochemically, surfactant is a molecular mixture composed mainly of structurally heterogeneous phospholipids. A major function of pulmonary surfactant is to reduce the surface tension at the air-liquid interface of the alveolus, thereby preventing alveolar collapse on expiration. It has also been demonstrated that the surfactant contains specific proteins . Four suMucins are major components of the mucus layer covering the airway epithelium. They consist of high-molecular-weight glycoproteins belonging to a broad family of mucin peptides . Mucins The Clara Cell secretory protein-(CC)-16 is a low-molecular-weight protein of 16 kDa is secreted in large amounts into the lumen of the respiratory tract by nonciliated bronchiolar Clara cells in humans and rodents . ImmunohCytokeratin is a specific cytoskeletal structure expressed in epithelial cells, including bronchial epithelia ,29. Of tThe cancer-associated antigens sialyl Lewis (a) (Ca 19-9) and sialyl Lewis (x) (SLX) are carbohydrate structures used as markers of cell differentiation and embryonic development . It has A number of cytokines probing different aspects of the immunopathogenesis of ILDs have been tested for their clinical usefulness as serum biomarkers for monitoring disease severity and predicting response to treatment and therefore leading to early diagnosis of progressive disease and determination of therapeutic interventions.Monocyte chemoattractant protein-(MCP)-1 and monocyte inflammatory protein- (MIP)-1a belong to the C-C subfamily of the chemokine family and appear to be important factors in the monocyte/macrophage-mediated inflammatory process in the ILDs ,40. It hIFN-inducible T cell-a chemoattractant-ITAC/CXCL-11 is a chemoattractant CXC chemokine with versatile properties including immunomodulatory, definsin-like antimicrobial, antiangiogenic and potentially antifibrotic activities. It has been reported to inhibit angiogenesis and thereby to reduce aberrant vascular remodeling resulting in diminished fibrosis ,44. StudIn addition, the activity and release of a large body of inflammatory mediators including tumor necrosis factor (TNF), antioxidant enzymes (glutathione), procollagen peptides (type III) and markers of cell damage such as lactate dehydrogenase (LDH) have been evaluated as prognostic and monitoring tools of the disease development, activity and progression in a variety of occupational and environmental studies.Other delineated serological parameters that have been scrutinized for their clinical efficacy as serum biologic markers of the disease severity and the activation of several inflammatory cells contributing to the immunopathogenesis of the disease include markers of T-cell activation and markers for the evaluation of macrophage/monocyte activity.Soluble IL-2 receptor (sIL-2R) represents a biomarker of the T-cell activation and can be found in BAL fluid and serum of sarcoidosis patients and it is released by activated alveolar immune cells. In addition to lymphocytes, activated sarcoid alveolar macrophages are capable of expressing increased numbers of sIL-2R upon activation . There aParameters suitable for gauging the activity of the macrophage lineage in ILDs and specifically sarcoidosis and occupational diseases have been delineated and comprise the angiotensin converting enzyme (ACE) a product of epithelioid cells that reflect the granuloma burden of the entire body, neopterin a metabolite of the guanosinetriphosphate pathway that is released by activated macrophages and monocytes under the control of IFN-γ produced by T-cells  and b-glIdiopathic pulmonary fibrosis (IPF) is a refractory and lethal ILD characterized by fibroblast proliferation, extracellular matrix (ECM) deposition and progressive lung scarring. The incidence of IPF is estimated at 15–40 cases per 100.000 per year, and the mean survival from the time of diagnosis is 3–5 yr regardless of treatment . On the One of the pioneering studies that set the foundations for the development of a new research field with massive clinical implications was published ten years ago by Honda et al . AuthorsMoreover, KL-6 serological levels have been tested by Kobayashi et al.  whether The first and to best of our knowledge the only so far comparative study testing the sensitivity and specificity of three groups of molecules reported to serve as sensitive markers for ILDs was conducted by Ohnishi et al . They geTowards this direction Takahashi et al. ,56 were In the first study , resultsFurthermore, Takahashi et al.  estimateSeveral biologic markers have been studied the past few years not only to evaluate the disease activity and for differential diagnosis of ILDs but also for prediction of the disease outcome and the effectiveness of the commonly applied treatment during the accelerated phase of pulmonary fibrosis.One of the most detailed and informative study in this field was published by Yokoyama et al . ScrutinCurrently, the hurdle faced by young physicians that limits the application of blood biomarkers in the daily clinical practice of IPF patients is whether alterations of their circulating concentrations are specific for IPF or they also reflect other lung parenchyma abnormalities seen in different ILDs. Several studies have tested the efficacy of blood biomarkers in differentiating IPF from other ILDs. Some of them are reviewed here.One of the first and most extensive studies addressing that important issue was conducted by Greene et al.  in the cTo this end Yanaba et al.  carried The latter observation concerning the utility of KL-6 as a biomarker in monitoring the clinical course of PF was further strengthened by the same group of scientists {Yanaba et al. } in the Alternatively, Ishii et al.  estimateTo gain a more comprehensive understanding on the activity of lung epithelial cell damage and repair characterizing IPF and other ILDs and to clear out whether these are related to the disease prognosis, investigators studied the significance of other lung specific epithelial proteins initially reported as tumour markers, in reflecting these pathological abnormalities. Some of the most extensively reported markers were CK19 and carbohydrate antigens sialyl Lewis .There have been several published papers reporting elevated levels of CK19 in the serum of patients with various types of ILD ,63. NeveFueled by this prospect, Nakayama et al.  were theElevated plasma levels of Ca 19-9 were first demonstrated by Bungo et al.  in 1988.Evidence presented give credence to the view that cancer-associated antigens could serve as valuable and reliable diagnostic and prognostic markers of ILDs. On the other hand, none of the studies provided us with established diagnostic cut-off values since they exhibited low statistical power and in most of them no adjustment with the disease severity was performed. Moreover, KL-6 has proven to have higher discriminative power than carbohydrate antigens. In addition, potential arguments include the lack of knowledge regarding the role of these markers in the pathogenetic pathway of IPF. So far, the only study addressed this crucial issue was published by Obayashi et al.  who repoThe past few years led by the same perspective idea to find a reliable and reproducible biologic marker for the diagnosis and prediction of ILDs, several group of scientists have tested the usefulness of a slew of cytokines to play that role.One of these reports was published by Suga et al . AuthorsOne of the most exciting roles cytokines can play is their use as indicators of the therapeutic effects of several drugs against IPF providing investigators with useful knowledge on the potential mechanisms of these drugs in terms of physiology and molecular biology highlighting novel therapeutic targets.The first study in humans to characterize the effects of IFN-γ1b on plasma and lung biomarkers, speculated to serve as critical mediators in the pathogenesis of IPF was recently published by Strieter et al . AuthorsThe concept of biomarkers is extensively developed in the field of occupational and environmental medicine. Historically, in epidemiologic research, the link between exposure and disease was often without knowing the mechanism or intervening events. The past few years, the identification of lower levels of exposures and the effective clinical and public health management of high risk populations has drugged much of attention. Borm  highlighBorm and co-workers ,80 evaluTo characterize the nature and extent of coal dust induced airway injury there is a need for biomarkers useful to monitor exposure effects. The potential of many cell mediators as monitoring tools i.e. CC16 , antioxiHypersensitivity pneumonitis also called extrinsic allergic alveolitis is an ILD that may be due to a wide variety of inhalative antigenic stimuli. To date, the conventional diagnosis of hypersensitivity pneumonitis is usually made on the basis of history of periodically recurring or permanent complaints upon exposure to a specific inhalative antigen, interstitial abnormalities in both lungs by chest radiography or HRCT and detection of precipitating antibodies. However, at present, there are few diagnostic procedures available to confirm the diagnosis and to estimate the disease activity and most of them are too invasive and costly for widespread and daily use. With this aim in mind, Takahashi et al.  evaluateWhile its direct role in pathogenesis of immune and inflammatory responses remains unclear, neopterin's ability to reflect monocyte and macrophage activation has been exploited and yet postulated as a marker of the beryllium-specific cell-mediated immune response that leads to a chronic granulomatous disease, a hypersensitivity disorder named chronic beryllium disease (CBD). In the study of Harris et al.  ROC curvSince lung specific-epithelium proteins reflect the epithelial damage and turnover it has been hypothesized and ultimately demonstrated that they can be used as effective circulating markers for the diagnosis and prognosis of the clinical course of various types of interstitial pneumonitis including drug-associated, radiation-induced and hypersensitivity pneumonitis. Furthermore, pneumoproteins have already been introduced as potential valuable biomarkers in the research field of pediatric ILDs.Numerous agents including cytotoxic and non-cytotoxic drugs exert pulmonary toxicity including interstitial pneumonitis which several times culminates to a fatal outcome . TherefoOhnishi et al.  in theirRadiation pneumonitis is the most common complication for thoracic tumours and is classified as an ILD. Since CT scanning that represents the gold standard diagnostic procedure for radiation pneumonitis is either not frequently repeatable or often exhibits non-specific findings hardly to be differentiated from the lung tumour manifestations, a lung specific laboratory test easily repeatable and reproducible is highly required for the early detection of radiation-associated lung injury. On the basis of this conception, Kohno et al.  were theAnother study searching for potential indicators for the early detection of radiation pneumonitis and for monitoring its clinical course was conducted by Takahashi et al . RemarkaHowever, these studies exhibit major caveats that must be addressed prior to their further application in the routine clinical practice. Briefly, we report the small number of patients studied, the application of chemotherapy prior to radiotherapy, the absence of serial measurements through the clinical course of the disease and the evidence that these proteins (SP-A and KL-6) are neither organ nor disease specific since they have also been used as markers for lung adenocarcinoma ,53. To cAs mentioned above the spectrum of ILDs includes a large heterogeneous group of disorders calculating over 200 members with IPF to be the most common form of idiopathic ILD. In contrast ILD in children occurs far less frequently and there are no dominant forms . AlthougSarcoidosis is a chronic systemic disorder characterized by the presence of non-caseating granulomas and accumulation of T-lymphocytes and macrophages in multiple organs . The mecOne of the first studies attempted to circumvent this problem was published by Ziegenhagen et al . AuthorsThe effectiveness of sIL-2R in evaluating sarcoidosis severity even in the early stages of the disease was further confirmed by another study of the same group of scientists  who cleaIn the study of Grutters et al.  authors To establish a more reliable association between sIL-2R and severity of sarcoidosis Rothkrantz-Kos et al.  applied Accumulated evidence from the data presented reveal that the predictive values of serum ACE in sarcoid patients are still under consideration and findings derived from these studies are controversial concerning its usefulness in assessing the disease severity and predicting the clinical outcome. Although some investigators suggest that a rising ACE serum level can predict radiographic relapses of sarcoidosis  others hThe role of chemokines in monitoring the disease behaviour and in reflecting the activity of granuloma formation in sarcoidosis patients has been evaluated and postulated. Hashimoto et al.  were theTo this end Iyonaga et al.  in paralApart from their application as prognostic and diagnostic tools of IPF and other ILDs the utility of lung-epithelium specific proteins including KL-6, SP-D and CC16 as biological markers of the disease activity has been separately evaluated in sarcoidosis patients. To date, several studies have addressed this issue and much good work has been done towards this direction.The potential usefulness of pneumoproteins and more specifically KL-6 as true marker in the assessment of sarcoidosis was initially suggested by Kobayashi et al . AuthorsMoreover Hermans et al.  scrutiniAlthough these markers have been studied separately ,119,120 ILDs represent a diverse group of lung diseases comprising over 200 different members that have been broadly classified into several categories . IPF accTherefore, a major challenge would be the determination of a reliable serologic marker reflecting the disease behaviour before it becomes obvious in clinical level, easily reproducible and feasible to be measured serially, meaning to be acceptable by the patient. The early serial measurement of this biomarker may lead to an early detection of relapse and thereby allow anti-inflammatory and other treatments to be evaluated or eventually modified before they have failed. The latter components can potentially compile a clinician's "wish list". On the basis of this conception, an important number of serum markers either lung-specific proteins and cancer-associated antigens or cytokines and other serological parameters probing different aspects of the immunopathogenesis of ILDs has been delineated. Initial reports of these markers shed further light into the pathophysiology of ILDs, ,120,122 Furthermore, the application of biological markers in the research field relevant to occupational and environmental lung disorders have raised some additional concerns. Most of the studies exhibited major limitations including the retrospective analysis, the heterogeneity of the studied population and the lack of serial measurements necessary for the adjustment to disease behaviour and the link to disease prognosis. The only so far follow-up study  identifiCollectively, these findings highlight the necessity for further validation prospective studies and the assessment of novel molecules  to serveCurrently, the application status for most of these biologic markers is still in its infancy and remains exploratory. Unfortunately, they do not yield indications for therapy or mark the end of the inflammatory process and their prognostic value still needs to be established. Although the majority of them have not yet lived up to the "great hype" that was generated, lung-epithelium specific proteins and mostly KL-6 and SP-D show the greatest promise in IPF and other ILDs whereas serum cytokines seem to be not ready for routine monitoring. As for sarcoidosis, despite the controversial aspects regarding the usefulness of ACE in reflecting the disease activity, it still remains the most reliable and well defined marker whereas sIL-2R represents one of the most fruitful applications. Finally, in occupational and environmental lung disorders the only so far follow-up study supports TNF release as a valuable predictor of the fibrotic disease progression, outcome and activity Table .Further prospective investigations, technical improvements and introduction of novel markers are warranted in order to elevate the association of serum biomarkers with the pathogenesis of ILDs in the same status as for tumour markers with lung cancer. Nonetheless, crossing the boundary from research to clinical application requires validation in multiple settings, experimental evidence supporting a pathophysiologic role, and ideally intervention trials showing that modification improves the outcome. The emergence of pioneering technologies including DNA and tissue microarrays which have already been applied with great success in the respiratory research field can help scientists to circumvent this problem and bridge this boundary. In the interim, these markers can be quite useful to supplement the clinical, radiological and physiological monitoring of the disease and identify high-risk patients who would benefit from aggressive management of established risk factors.Angiotensin converting enzyme (ACE)Bronchoalveolar lavage (BAL)Carbohydrate antigen sialyl Lewis a (Ca 19-9)Chronic beryllium disease (CBD)Clara Cell Protein (CC16)Collagen vascular disease-associated interstitial pneumonia (CVD-IP)Cytokeratin fragment 19 (CK19)Coal Worker's Pneumoconiosis (CWP)Enzyme-linked immunosorbent assays (ELISA)Extracellular matrix (ECM)Farmer's lung disease (FLD)High resolution computed tomography (HRCT)Idiopathic pulmonary fibrosis (IPF)Interferon-γ (IFN-γ)IFN-inducible T cell-a chemoattractant (ITAC)Interstitial lung diseases (ILDs)Krebs von den Lungen-(KL)-6Lactate Dehydrogenase (LDH)Monocyte chemoattractant protein-(MCP)-1Monocyte inflammatory protein- (MIP)-1aNon-specific interstitial pneumonia (NSIP)Progressive Massive Fibrosis (PMF)Pulmonary Function Test (PFT)Receiver-operating-characteristic (ROC)Sialyl Lewis x (SLX)Soluble IL-2 receptor (sIL-2R)Surfactant protein-(SP)Systemic sclerosis (SSc)Tumor Necrosis Factor (TNF)Usual interstitial pneumonia (UIP)The author(s) declare that they have no competing interests.AT and DB were involved with the study conception. AT, GK and SA performed the data acquisition and interpretation. AT prepared the manuscript. DB was involved in revising the article for important intellectual content. All authors read and approved the final manuscript.


Over the past several decades researchers have produced substantial evidence of a social gradient in a variety of health outcomes, rising from systematic differences in income, education, employment conditions, and family dynamics within the population. Social gradients in health are measured using deprivation indices, which are typically constructed from aggregated socio-economic data taken from the national census – a technique which dates back at least until the early 1970's. The primary method of index construction over the last decade has been a Principal Component Analysis. Seldom are the indices constructed from survey-based data sources due to the inherent difficulty in validating the subjectivity of the response scores. We argue that this very subjectivity can uncover spatial distributions of local health outcomes. Moreover, indication of neighbourhood socio-economic status may go underrepresented when weighted without expert opinion. In this paper we propose the use of geographic information science (GIS) for constructing the index. We employ a GIS-based Order Weighted Average (OWA) Multicriteria Analysis (MCA) as a technique to validate deprivation indices that are constructed using more qualitative data sources. Both OWA and traditional MCA are well known and used methodologies in spatial analysis but have had little application in social epidemiology.A survey of British Columbia's Medical Health Officers (MHOs) was used to populate the MCA-based index. Seven variables were selected and weighted based on the survey results. OWA variable weights assign both local and global weights to the index variables using a sliding scale, producing a range of variable scenarios. The local weights also provide leverage for controlling the level of uncertainty in the MHO response scores. This is distinct from traditional deprivation indices in that the weighting is simultaneously dictated by the original respondent scores and the value of the variables in the dataset.OWA-based MCA is a sensitive instrument that permits incorporation of expert opinion in quantifying socio-economic gradients in health status. OWA applies both subjective and objective weights to the index variables, thus providing a more rational means of incorporating survey results into spatial analysis. Research on health and place has produced substantial evidence that living in places with higher relative measures of socio-economic deprivation has a negative influence on a variety of health outcomes . A key oRarely, however, are social gradients in health quantified using survey-based data sources. Deprivation indices are most frequently constructed using variations of either Principal Component or Factor Analysis – both of which are highly computational structural detection and data reduction strategies designed to reduce the number of variables needed to measure socio-economic status (SES). However, these strategies minimize the opportunity to incorporate the knowledge of local health practitioners and of the day-to-day conditions that impact local health outcomes. This is problematic as the underlying conditions that influence neighbourhood variations in SES may go underrepresented when assessed without using their local knowledge. Previously, the author's had constructed a survey-based deprivation index for use in British Columbia using feedback from provincial Medical Health Officers (MHOs) . HoweverThe most relied on source for areal estimation of population SES is the national census. Researchers often rely on the national census for socio-economic information about the population because these datasets are freely available (or available at a low cost), broadly representative of all political jurisdictions, and contain a number of variables reflective of an individual's or area's socio-economic position relative to the surrounding population. They also have pragmatic value as aggregate data is used in absence of individual data to protect anonymity. Census-based deprivation indices date back to the early 1970's in the UK and have since been developed in Canada, the US, New Zealand, and elsewhere -16.There are three primary methods to quantify the effect of living under adverse socio-economic conditions. All three strategies are presented alongside their corresponding variable and scale components in table apriori weights to the index variables, but also provides a medium to compress individual socio-economic variables into their underlying social (i.e. family dynamics) or material  constructs. However, in compressing a set of n variables into a smaller number of components the resulting index explains only a partial amount of the variance exhibited in the entire dataset and so some information regarding the original correlation between the variables is removed.The second technique for index construction is the use of either Principal Component or Factor Analysis – both of which have been the principal method of index construction over the past two decades -20. PCA/Although less popular than the preceding strategies, a third approach is to construct the index using feedback from health experts. The most widely used deprivation index built on survey-based data was the Jarman UPA8 index ,22. The apriori weighting and the relative ease of constructing highly computational measures of deprivation using a wide number of statistical software packages . In other research areas, however, integrating the day-to-day knowledge of local health experts using survey-based approach is seen as a viable means for uncovering local SES conditions [Ensuing critique over the UPA8 reliance on the census; its geographic preference for London over the Northern Regions; and its weighting of the survey scores have since curtailed widespread popularity of survey-based deprivation indices -26. As anditions . In thisGIS-based MCA is one of the most common functions of geographical analysis but has not yet been thoroughly tested in social epidemiology – despite being introduced by Schuurman . In geogThe purpose MCA is to condense complex problems involving multiple criteria (e.g. variables) into an optimal ranking of the best variable scenarios from which an alternative is chosen ,37-40. Iunion (risk seeking) and intersection (risk adverse) operators. The scaling operator makes use of a pair of weights, one local, and one global. The local weights are assigned before hand and can be based on value judgments, proportional ranking, ratios, etc. The global weights are incrementally added and removed from one or all of the variables until reaching a full trade-off of equal weights on to the n variables. The order weights assigned between the full union and trade-off model favour a more risk-seeking modeling scenario, assigning larger weights to the highest ranking variables and lower weights to the smallest variables. The order weights assigned between the full intersection and trade-off model favour a more risk-adverse modeling scenario, where weights are maximized onto the variables with the smallest values and minimized on variables with higher initial values. The order weights are also assigned on a case-by-case basis. This allows the data to weight themselves according to their value relative to the other variables. The weighting flexibility of the OWA modeling environment is a robust tool for assessing how certain variables may compensate for other variables as weights are maximized, minimized, or allowed to trade-off equally. From a population health standpoint, this type of modeling framework may provide researchers a new vantage point from which to assess the SES indicator relationships .The OWA approach is among many possible techniques for ranking multiple criteria, which range from simple scalar methods to complex models based on fuzzy logic ,40-42. OIn this paper, we introduce an MCA model of socio-economic deprivation derived from a previous survey of British Columbia's MHOs. We employ 2001 Canadian Census data for the population of the Vancouver Census Metropolitan Area (CMA) to estimate SES. The utility of MCA is in synthesizing the variables and the weights assigned by the MHOs into a single, quantifiable model reflective of the socio-economic variables chosen by the experts, their weight, and the rank importance of their value existent in the dataset.In 2001, the Vancouver CMA contained 21 municipalities and just under 2 million people, which is over half of the total population of the province. The municipalities in the Vancouver CMA encompass some of the most and least privileged neighbourhoods in all of Canada. The West Point Grey, Shaughnessy, and Kitsilano neighbourhoods, which are some of the wealthiest neighbourhoods in the country, closely border the Downtown Eastside, Oppenheimer, and Chinatown neighbourhoods, widely considered amongst the poorest. Researchers have long investigated the impact of social gradients in health within a number of municipalities in the urban core and the outlying suburban regions of the Fraser Valley -45. HoweAt the time of this analysis the province was still without a census-based construct specifically designed to measure health and socio-economic deprivation of British Columbian's. Rather than construct an index based on combining numerous census indicators into a principal component analysis we chose to survey provincial Medical Health Officers. We selected the MHOs because they are trained physicians with an interest in public health and well being. Part of the job requirement of provincial MHO's is also to trace disease/outbreaks and they have the responsibility, in terms of their own agency, to provide direction or oversight in assisting those in the community.material wealth, housing tenure, family demographics, mobility, educational attainment, employment, or cultural identity.A complete description of the web survey and the particular variables selected for the analysis can be found elsewhere . BrieflyHousing tenure included four indirect measures socio-economic position, including the percentage of single-detached housing units, the proportion of renters and owners, and those residing in an apartment. These variables broadly measure similar socio-economic conditions that were previously found to be associated with health outcomes in Vancouver [The material wealth component was constructed from two census variables, including average income and average dwelling value. These variables represent a direct and indirect measure of material standing. Average income is perhaps the most robust factor representative of material deprivation and is widely used as an indicator of socio-economic position. Similarly, dwelling value is a multidimensional indicator of purchasing power and has previously been found to positively co-vary with health outcomes in British Columbia . Housingancouver . Each ofancouver ,47 and iancouver ,49. The MHO responses to each of the 21 indicators were assigned a score between 1 and 5 (Strongly Agree = 5 to Strongly Disagree = 1). From the original survey, seven indicators were selected for the final index based on the proportion of the MHOs agree and strongly agree responses, including; average income, home ownership, lone parent family, having secondary and post secondary education, the employment ratio and unemployment rate. This subset was selected by administering a cut-off score to only include variables that had summation values greater than 'neutral' response plus 1, which symbolized all respondents choosing a non-neutral positive response for that particular variable. The original and final indicators and their proportional weights are listed in table Health data were obtained from the Canadian Community Health Survey 2.1 (CCHS), a nation wide cross-sectional health survey of the population designed to allow comparison of health outcomes at a sub-provincial level across Canada. Data for the Cycle 2.1 database were collected between January and November of 2003. The target population of the CCHS is Canadians over 12 years of age who live in private dwellings. Individuals living on Indian Reserves, Crown Lands, institutional residents and full-time members of the armed forces are excluded. Data were collected primarily by telephone using three sampling frames, 48% from an area frame, 50% from a list frame of telephone numbers and 2% from random digit dialling. In our study, we use a sub-set of the CCHS response scores of respondents in the Vancouver CMA between the ages of 18 and 74 .good health component comprising the 'Excellent, Very Good or Good' responses and a poor health component comprising responses of fair or poor.Self-rated health data was assessed from the CCHS question "In general, would you say your health is: Excellent, Very Good, Good, Fair, Poor." Proxy measures of health status, such as self-rated health, are widely used as surrogate measures of population health and have shown a significant relationship with levels of mortality, morbidity and health care utilization ,51. For Confidence intervals for the prevalence estimates of the population reporting fair or poor self-rated health by neighbourhood SES quintile were obtained using 500 bootstrap weights provided by Statistics Canada using SAS software. The bootstrap weights are used to account for the complex design of the CCHS sampling frames. Sample weights were assigned to the self-rated health responses so that results were representative of the population living within the Vancouver CMA. The coefficients of variation (CV) produced using the bootstrapping weights were used to gauge the quality of estimates between the self-rated health responses and SES quintiles. The extent of the sampling error from the CCHS survey questionnaire is measured using the CV. The CV tables are prepared by Statistics Canada. Estimates less than 16.5% are deemed acceptable, estimates between 16.6% and 33.3% are flagged as marginal and estimates greater than 33.3% are flagged, but not released.Similar to traditional MCA spatial analysis, the OWA index was built from a series of overlay operations. Areal data obtained from the Canadian Census is geographically bound to set administration zones, which at the local level can range in size at the local level from Dissemination Area (DA), which are roughly the size of neighbouring neighbourhood blocks, to the municipal subdivision (CSD). Census DA boundaries were chosen for this analysis as they provide the finest lens to examine neighbourhood homogeneity. In the 2001 Canadian Census, the DA boundaries replaced the Enumeration Area (EA) as the basic unit of geographic dissemination. Each DA contains a target population ranging from 400 to 700 persons.order weights, are not assigned to the index variables according to the MHO ranking, but rather according to each indicator's position relative to the other variables in the dataset. Table intersection (⋂) model would carry 100% of the local weights. In DAUID59150002, however, income exerts the most influence on area SES. In this instance the variable representing education would receive 100% of the local weights. A number of weighting scenarios can be constructed using the OWA model, ranging from the classical risk-adverse Boolean intersection model to the risk-seeking Boolean union (⋂) model, with the classical weighted linear combination found in between. The benefit of this strategy is that the indicators most reflective of each area's SES position relative to the surrounding area are allowed to influence its rank. This enables the model to exhibit less influence from the MHO global weights, but also provides researchers with a theoretical base to assess the rank importance of the indicator variables selected by the MHOs.The OWA weighting logic is employed here to assess the degree to which the original weights assigned by the MHOs provides a more robust indication of neighbourhood SES than when local, data-driven weights are assigned to the indicator variables. The first set of weights assigned to the indicator variables are the global weights. These are universal weights (e.g. income is assigned the same weight throughout the entire study area) and represent the original ranked importance of the indicator variables assigned by the MHOs. The local weights are assigned on a case-by-case basis, where each case is represented by a unit of the census geography. The local, or intersection, or AND (⋂) OWA model assigns an order weight of 1 to the index variable within each Census unit that produces the lowest area score of the seven factors and a 0 to the remaining variables. In the full intersection model each Census unit is uniquely evaluated according to the least deprived of the seven factors within its own geography. The full intersection model may highlight severely deprived neighbourhoods much the same as a traditional deprivation index (e.g. areas experiencing multiple SES deprivation), but is designed to assess the strength of the SES variable relationships with a greater degree of control of the global weights assigned by the MHOs [intersection model is the Boolean union. The full union, or OR (⋃) OWA model assigns an order weight of 1 to the index variable that produces the highest area score of the seven factors and a 0 to all subsequent variables. This is similar in scope to the UK indices. Here, the deprivation score is assigned to census areas to maximize the level of deprivation within each area, although with some alteration as the full OR (⋃) model is represented by a single socio-economic variable, which implies single versus multiple deprivations. The remaining order weights are scaled across all SES variables between the crisp Boolean ANDing and ORing operators [Table the MHOs ,39. The perators . A full The full ANDing order weights were calculated byn is the number of criteria in the MCE, r is the position of each criterion, and wr is the importance assigned to the particular criterion, r [intersection of the order weights the first ranked SES criterion is multiplied by a value of 1 and the remaining SES criteria are multiplied by 0. Subsequent weights were gradually scaled across all SES criteria, with a weight of 0.142 assigned to each SES variable for a full trade-off of the order weights. Seven deprivation indices were constructed in total, three of which were synonymous with traditional OWA risk-adverse models, which were constructed from order weights from the full intersection to the trade-off model. The fourth index was a complete trade-off between the local and global weights. The remaining indices were synonymous with traditional OWA risk-seeking models, which were constructed using the weights between the trade-off and full union model.where erion, r ,39. Tablunion OWA model is the inverse of full intersection model and is calculated byThe full The level of trade-off between the local and global weights is calculated from the same weights and variables used to construct the ANDness model byThe OWA scores assigned to the census units were reclassified into quintiles. The least deprived areas were assigned a value of 1 and the most deprived socio-economic areas assigned a value of 5.Socio-economic Factor Index (SEFI) deprivation index [To test the robustness of the OWA weights that were assigned to the original MHO survey responses we examined the prevalence scores were against the on index . Brieflyintersection weighted indices contained discrepancies between SES quintile ranking and health rating, but the overall separation between the least and most deprived SES quintile and self-rated health scores remained. Maps of the Vancouver CMA for each OWA scenario are provided in figures Due to the sensitivity of the health data, self-rated health records were aggregated into their corresponding Dissemination Area and used as a marker representing instances of reporting 'fair or poor health' self-rated health for the individual DA. To further protect individual confidentiality, the specific DA boundaries where the individuals reside are suppressed. 53% of the DAs had at least one resident who completed the survey n = 3879) and an average of 2 residents per DA. Figure 9 and an intersection and union OWA models were constructed using a single itinerant SES variable rather than the amalgamation of the variables selected by the MHOs. The full intersection scenario allotted the most control over the global weights and constructed from the SES variable with the lowest value. The full union model is still assessed using the order weights although in this scenario the model maximizes the global weights using the largest SES variable. Table It is important to recognize that both the full intersection model, although the gap between the least and most deprived SES quintiles is the narrowest of all seven OWA indices at 4.7%. Closer examination of the full intersection modeling scenario revealed that nearly 60% of the SES quintiles were represented by the employment ratio in the Vancouver CMA. Several implications can be drawn from this finding. Unemployment trends, on average, have been declining in the CMA over the course of the past two decades so it is not so surprising to find that its significance is somewhat waning. Underemployment rather than employment ratios may provide a more significant relationship to self-rated health. Interestingly, this order weight is synonymous with the rank position of employment ratios in the global weights assigned by the MHOs. To that effect, the order weights were reflective of the significance placed on employment ratios by the MHOs towards characterizing relative health outcomes.A step-wise gradient in self-rated health is revealed within the full union scenario average income was the single most representative SES variable, representing nearly 75% of the DAs against the self-rated health data. In contrast with employment ratios, incidence of low and below average income rates are heavily concentrated within the CMA, especially in neighbourhoods surrounding the Downtown Eastside, Edmonds and south along the waterfront in New Westminster, and encompassing Whalley in North Surrey. Interestingly, only the employment ratio was ranked lower than average income by the MHOs. With the addition of the local weights onto average income, prevalence rates associated with the least deprived quintiles fell from 7.9% to 5.4%.When the order weights were maximized in the full most deprived SES quintiles both the full union and intersection models produced similar scenarios, but with prevalence rates slightly higher using the full union model at 13.8%. Overall, both of the extremity weighting scenarios revealed a social gradient in self-rated health in the Vancouver CMA. However, when greater decision uncertainty is placed on the MHO variable rankings the prevalence scores between self-rated health and neighbourhood SES are reduced. Although the full intersection model does not furnish as robust step-wise gradient in overall self-rated health as its antipode, it does provide a spatial filter for evaluating variable nuance of the MHO rankings.Within the most significant by the MHOs. In the full and semi-intersection model (b) unemployment and average income reaffirm their positive and negative extremes in addition to under weighting the original significance assigned to secondary and higher education. In the full trade-off OWA index, however, the order weights exert the least amount of control over the original MHO variable rankings. In this case, the results indicate that the steepest of all seven step-wise gradients between neighbourhood SES and self-rated health are found when the original responses and frequency weights assigned by British Columbia's MHOs are interpreted with minimum decision uncertainty and control. The method illustrated here is a means of assigning uncertainty to expert group weights in the event that validation and objective assessments of their selection is necessary.SES variables were included into the risk-adverse and risk-seeking OWA models until a full trade-off of the local and global weights was obtained. Until a combination of all seven SES variables were introduced into the risk-adverse OWA models, however, area SES was primarily analyzed by applying lower weighting schemas to variables previously ranked amongst the intersection and union  and the strongest when the OWA index was constructed from all seven variables (0.772 p < 0.01). This drop in correlation likely stems from the high proportion of single variable representation as the OWA weights near the full Boolean intersection and union.On average, both the SEFI and OWA indices produced nearly identical SES classifications throughout greater Vancouver. Only the OWA indices that utilized all seven of the indicator variables produced wider social gradients in self-rated health outcomes. The step-wise gradient revealed when using the SEFI index ranged from 5.2% in the least deprived quintile to 15.7% in the most deprived quintile, which was slightly narrower than the 4.9% and 16.2% range from the MHO weighted indicators. As expected, Spearman rank correlation coefficient between the SEFI and seven OWA indices revealed that the indices were more similar than dissimilar. Both utilize indicators for lone parent families, area unemployment rates, and secondary educational attainment. The correlation coefficient was the lowest between the OWA indices nearer the full Some attenuation between SES quintile rankings and prevalence of reporting 'fair or poor' self-rated health were observed between the original MHO weighted index and the OWA constructed indices. The original web-survey generated prevalence scores ranged from 4.0% in the least deprived quintile to 17.3% in the most deprived quintile, with rates rising step-wise across SES quintiles 2 – 4 from 8 – 9.8% at the DA spatial extent. The differences between the original and augmented MHO weights were small, with the gradient between least and most deprived quintiles attenuating by 1.0% in the least deprived quintiles and 0.6% in the most deprived SES quintiles. The variation in prevalence scores likely reflects smoothing of the indicator variables when multiplied by the constant averaging weight of 0.142. The similarity between the original MHO index and the OWA and SEFI weighted indices points to the well-known observation that different variable weights yield different results. As the OWA method was originally designed to validate the MHO response scores these results also suggest that the MHOs are well-versed in the conditions that characterize health inequalities within British Columbia.Although our analysis was not age-adjusted by individual SES variables contextual effects of community socio-economic characteristics are well-known indicators of population health independent of individual SES ,53. MoreThe location of individual self-rated health responses are concealed to protect anonymity, but given the prevalence scores between self-rated health and the SES variables selected by the MHOs it would not be surprising to find greater morbidity and mortality levels in these areas. Evaluating socioeconomic inequalities using OWA offers a flexible means of assessing systematic differences in neighbourhood SES as viewed by provincial MHOs and provides a mechanism for validating expert opinion using of local and globally defined weighting scenarios.The approach presented in this research is methodologically and conceptually distinct from traditional deprivation index construction. Unlike previous survey-based deprivation indices, the OWA index weights are simultaneously influenced by the experts and the data. This is a new method that may diminish the historic tension of incorporating highly subjective user assigned weights into spatial analysis. The local weights also provide leverage for controlling the level of uncertainty in the interpretation and weighting of the SES variables selected by the MHOs. Future research will focus on the use of OWA as a new vantage point for generalizing how variable weights influence one another (i.e. effect of education on income). This may offer a more robust understanding of neighbourhood variations in SES.The author(s) declare that they have no competing interests.NB drafted the manuscript, performed the statistical analysis, and constructed the maps and tables. NS and MH conceived early segments of the study and helped draft the original manuscript. All authors read and approved the final manuscript.This analysis was based on the Statistics Canada master file CCHS (Cycle 2.1) which contains anonymized data collected in 2003. All computations were prepared by the authors and conducted at the British Columbia Interuniversity Research Data Centre, University of British Columbia. The responsibility for the use and interpretation of these data is solely that of the authors. The opinions expressed in this paper are those of the authors and do not represent the views of Statistics Canada.


Angiogenesis is associated with follicular development and is regulated independently within each follicle potentially making the functioning of its vasculature critically important in determining its fate. This review examines the various ways in which follicular angiogenesis may be monitored, describes the follicular localisation and changes in pro- and anti-angiogenic factors that may regulate the process and how antagonists may be used to elucidate their physiological role in vivo. Thus, inhibition of vascular endothelial growth factor (VEGF), VEGF receptor-2, vascular endothelial cell cadherin or interference with the angiopoietin system can inhibit follicular development or prevent ovulation. Aside from wound healing and certain pathological processes, including neoplasia, the vascular system in the adult is generally quiescent. An exception takes place in the ovaries where there is intense angiogenesis and increased permeability of blood vessels during follicular development, ovulation and subsequent formation of the corpus luteum. Furthermore, angiogenesis is regulated independently within each individual follicle and depending on the extent of the vascular plexus and permeability of vessels, the supply of large molecular weight tropic factors, precursors and lipids can be controlled. This indicates the follicular vasculature could be intimately involved in the processes of follicular selection, dominance and atresia.in vivo. This review outlines the methods that are being used to study changes in the follicular vasculature, address the work on some of the angiogenic factors which have been studied in the ovary and seem of particular interest at this time, and examines the effects of manipulation of these factors on follicular angiogenesis and development in vivo.It is likely that some types of infertility are associated with disturbance of follicular angiogenesis resulting in inadequate development. In polycystic ovarian syndrome there is excessive angiogenesis while ovarian hyperstimulation syndrome (OHHS) is associated with an increase in capillary permeability. Thus, an understanding of the mechanisms of follicular angiogenesis and its regulation may lead to therapies for controlling inappropriate follicle development secondary to decreased or enhanced angiogenesis. The identification of putative angiogenic factors in the ovary and development of specific agonists or antagonists of angiogenic molecules, together with their application in animal models, presents novel opportunities to validate their physiological role While primordial and primary follicles receive nutrients and oxygen by passive diffusion from stromal blood vessels, follicular growth is associated with the development of an individual capillary network and continued angiogenesis to nourish the rapidly expanding follicle. The vascular sheath that develops around each follicle is confined to the thecal layer by the presence of the membrana propria until the breakdown of the basement membrane at ovulation. Some of the methods employed to monitor the progress of the follicular vasculature are outlined below.Measurement of ovarian blood flow can be achieved non-invasively by color and pulsed Doppler ultrasonography in species with sufficiently large and accessible ovaries such as humans, cattle and horses. This demonstrates increased flow to the ovary containing the dominant follicle. In addition, there is increased peak flow velocity with increasing follicular size and high vascularity and flow velocity of the dominant follicle before ovulation . In mareDynamics of ovarian blood supply to preovulatory follicles has been investigated by injection of radioactive microspheres into the ovarian artery and shows that the elevation in follicular blood supply associated with the preovulatory LH surge is followed by a fall in blood supply as the time of follicular rupture approaches . The spaThe most widely employed approach to study changes in angiogenesis during follicular development is to use ovarian sections in which endothelial cells are stained with a specific marker. Changes in endothelial cell area can then be quantified using image analysis. The most commonly used marker is platelet endothelial cell adhesion molecule (PECAM/CD31), a membrane protein that mediates cell-cell adhesion and is reliably detected in endothelial cells in the follicles of, e.g., the mouse ,6, rat  marmosetProliferating endothelial cells may be visualised and quantified by dual immunostaining using markers of endothelial cell proteins to localize to the cytoplasm together with proliferation markers localised to the nucleus. For example, nuclear incorporation of exogenous bromodeoxyuridine (BrdU) ,9 or locIn general, the above observations support the idea that vascular development plays a crucial role in the latter stages of follicular growth, and the selection of the ovulatory follicle. In particular, they suggest that efficient elaboration of competent blood vessels and maintenance of vascular permeability is necessary to maintain adequate delivery of gonadotropins, nutrients and oxygen necessary to sustain rapid follicular growth. A disruption of the follicular vasculature during the early stages of atresia would be a logical mechanism leading to a reduced supply of tropic factors and nutrients to the follicle, although such a cause and effect has yet to be established.in situ hybridization to measure mRNA and western blot and immunocytochemistry to investigate protein. The definition of changes in their temporal and spatial expression patterns in relation to specific stages of follicular development have implied a role for a large number of factors which suggests the process involves a series of complex interactions between numerous regulatory molecules. Although angiogenesis is confined to the thecal layer and the receptors for angiogenic factors are found predominantly on the ovarian vascular endothelium and associated cells, many of the factors that regulate the process are predominantly synthesised in the granulosa.The localisation and regulation of a large number of established and putative angiogenic factors and their receptors in the ovary has been described using a combination of northern blot, RT-PCR and Studies in which angiogenic factors, or specific antagonists, are administered over selected periods of follicular growth must be conducted in order to confirm the postulated physiological roles of these factors in the regulation of follicular angiogenesis and function. Most studies have employed systemic administration, but the ability to inject factors directly into the ovarian follicle has been also employed [e.g. 19]. Examples of some of the factors that are currently being investigated in the ovary are described below.Predominant among the candidates is the pro-angiogenic vascular endothelial growth factor (VEGF) family . The mosin situ hybridization [in vitro [in vivo [With respect to the localisation of VEGF mRNA and protein during the course of follicular development in the ovary, although there may be some species differences, a general pattern is apparent ,12. Typidization . A furthin vitro  and decr[in vivo .in situ hybridization [Surprisingly, in follicles that appear very close to ovulation, VEGF synthesis declined in the granulosa cells and exhibited a punctate expression in cells in the thecal layer at the border with the granulosa . The disdization  and immudization .in vivo, with resultant effects on follicular function, has been achieved using neutralising antibodies against VEGF itself [Specific inhibition of the VEGF pathway F itself , antibodF itself , a VEGFRF itself , the solF itself ,28 and rF itself ,23.VEGF Trap R1R2 comprises Ig domain 2 of VEGF-R1 and Ig domain 3 of VEGF-R2, fused with human Fc. It blocks all isoforms of VEGF A, VEGF B and PlGF but does not inhibit VEGF C and D. Marmosets treated with VEGF Trap from the beginning of the 10-day follicular phase had healthy pre-antral and small antral follicles in their ovaries, but large antral follicles, characteristically present in ovaries of late follicular phase control marmosets, were absent  into the pre-ovulatory follicle has been investigated . Half thIt is still not established whether the permeability-enhancing actions of VEGF are involved in gonadotropin access to the dominant follicle , the devThe effects of inhibition of VEGF at the early, mid- or late follicular phase on pituitary-ovarian hormone profiles in the blood have been described in macaques. When an antibody to VEGFR-2 was administered starting during the early follicular phase, marked changes in pituitary-ovarian function were observed . The folStudies with VEGF Trap in the stump-tailed macaque evaluated the hormonal response to a single injection of either 4, 1 or 0.25 mg/kg, i.v. administered during the mid-follicular phase, when the ovulatory follicle is being selected . The VEGWhen VEGF Trap was administered in the late follicular phase, the anticipated pre-ovulatory rise in estradiol was blocked and although this was followed by a rapid and sustained rise in LH and FSH, there was an absence of progesterone rises which occurred in controls a few days after injection of control protein . SubsequAll the studies in which VEGF has been inhibited in macaques have resulted in hypersecretion of LH and FSH without apparent stimulation of follicular development. This phenomenon has been studied directly in the mouse. Exogenous gonadotropin administered to hypohysectomized animals stimulated follicle development in controls, but had no effect when VEGF was inhibited using a VEGFR-2-blocking antibody . This suin vitro studies suggest angiogenic factors may also act on granulosa or thecal cells directly, in an autocrine or paracrine manner and VEGF receptors have been reported in bovine granulosa cells, suggesting VEGF also has a survival role in non-vascular cells [It may be that administration of angiogenic factors would be of benefit in cases of ovarian dysfunction characterized by impairments in follicular development and ovulation. Whether administration of VEGF itself might be beneficial in situations where follicular angiogenesis is inadequate is now being addressed. Injection of VEGF gene fragments into the ovarian medulla in pre-pubertal gilts increased VEGF concentration in follicular fluid, follicular vascular density, and the number of pre-ovulatory follicles when given together with gonadotrophin . Treatmear cells .In situ hybridization studies on rat ovaries showed that Ang-2 mRNA is absent from preantral follicles and did not become detectable until the pre-ovulatory stage, where its expression is associated with the thecal blood vessels. Ang-1 is expressed in the thecal layer uniformly during follicular development. A dramatic up regulation of Ang-2 mRNA in the granulosa cells of atretic follicles in association with reduced expression of VEGF was observed [in situ hybridization studies in the ovary. In the marmoset, Ang-1 mRNA was detected in the theca of tertiary follicles, but at a very low level while Ang-2 mRNA was not detected in follicles, but was present in a subset of stromal vessels [While VEGF is the prime initiator of angiogenesis, the formation and differentiation of a structurally and functionally mature vascular network probably requires the co-ordinated action of multiple factors. These include angiopoietin-1 and -2 (Ang-1 and Ang-2), which act via the tyrosine kinase receptor, Tie-2 . Follicuobserved . Surpris vessels . In cont vessels . Changes vessels .Long-awaited direct evidence for a role the angiopoietins in the ovary comes from observations in the rhesus monkey where local injection of Ang-2 into the pre-ovulatory follicle blocked ovulation  while thIn situ hybridization of human ovaries shows the mRNA in granulosa cells of primordial and primary follicles, while in antral follicles expression is highest in the theca, and declines in the granulosa [in vitro, but are differentially regulated and expressed in broadly complementary patterns in the ovary [in vivo to promote angiogenesis and the induction of the fenestrated vascular phenotype [If expression of VEGF is first detected after the initial recruitment of a vascular network to an individual follicle, it is likely that other pro-and anti-angiogenic factors have a crucial role in regulating the onset of follicular angiogenesis. Endocrine gland VEGF (EG-VEGF), also known as prokineticin-1, is a promising angiogenic factor that is expressed at earlier stages of follicular development. EG-VEGF was identified from the human ovary and proposed to function as a specific angiogenic regulator in steroidogenic tissues . EG-VEGFranulosa . Since Ehe ovary , it has henotype . There ahenotype . With rehenotype  it will Although the regulation of follicular vascular permeability has long been supposed to have a major impact on the access of hormones and precursors, there currently exists a dearth of information on its molecular regulation in the ovary and the endocrine and paracrine control of the process is just beginning to be addressed . Moleculin vivo has been demonstrated in the hypophysectomized mouse. VE-cadherin was inhibited by a neutralising antibody that suppressed gonadotropin-induced follicular angiogenesis and development [A critical role for VE-cadherin in follicular angiogenesis elopment .Natriuretic peptides are a family of small proteins that modulate salt and water balance as well as vascular tone . It has Connective tissue growth factor (CTGF) is a cysteine-rich secretory protein which is a member of the cysteine-rich 61/nephroblastoma-overexpressed family of genes . It has Thrombospondins -1 and -2 are large glycoproteins found in the extracellular matrix and act as autocrine factors. Included in the action of thrombospondins are anti-angiogenic and anti-cell migration properties. They are often produced at a rate inversely proportional to that of VEGF and counteract pro-angiogenic stimuli. Thrombospondin localisation in the ovary has been investigated in the rat  and cow Another way of gaining information on the role of follicular fluid and granulosa cells in the regulation of endothelial cell gene expression has been to use microarray technology to compare gene expression between unstimulated human umbilical vein endothelial cells treated with either human follicular fluid or culture medium conditioned by human granulosa cells . On the in vivo. The approach described in this review of identifying potential candidate molecules and inhibiting their action in vivo using specific antagonists to determine effects on follicular angiogenesis, development and function should provide a powerful tool to elucidate their physiological role and identify ways of either increasing or decreasing ovarian angiogenesis in the clinic.Angiogenesis plays a critical role in follicular development. The vasculature proliferates and regresses throughout the lifespan of the follicle, but its importance in governing follicle selection on one hand and the early onset of atresia have yet to be established. Many potential candidates involved in the regulation of angiogenesis, vascular permeability and maturation of the vasculature are present in the follicle. For some of these factors, such as VEGF, their functional role can be established by inhibiting their action


The effects of interferon-alpha (IFN) plus tamoxifen (TMX) in the treatment of advanced breast cancer were assessed. Changes of in vivo biologic determinants including hormone receptors, P24 protein, Ki-67 and growth factor expression were evaluated. Seven patients with advanced, heavily pretreated, breast cancer with accessible disease, underwent biopsy prior to and after sequential treatment with IFN and IFN plus TMX. Clinically 4/7 patients responded to treatment with one complete and three partial remissions. Apart from the favourable response rate the sequential in vivo changes in expression of tumour variables were of considerable interest. IFN treatment consistently increased the expression of the estrogen receptor (ER) and of the estrogen regulated protein P24 while decreasing the expression of the proliferation associated antigen Ki-67. Addition of TMX on the other hand resulted in a reduction of ER expression to pre-IFN levels and a rise in progesterone receptor (PR) expression. When the effect of either IFN or IFN plus TMX on the expression of two growth factors was assessed they were found to be somewhat variable. While PDGF expression tended to be suppressed, there was no clinical correlation with response to therapy. TGF beta expression was found in all patients prior to treatment and while all non-responders showed reduction of TGF beta following treatment, the alterations were variable amongst responders . It is concluded that both IFN and TMX exert multiple effects on the expression of tumour biologic variables and that while the study confirmed some of the predictions from in vitro models, the in vivo effect are more complex than has been appreciated from the models. From the clinical point of view, it might be expected that treatment which enhances the expression of ER in tumours should have a positive effect on the response to TMX.


Careful in silico design and evaluation of potential oligonucleotide probe targets is therefore crucial for performing successful hybridization experiments.Taxon specific hybridization probes in combination with a variety of commonly used hybridization formats nowadays are standard tools in microbial identification. A frequently applied technology, fluorescence in situ target accessibility patterns. The different types of sequence associated information (SAI) can be visualized by user defined background colors within the ARB primary and secondary structure editors as well as in the PROBE Match tool.The PROBE Design tools of the ARB software package take into consideration several criteria such as number, position and quality of diagnostic sequence differences while designing oligonucleotide probes. Additionally, new visualization tools were developed to enable the user to easily examine further sequence associated criteria such as higher order structure, conservation, G+C content, transition-transversion profiles and in silico probe design and evaluation can be performed with respect to in situ probe accessibility data. The evaluation of proposed probe targets with respect to higher-order rRNA structure is of importance for successful design and performance of in situ hybridization experiments. The entire ARB software package along with the probe accessibility data is available from the ARB home page .Using this tool,  The introduction and use of comparative sequence analysis of appropriate marker genes as a powerful tool in taxonomy has substantially contributed to the rapid growth of molecular sequence databases such as EMBL , GenBankin situ hybridization (FISH) technique became an integral part of the rRNA approach to microbial ecology and evolution [in silico plays a crucial role to ensure sensitivity and specificity of a potential probe in its practical application. Besides uniqueness of the target sequence, number, character and position of diagnostic residues, comprehensiveness with respect to the inclusion of members of the desired target group (taxon) and exclusion of non-members along with a target molecule or region accessibility in the real hybridization experiment, have to be taken into consideration. Recently, data on in situ accessibility of rRNA targets in several microorganisms have become available [Ever since the fluorescence volution , rRNA-tavailable -11.Since biology is a highly visual science, there is a general demand for tools to visualise the variety of biological knowledge as diagrams, illustrations, two-dimensional and three-dimensional reconstructions, and other types of graphical formats. Hence, the visualization of molecular data in an interactive and intuitive graphical user interface ideally will serve as third eye for a molecular biologist. In this paper, we describe how the ARB software package  providesPeriodically retrieved raw gene data comprising small subunit rRNA from public databases such as EBI , GenbankThe PT-Server  is a sufProbe design is carried out using the PROBE Design tool (PDT) of ARB software involving following steps:1. The user selects the target group or a species of interest.m) according to the 4°C GC, 2°C AT rule [2. The parameters such as size of the probe and the probable physico-chemical characteristics like %GC content, melting temperature  of ARB. PMT, by default evaluates the targets for the sequence (strand) stored in the database. Optionally, the complementary sequence (opposite strand) can be evaluated as well. Members of the target group are displayed in a separate PROBE Match window along with other information such as number of mismatches, weighted mismatches, s Figure .in silico evaluation and visualization of such probes and targets are necessary, particularly, when used for FISH experiments. Target accessibility is among the crucial criteria to be evaluated with respect to experimental success of the respective probe based identification and detection system [As the demand for oligonucleotide probes that can identify and quantify bacteria by nucleic acid hybridization is permanently increasing, n system -12. To fin situ hybridization approach the results of the accessibility studies conducted by Fuchs and co-workers on the 16S and 23S rRNA of Escherichia coli and other organisms are among such criteria. They showed that some regions of E. coli ribosome are virtually inaccessible for oligonucleotide probes when FISH is performed [in situ hybridization signals. Within the ARB program, these classes are coded in respective SAIs  and optionally visualized as background colors of the sequences in primary structure (ARB_Edit4), secondary structure (SEC_Edit), and probe visualization windows (PROBE Match) of ARB. All the displays produced by the ARB software are interconnected and any changes in one window are automatically updated in other windows as well. Simultaneous visualization and evaluation of oligonucleotide probes in different levels allows the user to look carefully and closely into the proposed probe candidates in silico, before carrying out further in situ or in vivo studies. More importantly, the user can perform a variety of sequence related operations such as importing sequence data, aligning, treeing, designing, evaluation and visualization of probes, performing statistical calculations and many other functions using interoperating and user friendly tools controlled from a common graphical platform within the ARB software package.Although a phylogenetic probe is primarily judged in terms of its taxonomic range to identify the members of its intended target taxon to the exclusion of non-target bacteria, for a practical consideration it must also fulfil certain other criteria with respect to its applicability depending on the particular hybridization format. In case of the fluorescence erformed ,9. They in situ target accessibility patterns, is possible at three different levels: the local alignment (PROBE Match tool), global alignment (ARB Primary Structure Editor) and secondary structure levels (Secondary Structure Editor).Visualization of potential probe candidates and the sequence associated information (SAI) such as higher order structure, conservation, G+C content, transition-transversion profiles and in situ accessibility maps for visualization  can be managed with the PROBE Match SAI window. The neighboring region up to nine nucleotides on either terminus of the potential probe target is retrieved from the database. A local alignment of the extracted rRNA sequence is established and displayed along with the respective unique identifier such as ARB short_name, accession number, or any other underlying database fields  Figure , 3, 4. TOn the global alignment level, the user selected oligonucleotide probe is visualized in different background colors in the primary structure editor window of ARB . The priin situ hybridization experiments, mapping of experimentally derived in situ accessibility patterns onto the primary structures of interest certainly provides valuable support to the users for probe evaluation. Part of a multiple 16S rRNA sequence alignment is shown in the figure Methanosaeta sedula [et al [As already described for the local alignment level, any type of SAI can be visualized by the user defined background colors for the individual alignment columns. Customized color selections can be assigned to the different types of SAIs mentioned before. By scrolling the mouse or the use of ARB search tools, the user gets an easy access to the information for any range or the selection of sequences. In the context of probe evaluation for a sedula  are mappa [et al .Thermus thermophilus which had been elucidated with atomic resolution by X-ray diffraction crystallography of ribosomal subunit [Theoretically as well as experimentally derived secondary structure information of SSU rRNA -17 is usin situ hybridizations [in silico probe design and evaluation can be performed with respect to in situ probe accessibility data. By identifying and excluding the probes targeting sites with a poor accessibility the number of time consuming empirical tests can be reduced.The evaluation of proposed probe target position with respect to higher-order rRNA structure is of more importance especially when probes are intended to be used for izations -12. Albeizations ,19, the izations ,19 takesThe entire ARB software and the periodic updates of well aligned and annotated ribosomal RNA databases are made freely available for the scientific community via World Wide Web . CurrentYK developed and implemented the tool and drafted the manuscript. RW participated in design and implementation. SB and BF provided the accessibility data and revised the manuscript. FOG, RA and HM critically revised the manuscript. WL initiated the development of the tool and supervised the ARB project.


Distant spread from breast cancer is commonly found in bones, lungs, liver and central nervous system. Metastatic involvement of peritoneum and retroperitoneum is unusual and unexpected.We report the case of a 67 year-old-woman who presented with gastrointestinal symptoms which revealed to be the clinical manifestations of peritoneal and retroperitoneal metastatic spread of an invasive lobular breast cancer diagnosed 15 years before.To the best of our knowledge, the case presented is the third one reported in literature showing a wide peritoneal and extraperitoneal diffusion of an invasive lobular breast cancer. The long and complex diagnostic work up which led us to the diagnosis is illustrated, with particular emphasis on the multidisciplinary approach, which is mandatory to obtain such a result in these cases. Awareness of such a condition by clinicians is mandatory in order to make an early diagnosis and start a prompt and correct therapeutic approach. Invasive lobular breast cancer (ILC) takes origin in the milk-producing glands of the breast and is the most common histological breast cancer after the ductal carcinoma (DC), accounting for 8–14% of cases ; the neo2, mitoxantrone 10 mg/m2, 5-fluorouracil 500 mg/m2, every 21 days) and was on tamoxifen. Ten years later a local recurrence occurred, and the patient underwent partial resection of the thorax wall followed by reconstruction by transverse rectus abdominis musculocutaneus (TRAM) flap technique. No adjuvant treatment was given. The patient came to our attention complaining of a 4-month history of diffuse abdominal pain associated to constipation, tenesmus and sporadic rectal bleeding. On physical examination, the patient was pale but moderately nourished. The mastectomy bed, the controlateral breast, and both axilla were normal. Abdominal examination showed no palpable mass or ascites. At digital examination the rectum appeared stenotic from about 6 cm above the anal verge, but without evidence of endoluminal masses. Haematological analysis and biochemical parameters including liver and renal function tests were within the normal range. The urine cytology revealed micro-hematuria. The patient was submitted to a rectosigmoidoscopy which showed a diffuse thickening of the anterior wall of the rectum, which determined mild stenosis beginning 7 cm above the anal verge, without evidence of endoluminal masses. The mucosa which lined the anterior rectal wall was hyperaemic and easily bleeding. The posterior wall of the vagina showed diffuse thickening at vaginal endoscopy. The histological examination of multiple biopsies taken during rectosigmoidoscopy, revealed an extensive infiltration by scarcely cohesive neoplastic cells with "Indian file" features and focal targettoid arrangement around rectal glands  scan of the abdomen and pelvis showed how the pelvic cavity was almost completely occupied by neoplastic tissue which infiltrated the rectal wall, causing marked stenosis, both the ovaries, the fundus of the vagina and the left lateral wall of the bladder, with involvement of the left ureter and concurrent hydronephrosis. Multiple enlarged lymph-nodes were identified in the perirectal fat, along the common iliac artery and the obturator chain and in the inter-aortocaval space. The liver and the chest appeared normal. Mammographic and ultrasonographic picture of the left breast and of both axilla were normal. On the basis of the diagnosis of wide peritoneal and extraperitoneal metastatic spread of ILC, the patient entered a protocol of systemic chemotherapy and hormonal therapy after the placement of a J ureteral stent to pass through the stenosis evidenced by the CT scan and confirmed by cystoscopy.It is well known that the pattern of metastatic spread differs dramatically between ductal and lobular breast cancer ,7. In paThe most representative series of metastatic ILC, focused on the specific sites of involvement, are summarized in table I [see additional file 1].The clinical manifestations in such cases are usually non-specific, and strikingly similar to that of primary gastrointestinal and genitourinary malignancies ,16. The The therapeutic approach to these patients is still object of debate. Surgery is mainly indicated to solve stenotic or hemorrhagic evolution of the colorectal locations. Although some authors suggest a potential benefit of surgical debulking procedures followed by chemotherapy , systemiThe case reported is peculiar, as it focuses on an extremely rare condition which, if ignored, leads to a delayed treatment, thus worsening the prognosis in terms of survival. The long and complex diagnostic workup described, draws the attention to the difficulty of making a correct diagnosis, also because the long interval after the initial diagnosis of breast cancer makes the occurrence of metastasic disease an unexpected event.This report stresses the importance for the clinician to be aware of the eventuality of a wide peritoneal/extraperitoneal metastatic diffusion, when dealing with a patient with previous diagnosis of ILC and presenting for gastrointestinal or genitourinary symptoms. Only bearing in mind such a possibility, the aggressive, multidisciplinary approach necessary to make diagnosis can be early undertaken and an optimal therapeutic approach promptly performed.The author(s) declare that they have no competing interests.Franceschini G Concept and designManno A Concept and designMulè A Histology and immunoassaysMaggiore C Histology and immunoassaysVerbo A Acquisition, analysis and interpretation of dataRizzo G Acquisition, analysis and interpretation of dataSermoneta D Drafting of the manuscriptPetito L Critical revisionD'alba P Critical revisionMasetti R Final revision and approvalCoco C Final revision and approvalAll authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Atrial fibrillation (AF) is the most common cardiac arrhythmia, and it increases in prevalence with advancing age to about 5% in people older than 65 years.We present a rare case of atrial fibrillation secondary to a mega-oesophagus occurring in an 84-years-old Caucasian woman. The patient had a history of progressive dysphagia and the accumulation of food debris lead to mega-oesophagus.The diagnosis was made by barium swallow and electrocardiogram; evacuations of 300 ml of the food debris lead to complete resolution of the arrhythmia. The possible aetiology leading to this AF is discussed. Atrial fibrillation (AF) is the most common cardiac arrhythmia, and it increases in prevalence with advancing age to about 5% in people older than 65 years Table . The chaWhen the atria are in fibrillation, contraction occurs at rates of 350–900 per minute. The AV node may conduct these impulses to the ventricles at 90–170 beats per minute, and often higher. There are several complementary and competing theories regarding the pathophysiology of AF initiation and propagation. The occasional impulses conducted by the atrio-ventricular node results in a totally irregular ventricular rhythm which is a characteristic of the condition which can be either continuous (acute or chronic) or paroxysmal .Achalasia is primarily associated with a degeneration of ganglion cells of Auerbach's plexus resulting in an absence of oesophageal peristalsis and failure of lower oesophageal sphincter relaxation. This results in oesophageal dilatation or mega-oesophagus .We present a rare case of a patient with achalasia in which accumulation of undigested food lead to mega-oesophagus and atrial fibrillation.An 84-year-old Caucasian women referred by her General Practitioner complaining of dysphagia and dyspnoea exacerbated by swallowing. She reported a 3 week history of progressive dysphagia, initially for solids and subsequently for fluids leading to total dysphagia. Other reported symptoms included regurgitation, cough and hoarseness of voice. There was no significant medical, family or social history.Clinical examination revealed a rapid, irregularly irregular pulse; no other cardiovascular-respiratory abnormalities were identified. Abdominal examination also revealed no abnormality; indirect laryngoscopy which was carried out by the same examining clinician showed pooling of saliva in the hypopharyngx.Lateral soft tissue radiograph Figure  revealedThe patient was initially resuscitated with intravenous fluids, followed by naso-oesophageal leverage and removal of 300 ml of retained food debris from the cervical oesophagus. An ECG performed 8 hours afterwards revealed reversion back to sinus rhythm Figure . EventuaThere is a correlation between achalasia and cardiac arrhythmia . Many paUsing 3 non-invasive tests, abnormalities of cardiovascular reflex function were found in 7 of 15 patients with achalasia. Abnormalities of heart rate responses to the valsalva manoeuvre, deep breathing, and standing were noted in patients with autonomic neuropathy defect. The findings are consistent with the hypothesis that an abnormality of vagal function may contribute to the pathogenesis of achalasia .Achalasia itself may be a result of an abnormality of vagal function due to an autonomic neuropathic defect and therefore abnormalities of cardiovascular response are not uncommon .In this patient, the removal of the food debris from the cervical oesophagus caused reversion of this arrhythmia back into sinus rhythm. This can be justified as either due to vagal repression or the relief of the pressure effect of the volume of the food debris transmitted directly to the atria. The evacuation may have resulted in either a sinus massage or a forced valsalva leading to reversion of the rhythm.There are several confounding factors as to the exact cause of the transient atrial arrhythmia in this patient. Previous electrocardiograms exhibit a degree of ischaemia which may predispose to transient arrhythmias in situations of vagal repression especially in the circumstances of extra-cardiac intra-thoracic pathology as in this case.The author(s) declare that they have no competing interests.TU: designed the study, carried out the literature research, clinical study and manuscript preparation.WJ: carried out the literature research, manuscript preparation, and manuscript review.ME: carried out the manuscript editing and manuscript review.SS: carried out the manuscript editing and manuscript review.CH: carried out the manuscript editing and manuscript review.JM: designed the study, carried out the literature research, clinical study and manuscript preparation.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: