The identification of potentially relevant biomarkers and a deeper understanding of molecular mechanisms related to heart failure (HF) development can be enhanced by the implementation of biological network-based analyses. To support these efforts, here we report a global network of protein-protein interactions (PPIs) relevant to HF, which was characterized through integrative bioinformatic analyses of multiple sources of "omic" information.We found that the structural and functional architecture of this PPI network is highly modular. These network modules can be assigned to specialized processes, specific cellular regions and their functional roles tend to partially overlap. Our results suggest that HF biomarkers may be defined as key coordinators of intra- and inter-module communication. Putative biomarkers can, in general, be distinguished as "information traffic" mediators within this network. The top high traffic proteins are encoded by genes that are not highly differentially expressed across HF and non-HF patients. Nevertheless, we present evidence that the integration of expression patterns from high traffic genes may support accurate prediction of HF. We quantitatively demonstrate that intra- and inter-module functional activity may be controlled by a family of transcription factors known to be associated with the prevention of hypertrophy.The systems-driven analysis reported here provides the basis for the identification of potentially novel biomarkers and understanding HF-related mechanisms in a more comprehensive and integrated way. Heart failure (HF) is a clinical syndrome that results from cardiac disease. HF can be characterized as the heart's inability to pump enough blood to meet physiological requirements. HF may be caused by cardiac injury  or by non-ischemic diseases (e.g. dilated cardiomyopathy). Independently of the etiology, HF is known to be the by-product of a large-scale, dynamic interplay of proteins, hormones and metabolites. These interactions are in turn brought about and controlled by a diversity of genes and molecular pathways responsible for different processes, which range from inflammation trough extracellular-matrix remodeling to angiogenesis. This motivates the development of approaches to the systematic, integrated analysis of protein interactions related to HF.Many questions connected to the elucidation of the complex molecular mechanisms spurring the emergence, progression and repair of cardiac malfunction remain to be answered. The increasing amounts of information about accepted and putative HF biomarkers and therapeutic targets, as well as of annotated datasets of protein-protein interactions (PPIs) in humans, offer new opportunities to understand HF within a systems biology framework .Advances in high-throughput technologies for the quantitative assessment of different "omic" information variables are fostering a more comprehensive, systems-level view of the PPIs involved in different physiological and pathological conditions. Over the past few years, larger amounts of experimentally-validated human PPIs -3 have bMajor steps forward in this area have been: a) the capacity to link clusters of highly-connected proteins (commonly referred to as "modules") within these networks and specific biological processes ,10 and bDespite the potential limitations related to knowledge incompleteness and uncertainty in the network inference process, the characterization of complex biological phenomena on the basis of functional modular architectures and topological parameters present us with new opportunities to improve our understanding of the evolution, operation and possible re-engineering of these systems ,13,14.Here we report the analysis of a PPI network in the context of human HF and in relation to diverse, complementary resources of "omic" information. This analysis aimed to characterize potential functional and structural patterns and associations, which may explain fundamental molecular mechanisms underlying HF, as well as the role of biomarkers, from a systems biology standpoint. The practical utility and potential biomedical relevance of the outcomes of this research are two-fold. First, the products of this research can be seen as a disease-specific knowledge reference for future research. Second, we offer testable hypotheses and predictions relevant to the discovery of potential novel biomarkers or targets based on an integrated network-based methodology. We aimed to uncover potential biologically-meaningful modules and inter-module relationships. Given that protein activity is influenced by different forms of regulation and between-process relationships, we investigated structural and functional features that can characterize the "centrality" or "traffic" mediation capability of network proteins. Finally, we addressed possible intra- and inter-module regulatory control mechanisms.Figure Before providing a more detailed description of these analysis phases, basic concepts relevant to network-based biology are defined as follows.x, its degree represents the number of interactions between x and any other proteins in the network. Nodes with large degree values are commonly referred to as "hubs". The traffic going through a network node, x, also referred to as its "betweeness centrality" [x, and which connect any two other nodes in the network. Thus, a "traffic value" can be seen as a numerical estimation of the communication-mediating capacity of a network node, and can be used to infer "cross-communication" hotpots or "bottlenecks" in the network.Networks are used here to represent direct, protein-protein interactions. Network "nodes" (or "components") and "edges" (or "links") represent proteins and interactions respectively. A group of highly-interconnected nodes can be defined as a "module". A module may be identified through network clustering, which meets specific statistical analysis criteria as explained below. There are various statistical or topological features that can be used to characterize the networks, its modules, and individual components. Two such fundamental features are the node "degree" and "traffic". The degree of a node refers to the number of edges associated with a node, i.e.,, given a protein, trality" ,16 is a Guided by recent reviews on cardiovascular disease biomarkers ,18, a liP values of those annotations that were found statistically-enriched in the set of known biomarkers. For each "omic" database, a list of functionally-related "candidate" genes was retrieved and ranked on the basis of their corresponding similarity scores. After performing a whole-genome search, a global ranking of candidate genes was obtained by combining all the database-specific similarity scores into a single similarity score based on order statistics, as proposed by [Genes and proteins functionally-related to the list of known biomarkers were searched in several external databases using the Endeavour system . These rposed by . Thus, tThe complete list of candidate genes obtained and their similarity rankings are available in the Additional file The combination of the list of HF biomarkers and the candidate genes defined the set of network seeds, which were used as inputs to the PPI network construction phase.PPIs associated with the network seeds were identified and retrieved from different public databases of annotated interactions. The PPI databases included in this analysis were: the Human Protein Reference Database (HPRD) , the genQ. The Q score measures the number of edges observed in a putative module in relation to the number of edges that would be obtained by randomly pairing the proteins in the module [Q score is defined as [The resulting network was digitally encoded, displayed and analyzed using different topological and functional features. Potential network modules were identified with a "greedy" network clustering algorithm . This ale module ,23. The fined as :numIME is the number of intra-module edges, and numE is the total number of network edges. The first term deals with the network investigated, while the second refers to a randomized version of the network: nodes are randomly connected with node degree preservation.Where Q score. This algorithm has allowed the identification of biologically-relevant modules, such as protein complexes and other functionally-related protein clusters, in different model organisms and humans [Thus, the module discovery algorithm searches for groups of inter-connected nodes that maximize the d humans ,5,10,24.To assess the potential application of the network-based predictions of putative biomarkers or targets, we analyzed their gene expression in the context of two groups of patients after acute myocardial infarction (MI): a group of 16 patients with HF, and a group of 16 non-HF patients. The non-HF patients exhibited preserved left ventricular (LV) systolic function and high ejection fraction (EF) after MI . The HF group presented impaired LV function and low EF . Patient characteristics are gathered in Table S3 (Supplementary Section).Acute MI was defined by the presence of chest pain <12 hours with significant ST elevation and increase in creatine kinase and troponin I to greater than 2-fold upper limit of normal levels. Blood samples were obtained at the time of mechanical reperfusion. EF was determined by echocardiography 1 month after MI. All patients signed an informed consent.Total RNA was extracted from 2.5 mL of whole blood by the PAXgene™ technology. Gene expression profiles of blood cells were obtained with genome-wide arrays .The protocol was approved by the local ethics committees  and written informed consent was obtained from all patients.The identification of genes functionally-related to the set of known HF biomarkers was implemented with the ENDEAVOUR system . Only fuP values describing the statistical strength of these associations were estimated with (two-tailed) Fisher's exact tests and corrected to account for multiple-hypotheses testing using the Benjamini & Hochberg adjustment procedure [P values are reported here and statistical significance is defined at the P = 0.05 level. Other GO annotation visualization tasks were carried out with GenNav v1.11 [Network module identification and traffic estimations were carried out with Polar Mapper . Networkrocedure . Unless av v1.11 . Whole-gav v1.11  and miRBav v1.11  databaset-test. Correlation between numerical variables was estimated with the Pearson's correlation coefficient. These calculations and statistical displays were implemented with the Statistica package [Independent, two-group statistical comparisons were performed with the Student's  package  and T-RE package . Classif package . Other nThe set of known HF biomarkers included 37 proteins (Table S1). Out of the top 100 genes that were found to be functionally related to the set of known biomarkers as a whole, 32 genes actually encoded known protein biomarkers. Thus, a total of 68 (unique) genes were found to be functionally relevant to the set of known biomarkers. The union of the set of known biomarkers and their functionally-related genes represented the "network seeds". The network seeds comprised a total of 105 proteins (Tables S1 and S2), and were used to assemble the PPI network as described in the Methods section.D(k)~k-γ, where k represents the degree values observed in the network, D(k) is the observed frequency of nodes with degree k, γ is known as the power-law exponent and ~ represents proportionality between these variables. On a logarithmic scale, as shown in Figure The PPI search and retrieval task generated a global network consisting of 772 nodes (proteins) and 1443 edges (PPIs). The resulting network comprises a single "core network" Figure , which iHereafter we focus our analyses on the core network. The core network not only concentrates the vast majority of nodes and edges, but also represents a single inter-connected network that preserves the degree distributions of the "global" set of nodes and interactions. By focusing on the core network, we also aim to identify biologically-meaningful interrelated groups of proteins based on the structural analysis of this network, e.g. detection of modules of inter-connected nodes.P values for multiple-testing. The vast majority of modules can be labeled with a variety of GO biological processes (16 out of 17 modules), and 14 of these modules can be assigned with confidence to specific cellular compartments. Module 17 did not show statistical enrichment of GO terms after making multiple-testing adjustments. Modules 10 and 14 did not include over-represented GO cellular localization terms at the nominal (P = 0.05) level. Overall, many of these modules tend to be implicated in cell adhesion and immune responses, with a significant number of proteins expressed in extracellular regions. However, a diverse range of specialized functional roles and cellular localizations were also observed. Examples of the former include: muscle contraction, tissue remodeling and signal transduction. Moreover, partial functional overlaps were observed. A more detailed description of these modules is available in the Additional file The clustering analysis of the core network generated 17 clusters of highly-connected proteins, which are hypothesized as potential functional modules in this network. Table These structural and functional features provide evidence of a hierarchical, module-oriented organization of the HF biomarker-centric network investigated here. This allows one to visualize their structure and functionality at a higher level of complexity, in which highly specialized, yet partially overlapping, functional modules interact and orchestrate their functional contributions to processes implicated in HF. Figure P = 1.35E-05), organismal catabolic process (P = 1.35E-05) and protein digestion (P = 1.35E-05). In addition, the extracellular space was the localization with the most significant enrichment of biomarkers (P = 1.09E-16). The module-based analysis detected significant associations that were not found in the list of biomarkers, e.g., tissue remodelling . We also observed that the statistical significance of the biological process terms overrepresented in the set of known biomarkers was weaker than that observed in the module-based analysis. For example, "blood pressure regulation" (P = 1.35E-05) was the most over-represented biological process in the set of biomarkers. In the network-based analysis, the enrichment of this term (Module 4) was statistically detectable at P = 3.33E-11. This indicates that our network-based approach was capable to recognise known relationships, as well as more specific ones, which could not have been detected by solely looking into the list of known biomarkers.A GO-based functional analysis of the set of known biomarkers reported the following biological processes as statistically detectable (strongest enrichments below level 3 of GO): blood pressure regulation (P = 2.7E-18), anatomical structure development (P = 9.8E-09) and coagulation (P = 2.4E-07). This module appears to mainly operate in the extracellular region part (P = 9.9E-47) and collagen (P = 6.9E-33), as defined by the GO.These modules can also be characterized on the basis of their cross-communication capability. This is important to identify potential communication "coordination" or "routing" centers, which may play major roles in the regulation of functional activity at a systems-level. The modules incorporate different numbers of inter-module interactions, ranging from 1 (Module 17) to 46 (Module 7) .Based on an estimation of "traffic" values Table , Module r = 0.92, P = 0.0001). Figure We found a strong linear correlation between node degree and traffic  and tend to exhibit greater traffic levels  in comparison to the other proteins included in the core network.A comparison between the group of known HF biomarkers and the other nodes in the network showed statistically detectable differences in terms of node degrees and traffic levels. Known biomarkers, in average, have more connections (P = < 1E-5) and 0.84 (P = <1E-6) for traffic and degree values respectively.Based on the evidence that HF biomarkers tend to be high-traffic nodes, we suggest that such a network-based feature may be used to point out to potential new biomarkers or targets. Table t-tests) reported very low differential expression of these genes between the clinical classes  and PTEN . Traffic values and levels of differential expression (as estimated by the t-statistic) are indeed uncorrelated . This shows how these potentially influential proteins could not have been detected on the basis of their gene expression alone. However, the potential of gene expression information of high-traffic proteins for biomarker or therapeutic target applications deserves further investigations. We found that patient classification models built on the expression values of TGFB1 and PTEN could correctly classify 72% of these samples using a linear support vector machine classifier (SVM) and leave-one-out cross-validation (LOO). To further explore the potential prognostic accuracy of these genes, we implemented SVM classifiers using high-traffic, known biomarkers as inputs . Table PTF-Module, of the associations between individual transcription factors and modules. Muscle creatine kinase (CKM) and interleukin 6 (IL6) are examples of HF biomarkers included in the regulated modules. ITGB1, four and a half LIM domains 2 (FHL2), collagen type VIII alpha 1 (COL8A1), CKM, and integrin alpha 2 (ITGA2) represent the highest traffic nodes controlled by the E2F family.Table This research shows how a biomarker-centric interactome related to HF exhibits a functional architecture of well-specified interconnected modules, which tend to be distributed across different cellular compartments. Inter-module communication is mediated by high-traffic or central proteins, which establish relatively short interaction paths between other proteins. Such a functional modularity and inter-module interplay suggest a coordinated, yet sometimes redundant, functional co-operation of processes and proteins relevant to the emergence and development of human HF. This coordinated cross-communication tends to be established by proteins that represent both high-degree and high-traffic network nodes. Moreover, these inter-module mediators appear to be responsible for linking, and possibly coordinating, the activities of multiple network modules associated with diverse functional processes and cellular localizations.Within the proposed methodological framework and disease investigated, the systems-level analysis is promising because it can detect potentially novel genes beyond the traditional differential gene expression analysis. Our results indeed proved that this was the case by offering potentially novel biomarkers that were not only biomedically-meaningful, but also capable to enable relatively accurate patient classification.Our results indicated that HF biomarkers implicated in different biological processes tend to represent high-traffic network nodes. High-traffic nodes can also be seen as bottlenecks or cross-talking hotspots because they represent components inter-linking different network regions and proteins. High-traffic nodes are found in a significant number of shortest paths connecting different network nodes. Previous studies have correlated high-traffic proteins with cell essentiality, pleiotropy and mediating communication roles between biological processes ,37.th and 29th respectively. Interestingly, ITGB1 is a receptor for FN1 and this ligand-receptor couple regulates leukocyte adhesion. Taking into consideration that inflammation, mediated by recruitment of circulating leukocytes to the heart through adhesion to the vessel wall, is an essential component of the pathogenesis of HF, our results suggest that ITGB1 and FN1 play key roles in the development and progression of HF. Indeed, accumulation of extracellular matrix proteins, such as laminin and fibronectin, is a hallmark of the development of HF [We also determined different proteins that deserve further research as potential novel HF biomarkers or therapeutic targets. These proteins have been shown to be involved in a wide range of processes, including blood vessel development and regulation of protein kinase activity. ITGB1 and FN1 were identified among the top-3 high-traffic proteins. Note that the rankings of ITGB1 and FN1 in the Endeavour-based prioritization were 66nt of HF , and ITGnt of HF . In addint of HF  and the nt of HF .FN1 is an acute-phase reactant synthesized by hepatocytes following injury, such as MI or ischemic stroke. This may explain why transcriptomic analysis of blood cells performed in the present study failed to readily detect FN1 expression and a potential association with clinical outcome of MI patients . Since FN1 does not belong to the family of known biomarkers of HF and is released in the plasma following injury, it will certainly be interesting to determine its prognostic performance and therapeutic utility in the context of HF.Matrix metalloproteinases (MMPs) constitute a family of matrix degrading enzymes that contribute to the development of HF . In the Here we put forward a hypothesis about why biomarkers tend to reflect relevant states in the emergence or progression of the disease. Biomarkers can mirror inter-module and global functional activities in an integrated, network-oriented fashion. We showed how the role of potential biomarkers or therapeutic targets may be explained on the basis of network architecture features. We found that potentially important proteins are encoded by genes with relative low differential expression between HF and non-HF samples (e.g. ITGB1 and FN1). Thus, these key proteins could not have been detected using standard gene expression analysis alone because of the subtle differences in expression levels between these prognostic classes. This underscores the importance of alternative, post-genomic views of the biomarker and target discovery process based on the analysis of multiple resources of "omic" information. Previous research has shown that high-degree and high-traffic network nodes may be encoded by genes that are not necessarily highly expressed or differentially expressed across case-control samples in different diseases ,8. This The lack of differential gene expression of the known biomarkers may be explained by the following reasons:1. Changes in the expression of these biomarkers do not necessarily rely on transcriptional modifications. Post-transcriptional and translational modification mechanisms may be also responsible for their differential expression. Many of these biomarkers are known to be differentially regulated at the protein expression level.2. Strong differential gene expression may not be possible to detect due to the small size of our dataset. Also note that the new putative biomarkers have levels of differential expressions that are not strong enough to have allowed their discovery through standard gene expression analysis alone.This motivated our systems-based discovery framework based on the integration of gene expression, protein-protein interactions, biological process annotations and DNA-gene interaction information.This investigation also suggests that intra-module functional activity may be regulated by a family of transcription factors previously associated with cancer progression, blood pressure regulation and the prevention of hypertrophy ,35,40. Mde novo predictions of interactions or functional annotations for specific proteins, we expect this potential deficiency to have a less significant effect on our findings. Our main research objective was to establish system-level associations and statistically detectable functional patterns, which may be used to characterize PPIs and processes relevant to HF. In addition, our analysis was not based on prior hypotheses or assumptions about the structure of the network. To reduce the potential number of false-positive interactions, we also focused on human-specific, experimentally-validated interactions. The strong statistical relationships found between network modules and known functional processes also suggest that the possible inclusion of false-positives interactions, as well as the possible exclusion of important biomarker-related proteins, would not represent a major drawback of this network.Key limitations of this research relate to information incompleteness and uncertainty. This investigation is based on a sample of current knowledge about HF biomarkers and the human interactome. Both information sources may represent either biased or incomplete information. Although we aimed to include a relatively large number of known biomarkers and functionally-related proteins to assemble the network, it is evident that false negative and false positive relationships may have influenced our findings. Nevertheless, because we do not use network topology information to make The notion that known biomarkers tend to be relatively more studied than other proteins may suggest a potential source of bias in our estimations of the correlation between the traffic and degree values of the biomarkers. However, it is important to stress that in the list of known biomarkers, there are proteins that exhibit high traffic values despite their relative low degrees. The biomarkers IL6 and CHGB illustrate this scenario. On one side, IL6 exhibits a traffic level higher than that obtained for CHGB. On the other side, CHGB has more interacting partners than IL6 (45 vs. 6 connections). Furthermore, only 6 out of the 20 highest-traffic proteins in the global network originate from the list of known biomarkers. These observations indicate that the potential bias toward well-studied proteins did not significantly impact our findings.In any case, we cannot rule out the possibility that a relative high number of false-negatives, i.e., true interactions or proteins relevant to HF that were not included in the network, may have influenced or biased the results reported here. In the long-term, the generation of more detailed descriptions of PPIs and the incorporation of emerging biomarkers, can provide conclusive confirmation or refutation of the results presented here. Future research should also incorporate additional information on biological context, such as tissue specificity or binding affinities.Another limitation relates to the transcriptomic analysis of blood cells from MI patients. First, this analysis included only a low number of MI patients and it will be necessary to validate the gene expression data in a larger population. Second, the use of readily available blood cells instead of cardiac biopsy to draw a biosignature of HF can be questionable. However, several studies have demonstrated the usefulness of blood cell transcriptomes to identify biomarkers in both the oncology ,49 and cOnly GO biological process and cellular localization annotations were examined to describe modules. This was done to emphasize the involvement of genes in entire, well-defined biological processes rather than in unique "activity" terms, as defined in the molecular function hierarchy (MF). Unlike MF terms, biological process terms can define involvement in more than one activity. In addition, MF terms tend to be good estimators of cellular localization terms, as reported in empirical studies . MoreoveWe also acknowledge that the biological relevance of network concepts, such as betweenness centrality or traffic, and their direct correspondence with biological information transfer phenomena remains a topic of discussion. Despite the need for additional research in this area, network-oriented concepts have proven to be powerful approaches to the generation of testable predictions about the functionality of complex biological systems in different model organisms and diseases ,50,52-54Key questions that deserve to be investigated are: What is the effect of incorporating multi-level interaction networks ?; how the HF-specific network relates to other phenotype-specific networks?; what place the HF-specific network occupies in a hierarchical organisation of the human interactome?; how such knowledge may facilitate the discovery of new key mediators of HF?. Answers to these questions will require larger, high-quality annotated PPI networks, with proven implications in human heart failure. Another relevant topic is the role of network-based differential expression for prognostic applications, as investigated in  for breaThe main findings of this investigation can be summarized as follows:1. Well-defined functional modularity characterizes a HF biomarker-driven PPI network. Such modules can be assigned to specific cellular regions and their functional roles tend to partially overlap.2. Inter-module relationships are established and facilitated by nodes exhibiting high-traffic or centrality betweenness in the network.3. HF biomarkers tend to represent network hubs and high-traffic nodes in this network.4. Putative biomarkers or targets may be predicted on the basis of their traffic level properties.5. High traffic proteins may be encoded by genes with relatively low differential expression between HF and non-HF in post-MI patients.6. The integration of expression data from high-traffic genes may provide the basis for potentially accurate prognostic systems.7. Intra- and inter-module functional activity may be regulated by a family of transcription factors known to be implicated in cancer progression, blood pressure regulation and the prevention of hypertrophy.In conclusion, this investigation contributes to the development of new global insights into complex molecular interactions and processes relevant to human HF. These results offer alternative views of crucial mechanisms driving biomarker-related functionality and organization at a systems level. Based on our analyses, HF biomarkers may be characterized as key coordinators of intra- and inter-module communication.FA conceived the integrated approach, implemented the computational methodology and wrote the manuscript. YD and DW provided biomedical advice, contributed interpretations and co-wrote the manuscript. All authors read and approved the final manuscript.Supplementary Data. File format: PDF Size: 123KClick here for file


A multicountry cohort study in sub-Saharan Africa by Landon Myer and colleagues reveals higher pregnancy rates in HIV-infected women on antiretroviral therapy (ART). With the rapid expansion of antiretroviral therapy (ART) services in sub-Saharan Africa there is growing recognition of the importance of fertility and childbearing among HIV-infected women. However there are few data on whether ART initiation influences pregnancy rates.We analyzed data from the Mother-to-Child Transmission-Plus (MTCT-Plus) Initiative, a multicountry HIV care and treatment program for women, children, and families. From 11 programs in seven African countries, women were enrolled into care regardless of HIV disease stage and followed at regular intervals; ART was initiated according to national guidelines on the basis of immunological and/or clinical criteria. Standardized forms were used to collect sociodemographic and clinical data, including incident pregnancies. Overall 589 incident pregnancies were observed among the 4,531 women included in this analysis . The rate of new pregnancies was significantly higher among women receiving ART (9.0/100 PY) compared to women not on ART (6.5/100 PY) . Other factors independently associated with increased risk of incident pregnancy included younger age, lower educational attainment, being married or cohabiting, having a male partner enrolled into the program, failure to use nonbarrier contraception, and higher CD4 cell counts.ART use is associated with significantly higher pregnancy rates among HIV-infected women in sub-Saharan Africa. While the possible behavioral or biomedical mechanisms that may underlie this association require further investigation, these data highlight the importance of pregnancy planning and management as a critical but neglected component of HIV care and treatment services.Please see later in the article for the Editors' Summary Human immunodeficiency virus (HIV) causes Acquired Immunodeficiency Syndrome (AIDS), which is a major global cause of disease and death. More than 33 million people around the world are infected with HIV, with nearly 5,500 dying daily from HIV and AIDS-related complications. HIV/AIDS is especially problematic in sub-Saharan Africa, where it is the leading cause of death. There is no cure for HIV/AIDS, but medicines known as “antiretroviral therapy” (ART) can prolong life and reduce complications in patients infected with HIV. 97% of patients with HIV/AIDS live in low- and middle-income countries. According to the World Health Organization, nearly 10 million of these patients need ART. As patients' access to treatment is often hindered by the high cost and low availability of ART, global health efforts have focused on promoting ART use in resource-limited nations. Such efforts also increase awareness of how HIV is spread . ART reduces, but does not remove, the chance of a mother's passing HIV to her child during birth.By the end of 2007, 3 million HIV-infected patients in poor countries were receiving ART. Many of those treated with ART are young women of child-bearing age. Childbirth is an important means of spreading HIV in sub-Saharan Africa, where 60% of all HIV patients are women. This study questions whether the improved health and life expectancy that results from treatment with ART affects pregnancy rates of HIV-infected patients. The study explores this question in seven African countries, by examining the rates of pregnancy in HIV-infected women before and after they started ART.The authors looked at the records of 4,531 HIV-infected women enrolled in the Mother-to-Child-Transmission-Plus (MTCT-Plus) Initiative in seven African countries. MTCT -Plus, begun in 2002, is a family-centered treatment program that offers regular checkups, blood tests, counseling, and ART treatment (if appropriate) to women and their families. At each checkup, women's CD4+ cell counts and World Health Organization guidelines were used to determine their eligibility for starting ART. Over a 4-year period, nearly a third of the women starting ART experienced a pregnancy: 244 pregnancies occurred in the “pre-ART” group (women not receiving ART) compared to 345 pregnancies in the “on-ART” group (women receiving ART). The chance of pregnancy increased over time in the on-ART group to almost 80% greater than the pre-ART group, while remaining relatively low and constant in the pre-ART group. The authors noted that, as expected, other factors also increased the chances of pregnancy, including younger age, lower educational status, and use of nonbarrier contraception such as injectable hormones.This study suggests that starting ART is associated with higher pregnancy rates in sub-Saharan Africa, nearly doubling the chances of a woman becoming pregnant. The reasons for this link are unclear. One possible explanation is behavioral: women receiving ART may feel more motivated to have children as their health and quality of life improve. However, the study did not examine how pregnancy desires and sexual activity of women changed while on ART, and cannot discern why ART is linked to increased pregnancy. By using pregnancy data gathered from patient questionnaires rather than laboratory tests, the study is limited by the possibility of inaccurate patient reporting. Understanding how pregnancy rates vary in HIV-infected women receiving ART helps support the formation of responsive, effective HIV programs. Female HIV patients of child-bearing age, who form the majority of patients receiving ART in sub-Saharan Africa, would benefit from programs that combine starting HIV treatment with ART with education and contraception counseling and pregnancy-related care.http://dx.doi.org/10.1371/journal.pmed.1000229.Please access these Web sites via the online version of this summary at HIV infection and AIDSInformation is available from the US National Institute of Allergy and Infectious Diseases on HIV InSite has comprehensive information on all aspects of HIV/AIDS, including a list of articles and other sources of information about the primary care of adolescents with HIVglobal AIDS epidemicA UNAIDS 2008 report is available on the sexual and reproductive health and HIVThe International Planned Parenthood Foundation provides information on HIV care and treatment programs in resource-limited settingsThe International Center for AIDS Care and Treatment Programs at the Columbia University Mailman School of Public health provides information to assist  By the end of 2007 there were almost 3,000,000 HIV-infected individuals receiving antiretroviral therapy (ART) in resource-limited settings Studies from Europe and North America indicate that HIV-infected women frequently become pregnant HIV care and treatment services are in a unique position to address the childbearing desires of HIV-infected individuals as well as to ensure safe pregnancy and delivery Despite the importance of fertility and childbearing in HIV-infected individuals, little is known about whether initiation of ART alters pregnancy rates among women in HIV care and treatment programs in resource-limited settings. One study from the United States suggested that ART use did not impact pregnancy rates www.mtctplus.org) Data for this analysis come from the MTCT-Plus Initiative, a multicountry HIV care and treatment program that utilizes a woman-centered, family-focused service model. The design and progress of the Initiative has been previously described  guidelines Upon enrollment women completed a short, standardized provider-administered questionnaire, which included demographic and socioeconomic information, partnership status, and obstetric history. At enrollment and each follow-up visit, clinicians completed a standardized medical survey including history of opportunistic infections, WHO stage, initiation or continuing use of ART, contraceptive use (including the use of specific barrier and nonbarrier methods), and pregnancy status (including pregnancy loss since the previous visit). The gestational age of pregnancies at detection was estimated clinically , and estimated delivery dates were calculated accordingly. CD4 cell enumeration was conducted at local laboratories.The conduct of the MTCT-Plus Initiative as a service delivery program with data collection for monitoring and evaluation purposes was approved by the Institutional Review Board of Columbia University.Data were analyzed using Stata version 10.0 (Stata Corporation). Timing of incident pregnancies was based on estimated conception dates, calculated 280 d before the date of delivery (estimated or observed). Pregnancy-free survival was calculated from 60 d following the delivery of the index pregnancy (during or after which women were enrolled into the program) until either: incident pregnancy, death, loss to follow-up, or censoring at the last clinical visit recorded before 31 January 2007. Participants were censored after their first new pregnancy during follow-up. Women who reported being sterilized or having a hysterectomy were excluded. Follow-up of women prior to initiation of ART was designated as the “pre-ART period,” while follow-up after initiation of ART is referred to as the “on-ART” period.Median values were compared using the rank-sum test and proportions were compared using chi-square tests replaced by Fisher's exact test for sparse data. Pregnancy rates were calculated with 95% confidence intervals (CIs), and crude rates were compared as incident pregnancy rate ratios (RRs). The time to pregnancy was compared between subgroups using proportional hazards models with the Breslow method for tie handling. The results of these models are expressed as hazard ratios (HRs) with 95% CI. In these models, the clustering of women within sites was accounted for using the sandwich-Huber-White robust variance estimation method n = 1,755) reported contraceptive use, including barrier methods , injectable hormonal methods , oral hormonal contraceptives , intrauterine device (IUD) use , or other methods . 259 women (6%) reported using condoms as well as a nonbarrier method.A total of 4,531 women enrolled at 11 sites between February 2003 and January 2007 were eligible for analysis. The median number of women enrolled per site was 410 . n = 2,551) were observed only during the pre-ART period; 580 women (13%) were observed only on-ART, and another 1,400 (31%) were observed both pre-ART and on-ART.Approximately half of the women . This total was comprised of 244 incident pregnancies over 3,747 person-years of observation during the pre-ART period  and 345 incident pregnancies over 3,817 person-years of observation during the on-ART period .Crude rates of incident pregnancy varied by site and country, with rates ranging from 3.29 pregnancies per 100 person-years in South Africa (95% CI 2.60–4.18) to 21.68 per 100 person-years in Rwanda (95% CI 17.10–27.50); the incidence of pregnancy was between 6 and 9 per 100 person-years in each of the remaining countries.p<0.001).Women who reported any contraceptive use during follow-up had lower pregnancy rates than those who did not. This association was due largely to the effect of nonbarrier methods, particularly injectable hormonal contraceptive use . Condom use was associated with a higher pregnancy rate compared to nonbarrier method use, but the rate was still lower than the rate among women reporting no contraception. Overall, 89 pregnancies (15%) occurred while women reported using a nonbarrier method, and 201 while women reported using condoms (34%).Lower current CD4 counts during follow-up were associated with lower pregnancy rates in both the pre-ART and on-ART periods. Compared to women with current CD4 counts <200 cells/µl, women with current CD4 counts ≥500 cells/µl had more than five times the rate of pregnancy during the pre-ART period ; this association was smaller but still significantly higher during the on-ART period .In a model examining pregnancy during the overall observation period (combining pre-ART and on-ART periods) adjusted for participant demographic and clinical characteristics, use of ART was associated with an almost 80% increased risk of pregnancy  . In the In separate proportional hazards models including the overall observation period stratified by country and adjusted for all covariates shown in The incidence of pregnancy among women enrolled in HIV care and treatment programs in sub-Saharan Africa has important implications for the health of women and their infants. Our findings indicate a high overall incidence of pregnancy (more than seven pregnancies per 100 woman-years of observation) and a significant association between the use of ART and increased incidence of pregnancy . Within 4 y of follow-up, one-third of women who initiated ART experienced a pregnancy, highlighting the urgent need to make pregnancy-related services a central component of HIV care and ART programs.A series of biological and behavioral factors may influence the association between the use of ART and increased incidence of pregnancy. It is possible that the rapid improvements in health and quality of life that take place with ART initiation lead to increased sexual activity, particularly for those with stable partnerships We do not have data on sexual activity or fertility intentions to help elucidate the reasons why ART use may increase fertility. There is evidence that fertility intentions change over the natural history of HIV infection. In keeping with the findings of qualitative studies, a recent analysis of HIV-infected and -uninfected women from Malawi Nonbarrier contraceptives, although used by only approximately one-fifth of women, were strongly associated with a reduced incidence of pregnancy. This reduction was driven chiefly by use of injectable hormonal contraceptives and the intrauterine device (IUD), underscoring the valuable role of these methods for pregnancy prevention. A substantial proportion of pregnancies in this cohort occurred during self-reported method use, suggesting that method failure, noncompliance, or over-reporting of method use may occur in many instances. Among women reporting any contraceptive method, the highest pregnancy incidence was observed among those using condoms, highlighting the limited effectiveness of condoms as a stand-alone contraceptive method and emphasizing the importance of dual method use. The association between oral contraceptive use and higher pregnancy rates compared to other nonbarrier methods may reflect nonadherence with oral contraceptive pills or drug interactions between antiretroviral and oral contraceptive drugs The high incidence of pregnancy, coupled with the low prevalence of contraceptive use, underscore the importance of addressing fertility-related issues within HIV care and treatment programs in sub-Saharan Africa. The increasing rate of new pregnancies we observed with duration of follow-up on-ART is consistent with findings from prior studies The data presented in this paper were derived from systematic, standardized data collection in an HIV care and treatment program across seven countries in sub-Saharan Africa. The clinical sites that participated in this initiative are broadly representative of public sector HIV care and ART delivery programs, and thus the findings are likely to be more generalizable than evidence from small research cohorts A distinctive strength of these data is that the analyses included women followed up for incident pregnancies both before and after ART initiation, as sites enrolled women into ongoing HIV care regardless of disease stage or ART eligibility. However, our findings should be interpreted in light of several limitations. Although data were collected on standardized forms, the ascertainment of new pregnancies was based on patient self-report and clinical assessment rather than laboratory assays, a factor that may have led to the underdetection of pregnancies. A proportion of pregnancies are likely to be lost prior to clinical detection, which may occur more frequently in HIV-infected women In summary, our findings indicate that the incidence of pregnancy increases significantly after the initiation of ART. Although the precise reasons for this increase require additional research, HIV care and treatment programs have an important opportunity to address women's fertility intentions and to shape their services to address the needs of the women and their families over time.


Although work related risk factors associated with Cardiovascular Diseases (CD) have been well researched, there is no detailed knowledge regarding disparate occupational groups each with a different risk exposition. Therefore, two occupational groups (chefs and office workers) were compared with a focus on nutritional and psychosocial factors.12, folic acid; C-reactive protein, uric acid, red blood cell fatty acids, plant sterols, antioxidative capacity and oxidative stress.Two groups of subjects were tested for work and diet-related risks of CD (45 chefs and 48 office workers). The groups matched both for gender  and age (30 to 45 years). The study included a medical check-up, bioelectrical impedance analysis as well as an evaluation of questionnaires on health, nutritional behaviour and coping capacity. In addition, volunteers were required to compile a 7-day-dietary-record and collect their urine 24 h prior to their check-up. Blood samples drawn were analysed for glucose and lipid metabolism, homocysteine, vitamin BOn average, the chefs showed one risk factor more compared to the office workers. The most frequent risk factors in both groups included overweight/obesity  and elevated TC , more office workers suffered from hypertension (37.5%).trans fatty acids were found more frequently in the red blood cell membranes of office workers. While there were no significant differences in analysed plant sterols between the two occupational groups, 7,8-dihydro-8-oxo-2'-deoxyguanosine was significantly increased in office workers.Chefs showed significant higher concentrations of saturated fatty acids and oleic acid, whereas docosahexaenoic acid, Omega-6- and Concerning the work-related psychosocial factors, the chefs were characterised by a stronger subjective importance of work, a greater degree of professional aspiration and enhanced efforts at perfectionism at their workplace.The chefs in the study bear a higher risk of CD compared to the office-workers. Although, CD is not exclusively a result of workplace-conditions, study results show that work-related influences can not be ignored. Thus, prevention of CD may be an important task attributable to occupational physicians. Atherosclerosis due to inappropriate nourishment together with a lack of physical activity is responsible for of approximately half of all the deaths of adults aged over 60 in industrialized nations worldwide .Compared to former generations, today there is a readily available greater food supply and less physical activity in leisure and labour time. However, people today are confronted more frequently with complex psychosocial demands .In addition to genetic and lifestyle factors, work-related influences are linked to a higher risk for diseases. Disability-statistics available from German Health Insurance Funds provide an insight into the prevalence of work-related risks of CD, yet such data gives no evidence of disease pathogenesis.Chefs were chosen as a subject group for this study in the interest of the Preventive Department of the German Employers' Liability Insurance Association for Food Industry and Restaurants (Berufsgenossenschaft Nahrungsmittel und Gaststätten). The aim was to investigate whether chefs have a higher cardiovascular risk due to their job requirements. Certain aspects of their occupation such as the irregular working hours, erratic mealtimes and consumption of rich food may lead to a higher disease risk due to a higher energy intake, in particular of fat. Furthermore, psychosocial factors leading to mental stress instigated by working under time pressure during peak periods and complaints from discontented customers may additionally add to the risk of disease.Regarding the work-related exposition of chefs, the focus was placed on atherosclerotic risk factors affected by nutrition. In addition to the analysis of the common risk parameters such as abnormal fasting glucose value and lipid metabolism, there are a large number of blood tests which can demonstrate the CD risk. Detailed parameters of lipid metabolism, especially lipoprotein (a) (Lp(a)) and apolipoprotein B (Apo B) were of interest to the study. The thrombogenic and atherogenic effects of Lp(a) are due to its close homology with plasminogen , whereas12). Therefore, an intake of both vitamins is necessary to prevent the accumulation of higher levels of homocysteine [12) [High concentrations of the amino acid homocysteine have been implicated in the progression of CD owing to its association with a large number of atherogenic effects such as degenerated vascular architecture and endothelial function, elevated oxidative stress, and higher risk of thrombosis. Homocysteine, resulting as an intermediate product of the methionine metabolism, is re-methylated in presence of folic acid and cobalamin (vitamin Bcysteine  since inine [12) .Uric acid is also discussed as a risk factor as it increases the blood pressure due to its stimulating effect on the proliferation of smooth vascular muscle cells and the activation of circulating platelets .Finally, inflammatory processes are believed to play a role in the development of atherosclerosis. The measurement of C-reactive protein, a highly sensitive marker of inflammation provides a quick and a simple method of risk prediction .Other risk factors associated with nutrition, include the fatty acids that are a part of the lipids regulating the structure and function of biological membranes . TherefoFor a long time saturated fatty acids (SFA) have been categorised as atherogenic because of increasing LDL-C levels although current case-control-studies give no significant evidence for this consideration. In fact, SFAs even seem to have a positive influence on HDL-C, when they are exchanged with carbohydrates on an isocaloric basis ,13.Omega-3 fatty acids are the most important fatty acids having a preventive function for CD. Both eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) are e.g. found in fish oil or can be synthesised from the essential alpha linoleic acid (ALA). The synthesis of the two Omega-3 fatty acids is dependent on the nutritive supply of ALA, found in vegetable oil, e.g. in linseed, hempseed and walnut oil . The protrans fatty acids (TFAs) are associated with a higher risk of CD due to hazardous effects such as negative influences on lipoprotein metabolism, pro-inflammatory effects, elevation of oxidative stress as well as deteriorated fluidity of membranes and insulin sensitivity et cetera[In contrary et cetera.Sterols are essential components of cellular membranes of plants differing from cholesterol by possessing an additional methyl or ethyl group. They have positive influence on human cholesterol levels as they compete for the same resorption protein (Nieman-Pick C1 like 1 protein) and are selectively removed back to the intestine .Both oxidative stress and the concentrations of antioxidants provide additional information concerning the risk of CD. Oxidative stress contributes to atherogenesis due to oxidation of LDL-C, thereby influencing the genesis of many degenerative diseases . The humThe study groups consisted of chefs (N = 45) and office workers (N = 48). All subjects were male and aged between 30 and 45 years. The volunteers were informed of the purpose, course and possible risks of the study and all subjects signed the consent form. The study was approved by the ethics committee of the Friedrich Schiller University .Data from the chef group were collected between April 2004 and April 2005. The data form the group of office workers were collected between August and November 2005. All subjects underwent a detailed medical check-up with a bioelectrical impedance analysis. The volunteers were interviewed with regards to their common health state and nutritional pattern, including filling-out a food-frequency-questionnaire. By using the measure of coping capacity questionnaire (MECCA), items of work-related behaviour and work experience were determined. The subjects were required to compile a 7-day-dietary-record and to collect their urine 24 h in advance to the medical check-up.Blood samples were drawn between 7:00 and 8:00 a.m. by venipuncture into 9 monovettes  after overnight fasting. Four of the monovettes contained EDTA as an anticoagulant for plasma preparation. Blood samples were prepared and stored at -25° or -80°C until analyses.Three urine-monovettes  of 24-h urine were collected and stored at -25°C until analyses.12, folic acid; C-reactive protein (CRP) and uric acid.The following blood parameters were analysed in a clinical laboratory by using standardised methods: fasting glucose and HbA1c; total cholesterol (TC), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), triacylglycerides (TAG), Lp (a), ApoB; homocysteine, vitamin BBLIGH & DYER procedure [The analysis of red blood cell fatty acids was carried out qualitatively as follows: after initially isolating red blood cell membranes (RBCM) in phosphate buffer , the samrocedure  with metrocedure .The preparation and measurement of plant sterols in human plasma is described elsewhere .The measurement of hydropholic Trolox equivalent antioxidative capacity III (TEAC III) was carried out as described by Re et al. Total pIn order to determine the styles of coping with occupational burden, the measure of coping capacity questionnaire (MECCA)  was emplth or the 75th percentile (P25-P75), respectively. The comparison of the professional groups was analysed using Mann-Whitney-U test. Pearson's χ2 test was used to estimate the frequency of distribution in subgroups. Fisher's exact test was applied in subcategories with n < 5. Coefficient of rank correlation was calculated by Spearman.Statistical analysis were performed using SPSS for Windows, Version 11.5.1 . The assessment of normal distribution was carried out by Kolmogorov-Smirnov test. P < 0.05 was considered as significant. Due to not normally distributed variables, values are presented as the median and the 25There were no significant differences in BMI, body fat and lean body mass  were accumulated, a significant difference between the occupational groups was revealed . On average, chefs showed one more risk factor than office workers (5 versus 4). The most frequent risk factors in both groups were overweight/obesity and increased TC. Furthermore, chefs often had higher CRP-concentrations whereas more office worker suffered from hypertension Table .12 and folic acid-states and therefore, showed fewer cases of increased homocysteine-concentrations . In contrast, oleic acid (OA), the most prevalent Omega-9-fatty acid occurred more significantly in the red blood cells of chefs Table . There wChefs showed significant lower hydrophilic Trolox equivalent antioxidative capacity (TEAC III) in plasma than office workers, but in contrast significant higher total phenolic (GAE) levels. Significant higher concentrations of plasma α-tocopherol were detected in the chef group Table . The bio2 test; Table A remarkable difference between the occupational groups was found on evaluating the MECCA questionnaire. Although 50% of chefs showed the healthy-ambitious coping style type G, they were significantly more often assigned to the CD-related type A than office workers  while their intake of fat, SFAs as well as carbohydrates, especially simple sugars are considerably higher. All these facts indicate an inferior nutritional behaviour with all its negative effects on the risk of CD such as metabolic syndrome etc -34.The higher percentage of DHA in the RBCM of office workers might be explained by the higher socio-economic situation in this group. Dietary supplements can be ruled out as being causal since no subject supplemented with Omega-3-fatty acids.Heart and Soul Study. Patients with coronary artery diseases and a lower socio-economic status defined according to household income, education, occupation, and housing status showed lower concentrations of DHA and EPA in RBCM. The authors state that this was due to a lower fish consumption [Similar results were found in the sumption . HoweverThe Omega-6-fatty acids in RBCM were also significantly different between the two observed occupational groups. A higher percentage of LA as well as AA were revealed in RBCM of office workers. Increased levels are caused by a higher intake of fatty acids in food or by the conversion of LA to AA. In a normal Western diet, the conversion too AA usually exceeds the dietary supply of AA . HoweverA higher intake of TFA-rich food  and therefore, an increased concentration of TFA were expected in the RBCM from the chefs. On the contrary, the chefs showed a significant lower sum of TFA compared to the office group. Thus, there is no higher risk resulting from TFAs for chefs.The same levels of plant sterol concentrations found in the two groups might be explained by a lesser influence of nutrition and lifestyle on plant sterol concentrations in plasma. Due to the fact that the intake of plant sterols in a common Western diet, estimated to be between 200 - 400 mg/d, is potentially to low for the accumulation of plant sterols in plasma, assumed that individuals are not suffering from genetic disorders like phytosterolemia ,38.However, is there at all a need to enrich food with plant sterols? Current research in this field provides controversial data. For example, individuals with phytosterolemia have a higher risk of CD because of the atherogenic potential of plant sterols in higher plasma concentrations ,40. It iThe results correlating to the analysis of oxidative stress and the concentrations of antioxidants in plasma were unexpected. Office workers either smoked less or only occasionally. Moreover, they consumed more vegetables and fruits in comparison to chefs. Nevertheless, this group showed significantly higher concentrations of 8-oxodG in the 24-h-urine samples. The Spearman correlation did not show an association between 8-oxodG and the hydrophilic TEAC III in plasma (data not shown). Thus, antioxidative capacity in food does not correlate very well with those of individuals and may be influenced by several as yet not well understood factors such as bioavailability, absorption, metabolism and the antioxidative capacity of different total phenols in vivo. Further studies are needed to determine the effect of antioxidants .The inverse relationship of TEAC III and total phenols between the two occupational groups can be explained by an increased antioxidative capacity without a concomitant increase in total phenol levels. The last one may depend on varying composition of polyphenols in plasma . FurtherThe remarkably high score of MECCA type A in the chef group was comparable to the teaching profession, which has scored the highest percentage of MECCA risk types A and B (30% in each case) of all occupational groups analysed to date .The results indicate that chefs encountered workplace-related stressors. This is confirmed by the results from the stress self-assessment test. The stress suffered by the chefs was more frequently job-related rather than resulting from other causes. They apparently responded to this stress-related situation by increasing their efforts at work which in turn, lead to a lesser ability to distance themselves from their work. The fear of job loss in the group of chefs seemed to be another reason for their tending to MECCA type A.It is worth noting that a meta-analysis of fourteen prospective cohort studies demonstrated an average 50% excess risk for coronary heart diseases among employees with workplace stress ,46.The chefs involved in the study carry a higher risk of CD than the comparison group of office workers. The nutritional behaviour of chefs consisting of food rich in fats, particularly animal origin is associated with higher TC, Apo B and higher rates of SFAs in red blood cell membranes as well as higher concentrations of uric acid. In addition, there were more smokers in the chef group.Chefs have a stronger work-related dedication, which is displayed by a significant higher effort at achieving perfection, more willingness to perform strenuous work and an increased occupational aspiration. These factors can lead to psychological stress, which is strongly associated with a high risk of CD.The differences which where found between the two groups are probably not only work-related. The study design and method were not able to appraise a clear relationship between workplace-conditions and analysed risk parameters. However, according to statements of the investigated chefs, their nutritional behaviour is not only work-related influenced but also chosen willingly.Although CD are not only caused by workplace-conditions, work-related influences can not be ignored. The cooperation with Employers' Liability Insurance Associations and Health Insurance Companies as well as employers and employees is the base for a multi-causal approach for the prevention of CD. Occupational physicians provide the necessary link between the involved parties and are in an excellent position to give advice to both employers and employees regarding better working conditions, a healthier lifestyle  and provide an effective job motivation.Finally, research on other occupational groups with reference to the risk of CD is necessary. A subsequent follow-up study after ten years is also essential to provide data on the progression of the risk in the reviewed groups.(ALA): alpha linoleic acid; (AA): arachidonic acid; (CD): cardiovascular diseases; (DHA): docosahexaenoic acid; (EPA): eicosapentaenoic acid; (FAME): fatty acid methyl ester; (GAE): gallic acid equivalents; (LA): linoleic acid; (OA): oleic acid; (RBCM): red blood cell membranes; (SFA): saturated fatty acids; (TFA): trans fatty acids; (TEAC III): trolox equivalent antioxidant capacity III; (8-oxodG): 7,8-dihydro-8-oxo-2'-deoxyguanosine.The authors declare that they have no competing interests.DH and MS carried out the study, participated in the sequence alignment and drafted the manuscript. RG participated in the design of the study. SK participated as a counsellor relating to laboratory techniques and scientific background. GJ contributed to the study design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Accumulating genetic and functional evidence point to ETV6 as being the tumour suppressor gene targeted by the deletions at chromosome 12p12-13 found in various cancers, particularly childhood leukemia. ETV6 is a ubiquitously expressed transcription factor (TF) of the ETS family with very few known targeted genes. We recently compiled a list of 87 ETV6-modulated genes that can be classified into a number of subgroups based on their coordinated expression patterns. In the present report, we hypothesized that genes presenting a similar profile of modulation could also share biological features, promoter sequence similarities and/or, common transcription factor binding sites (TFBSs). Using an exploratory approach based on hierarchical clustering of expression data, Gene Ontology (GO) terms, sequence similarity and evolutionary conserved putative TFBSs, we found that many genes presenting a similar expression profile also share biological features and/or conserved predicted TFBSs but rarely show detectable promoter sequence similarities. We also calculated the proportion of ETV6-modulated genes that have any conserved TFBSs of the Jaspar database in their regulatory sequence and compared these proportions to those calculated for two other gene lists, ETV6 non-modulated and ETS-regulated. We found that the NF-kB, c-REL and p65 TFBSs, which all bind TFs of the REL class, were under-represented among the ETV6-modulated genes compared to the ETV6-non-modulated genes, while the Broad-complex 1 TFBS appeared to be over-represented. NF-Y and Chop/cEBP TFBSs were over-represented in the promoters of ETV6-modulated genes compared to ETS-regulated genes. These analyses will help direct further studies intending to understand the role of ETV6 as a transcriptional regulator and aid in constructing the ETV6-regulatory gene network. Transcription is one of the main levels of regulation of gene expression. Amongst the players involved in this process, transcription factors (TFs) are trans-elements regulating the rate of transcription of particular genes by binding to cis-acting elements known as transcription factor binding sites (TFBSs). However, the whole repertoire of genes targeted by a single TF is not necessarily modulated at the same time or in the same direction .The mechanisms underlying the co-regulation of subsets of genes are still largely elusive. It is currently thought that particular TFs act together in different combinations and that, depending on these combinations, they co-ordinately regulate particular groups of genes.e.g. . It is aIn earlier studies, we and others provided evidence that ETV6, a TF of the ETS family, was inactivated in childhood pre-B acute lymphoblastic leukemias . In an aIn this study, we used the ETV6-modulated gene list along with computer-based tools such as ConSite, Dialign as well as various clustering methods to address the following questions: first, do genes with similar modulation profiles share 1) biological features, 2) sequence similarity in their promoter, and/or 3) related TFBSs?; and secondly, are certain TFBSs over- or under-represented in ETV6-modulated genes compared to ETV6-non-modulated and ETS-regulated genes? The methodological approaches presented in this exploratory study should be useful in providing the groundwork for future studies aiming at deciphering the functional and regulatory networks of TFs.Taking advantage of an ETV6 inducible system and of microarray analyses, we recently reported a list of 87 genes modulated in time (48 h) after the induction of ETV6 . AccordiIn order to group the genes according to their modulation of expression over time, we chose hierarchical clustering (HC) since, as opposed to other methods like K-means clustering, the resulting trees do not change over repeated analyses with the same data. The expression trees were then divided into expression groups and a colour code was assigned to each group . Two difIn addition, we also built trees with terms corresponding to each of the GO organizing principles  taken independently or in a pair-wise combination. A summary of the resulting significant associations are listed in To determine the nature of the GO terms responsible for the co-clustering of genes by expression profile, we selected the terms that were related to at least two genes in every association groups . SupplemTo assess whether genes with similar expression profiles also shared promoter sequences we clustered ETV6-modulated genes according to their promoter sequence (1 kb upstream of exon 1) similarities using the Dialign alignment tool , and theNext we wanted to test the possibility that ETV6-modulated genes share common TFBSs. Since the short and degenerate nature of TFBSs leads to the prediction of a high rate of false positive putative binding sites . For each gene list, the proportion of genes presenting any particular TFBS was calculated and compared between groups in a pairwise fashion using the Fisher’s exact test. Again, FDR was used for multiple testing correction, but note that fewer results remained statistically significant compared to the clustering association studies. CF2-II as well as TFBSs from the REL family  were under-represented in the ETV6-modulated group compared to the ETV6-non-modulated genes and NF-kB and p65 were also under-represented in the ETV6-modulated genes vs. ETS-regulated genes . We obsein silico exploratory analyses to assess whether genes with similar expression profiles also share either biological or promoter features. Three different clustering methods were used to group the genes according to their expression profiles. Since each method employs a different distance metric, reflecting different yet complementary concepts when used in combination, they should allow for a more stringent analysis of the data  following induction of ETV6 in HeLa cells . The useHierarchical clustering of ETV6-modulated genes based on expression data was done using three different methods, each one providing different conceptual information . The firGO terms corresponding to various organizing principles , were retrieved from the GO database . Binary In each gene, a portion of the DNA sequence corresponding to 3 kb upstream and 0.5 kb downstream of the transcriptional start site from mouse and human (Genbank database) were submitted to ConSite (asp.ii.uib.no:8090/cgi-bin/CONSITE/consite/)  in orderhttp://bibiserv.techfak.uni-bielefeld.de/dialign/) has the option to give back coordinates, which were used to build a hierarchical tree by using the Phylip program (http://evolution.genetics.washington.edu/phylip.html). Same analysis was also performed using 1.5 kb of alternative promoter sequences (sequences with highest confidence and clone numbers were used) from DBTSS (http://dbtss.hgc.jp/index.html).When data was available in GenBank, up to one kb of DNA sequence upstream of exon 1 of the ETV6-modulated genes was submitted to Dialign  for promGenes were grouped based on their expression data and a colour code was assigned to each group  and 2. Ghttp://genome.ucsc.edu/) combined with the Galaxy tools (http://main.g2.bx.psu.edu/) and their proportions in the lists were also statistically compared. FDR was used for multiple testing corrections of probabilities.The proportion of genes presenting any TFBS in the 5’ regulatory region (3 kb upstream and 0.5 kb downstream of exon 1) of the ETV6-modulated gene list was compared to that of ETV6-non-modulated and ETS-regulated gene lists. All putative TFBSs of the 5’ region that were conserved between mouse and human were retrieved for all the genes from all three lists using ConSite as mentioned above. The proportion of genes sharing a given TFBS as identified by ConSite (JASPAR database) was assessed for each gene list. When these proportions appeared to vary from one gene list to another, they were compared in a pair wise fashion using the Fisher’s exact test to test for statistical significance. Conserved TFBSs found to be differentially represented in any list using ConSite were also probed independently in each list using the Tables Browser tools from the UCSC Genome Browser  were used to build a hierarchical tree in which genes that co-cluster together are found closer together in the tree. B. Terms involved in the associations (Supplementary Table 1S) were counted and plotted by frequency of occurrence.Table 1S.


Complete resection seemed to be curative in patients with Castleman disease of any location but the disease is likely to be reactive in its pathogenesis. The relation between Epstein-Barr virus and Castleman disease has not been elucidated. We tried to define the role of Epstein-Barr virus in the pathogenesis of Castleman disease.20 cases of Castleman disease were retrospectively reviewed from 1993 to 2006. At least 2 to 4 representative sections of formalin-fixed, paraffin-embedded specimens from each patient were obtained to examine the presence of EBV and its localization by hematoxylin-eosin stain, immunohistochemistry, polymerase chain reaction and In-situ hybridizationHyaline-vascular type was diagnosed in 18 cases, plasma cell type in 1 and mixed type in 1 case. All of them were positive for Epstein-Barr virus confirmed by PCR. For tumors that EBER(Epstein-Barr early region) signals mainly localized in the germinal centers have increased vascularity than cases with EBER detected in inter-follicular areas.There is a strong association between Castleman disease and Epstein-Barr virus. EBV may have a potential role in angiogenesis of Castleman disease. For smaller lesion with high activity of angiogenesis but not amenable for curative resection, anti-angiogenesis medications may have a potential role to control the disease. Castleman disease (CD) is a rare and usually benign lymphproliferative disease. It has many synonyms, including giant lymph node hyperplasia, angiofollicular hyperplasia, lymph node harmatoma, benign giant lymphoma, and follicular lymphoreticuloma. Castleman et al first described it as a new disease entity in 1956 It has 2From January, 1994 to December, 2005, we retrospectively reviewed 20 cases with the diagnosis of Castleman disease managed in Mackay Memorial hospital in Taiwan. We retrieved all formalin-fixed, paraffin-embedded specimens of these cases from Pathology specimen bank. The diagnosis of these cases was confirmed both clinically and histologically using the criteria described by Keller in 1972[For each case, we selected at least 2 to 4 representative formalin-fixed, paraffin embedded specimens. For each specimen, 7 to 8 sections of 5 to 10 μm thickness were obtained for crude extracts of subsequent study. One section of each specimen was stained by Hematoxylin-Eosin, one for immunohistochemistry stain while another one section was for In-situ hybridization. Immunohistochemistry was performed in selected cases. After dissolving in xylene, DNA was then extracted from tissues of the remaining 4 sections by alcohol-phenol extraction method (QIAamp DNA Mini Kit).For hyaline-vascular type and mixed type, we divided these cases into highly vascular and less vascular according to HE stain. If more than 50% follicles were surrounded by proliferated vessels, these cases are defined as hypervascular cases. If less than 50% follicles are surrounded by the proliferated vessels, these cases were defined as less vascular cases.For confirming the acceptability of the DNA as amplification template after extraction, we used polymerase chain reaction (PCR) assay by one primer pair designed to amplify the genes corresponding to β-globin region of human genomic DNA. The expFor detection of EBV in our specimens, we selected 2 target genes for amplification by PCR. They are EBER (EB early region) and EBNA-2 (EB nuclear antigen),10 PrimeBecause of high copy number of EBER during latent EBV infection, we selected EBER as the target for In-situ hybridization, a method previously described. The posChi-square test and Student T-test were used to compare the characteristics of both groups. A P-value less than 0.05 is considered as significant.There are 19 cases included in this study with 17 hyaline-vascular CD, 1 plasma cell type and 1 mixed type. The only one plasma cell type is also a multicentric type while the remaining 18 cases are of unicentric type. There are 10 males and 9 females with mean age of 30.7 years (range: 9 to 68). There are 8 patients having CD in the thorax, 5 in the neck, 2 in the retroperitoneum and 4 in other locations . All specimens were found to be EBV-positive by PCR amplification of both EBER and EBNA-2. 10 specimens were classified as hypervascular CD and 8 specimens were classified as less vascular CD. The demographics, results of EBV detection and localization and extent of vascularity of each case are listed in table We excluded one case of plasma cell type when we compare the localization of EBV and the extent of vascularity because it lacks vascular component. We divided these cases into 2 groups, with one that EBER signals were mainly localized in the germinal centers and another one having signals mainly in inter-follicular areas. There are 12 cases having EBER signal in areas of germinal centers and 6 cases having signals in inter-follicular areas. In the group of EBV localized within germinal centers (GC-EBV), patients tend to be older , the size of the tumor is smaller p = 0.003) and more likely to be highly vascular (p = 0.01). There is borderline significance that females tend to have EBV in the follicular space.(p = 0.046) However, there is no significant difference in the locations of CD. The results of comparison are listed in Table  and moreEpstein-Barr virus is a lymphotropic herpesvirus and its infection rate varies geographically. Usually, the clinical course after infection by EBV is indolent. However, there are strong associations between EBV and infectious mononucleosis, nasopharyngeal carcinoma, Hodgkin's disease and Burkett's lymphoma. In addi7 copies per infected cell during latency, which makes them excellent targets for detection[There are many strategies to detect the presence of EBV in formalin-fixed, paraffin-embedded specimen. The role of polymerase chain reaction in detection of EBV in paraffin-embedded specimen is quite limited because EBV is ubiquitous. Without the information of its localization, interpretation of positive result becomes difficult and is often meaningless. In-situ hybridization provided additional information about the localization of EBV and may help us to clarify its relationship with 2-dimensional structures within pathologic lymph node. Gene expressions of EBV during latent phase are so limited that detection is difficult. EBERs, however, are polymerase III transcripts and abundantly expressed in EBV-infected cells, up to 10detection.Interleukin-6 production in abnormal lymphoid follicles by germinal center cells was identified by both IHC stain and ISH in previous studies,16. In aMurray et al published their experience of 15 cases of Castleman disease studied by IHC stain and in situ hybridization. They demonstrated that only 6 out of 15 cases were found to have scanty EBV-positive cells. And these EBV-positive lymphocytes are few, confined to the inter-follicular areas and never seen in germinal centers. Thus, they concluded that EBV is not associated with Castleman disease. In this study, the results of our observation are greatly different. EBV-positive cells were abundantly found in 19(100%) cases by both PCR and ISH. 12 out of 19 cases were found to have rich EBV-positive cells in germinal centers while remaining 7 cases in the inter-follicular areas. We also observed that more EBV-positive cells in germinal centers accompanied with more vascular proliferations in these tumors, suggesting that localization of EBV in germinal centers may relate or contribute to some extent of vascular proliferation in hyaline-vascular CD. EBV may promote angiogenesis through expression of Latent Membrane protein-1 (LMP-1) induced production of Interleukin-8, as in the case of nasopharyngeal carcinoma[For surgeons, complete resection is not possible in every case. Knowing the underlying pathogenesis may reveal potential alternative or adjuvant treatment when complete resection is not possible or a patient could not tolerate major resection. Most lesions that could not be completely resected are those deep in the thoracic cavity. The new findings may help thoracic surgeons to consider the applications of either alternative or adjuvant therapy. Since many medications and even radiation therapy could block angiogenesis. This may explaine why some reports showed radiation and chemotherapy can control but not cure the disease.One limitation of this study is lack of non-CD tissues in each case because no or only scanty non-tumor tissues were preserved in our Pathology specimen bank. Near all formalin-fixed, paraffin-embedded tissues we obtained contain only pathologic lymph nodes, which limited us to evaluate the role of EBV in both tumor parts and non-tumor parts of each patient.Epstein-Barr virus is highly associated with Castleman disease. More EBV-positive cells in germinal centers are associated with increased vascularity and smaller tumor size. EBV may play a role of angiogenesis in early stage of Castleman Disease. For lesions with high activity of angiogenesis but not amenable for curative resection, strategy for anti-angiogenesis may have a potential role to control the disease.The authors declare that they have no competing interests.CH Chen is the main author to design the study, write the article and submit the manuscript. TT Hung helped analyze the results of the study. HC Liu and TP Liu participated in reviewing the manuscript. All authors have read and approved the final manuscript.Written informed consent was obtained from the patient for publication of this report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal


In the field of auditory neuroscience, much research has focused on the neural processes underlying human sound localization. A recent magnetoencephalography (MEG) study investigated localization-related brain activity by measuring the N1m event-related response originating in the auditory cortex. It was found that the dynamic range of the right-hemispheric N1m response, defined as the mean difference in response magnitude between contralateral and ipsilateral stimulation, reflects cortical activity related to the discrimination of horizontal sound direction. Interestingly, the results also suggested that the presence of realistic spectral information within horizontally located spatial sounds resulted in a larger right-hemispheric N1m dynamic range. Spectral cues being predominant at high frequencies, the present study further investigated the issue by removing frequencies from the spatial stimuli with low-pass filtering. This resulted in a stepwise elimination of direction-specific spectral information. Interaural time and level differences were kept constant. The original, unfiltered stimuli were broadband noise signals presented from five frontal horizontal directions and binaurally recorded for eight human subjects with miniature microphones placed in each subject's ear canals. Stimuli were presented to the subjects during MEG registration and in a behavioral listening experiment.The dynamic range of the right-hemispheric N1m amplitude was not significantly affected even when all frequencies above 600 Hz were removed. The dynamic range of the left-hemispheric N1m response was significantly diminished by the removal of frequencies over 7.5 kHz. The subjects' behavioral sound direction discrimination was only affected by the removal of frequencies over 600 Hz.In accord with previous psychophysical findings, the current results indicate that frontal horizontal sound localization and related right-hemispheric cortical processes are insensitive to the presence of high-frequency spectral information. The previously described changes in localization-related brain activity, reflected in the enlarged N1m dynamic range elicited by natural spatial stimuli, can most likely be attributed to the processing of individualized spatial cues present already at relatively low frequencies. The left-hemispheric effect could be an indication of left-hemispheric processing of high-frequency sound information unrelated to sound localization. Taken together, these results provide converging evidence for a hemispheric asymmetry in sound localization. In the field of auditory neuroscience, an enduring topic of interest is the study of the neural processes underlying the perception of spatial sound properties and the localization of sound sources in the three-dimensional environment. Stimuli with spatial properties have been used to study sound localization with both psychophysical and neuroscientific measures. A century of psychophysical research has so far meticulously documented the psychoacoustic and behavioral aspects of sound localization . The When performing localization of spatial sounds, the human auditory system can utilize several types of auditory cues, and previous brain measurements have suggested that the different cues are processed by different cortical mechanisms  and to be less important in horizontal localization ,2. HowevSo far, many studies on the neural basis of sound localization have utilized sounds containing ITD and ILD cues as stimulus material. However, as described above, interaural differences only represent a subset of all natural sound localization cues. Loudspeakers can be used to generate genuine spatial sounds, but they cannot be used in magnetoencephalography (MEG) or functional magnetic resonance imaging (fMRI) measurements due to their magnetic interference. However, spatial stimuli presentable through non-magnetic earphones, and thus eligible for MEG and fMRI experiments, can be constructed using head-related transfer functions (HRTFs) , which sMany previous studies have found an interesting hemispherical asymmetry in human perception and localization of spatial sounds, indicating a dominant role for right-hemispheric auditory areas .All stimuli elicited prominent bilateral N1m responses, with equivalent current dipole (ECD) modeling indicating sources in the vicinity of the auditory cortices of both hemispheres. Fig. F = 9.33, p < 0.001) and latency  exhibited directional tuning, being respectively larger and shorter for contralateral than ipsilateral stimulation. The mean N1m amplitude was affected by the bandwidth of the stimuli, being larger for all stimuli with reduced bandwidth and largest for the stimuli with a bandwidth of 3 kHz. Separate analysis of each hemisphere revealed that while the effects of narrowing the bandwidth on the right-hemispheric N1m amplitude were statistically significant , the effects on the left-hemispheric N1m amplitude were not. In both hemispheres, the peak latency of the N1m was, on average, 14 ms longer for the stimuli with a bandwidth of 600 Hz than for the other stimuli . The right-hemispheric N1m source location for the 600 Hz stimuli was on average 2 mm more anterior than for the other stimuli  = 5.54, p < 0.01). No other significant differences in N1m source location between stimuli were observed.Overall, the N1m tended to be larger in amplitude and shorter in latency in the right hemisphere, but the differences did not reach statistical significance due to interindividual variation. Both N1m amplitude . The narrowest bandwidth made especially the localization of the oblique direction angles (-45° and 45°) very difficult, with performance at chance level  and the subjects tending to localize these sounds to their corresponding orthogonal direction (-90° and 90°) instead.In the behavioral experiment, subjects made only occasional errors in discriminating the direction of the broadband stimuli. Reducing the bandwidth of the stimuli had no significant effect on the ability of the subjects to discriminate the sound direction until the bandwidth was only 600 Hz . The inEven though the removal of high-frequency spectral information from binaural spatial sounds did not affect horizontal direction discrimination and related brain activity in the present experiment, the fact remains that spectral localization cues are most likely to have some function in the perception of any environmental auditory object. While interaural cues are sufficient for accurate discrimination between horizontal sound directions, they alone do not create the subjective perception of the sound originating from outside of the listener's head. On the contrary, binaural spectral information appears to be necessary for the listener to localize a sound source as being external . In The stimuli with the most reduced bandwidth (600 Hz) seem to fall into a class of their own with regard to elicited brain activity and direction discrimination accuracy. Firstly, the N1m responses to these stimuli were considerably delayed in both hemispheres, and the right-hemispheric N1m sources were slightly more anterior than for stimuli with a wider bandwidth. It is questionable, however, whether the longer latency was related to spatial properties; for similarly delayed N1m responses to non-spatial low-frequency stimuli, see . SecondlExtending on the wealth of psychophysical, neuropsychological and animal model research on sound localization, the cortical activity underlying the human perception and localization of naturally occurring sounds can now be investigated in detail with MEG experiments utilizing natural spatial sound stimuli. Neuromagnetic experiments utilizing realistic spatial stimuli can be especially beneficial for investigating the neural processing of binaural spectral cues, whose contribution to sound localization and integration with the interaural (ITD and ILD) cues is not yet completely understood. In particular, the contribution of high-frequency spectral cues to the discrimination of elevation differences can be seen as a promising subject of such experiments , as speThe present results indicate that the cortical processes underlying frontal horizontal sound localization, indexed by the dynamic range of the right-hemispheric N1m amplitude, are insensitive to the presence of high-frequency spectral information. The results are thus in accord with previous psychophysical findings on human localization of sounds in the horizontal plane. The previously described changes in localization-related brain activity, reflected in the enlarged N1m dynamic range elicited by natural spatial stimuli, can most likely be attributed to the processing of individualized spatial cues present already at relatively low frequencies. Interestingly, the dynamic range of the left-hemispheric N1m was affected by the removal of high frequencies over 7.5 kHz, perhaps suggesting left-hemispheric processing of high-frequency sound information not related to localization. Taken together, these results also provide converging evidence for a hemispheric asymmetry in sound localization.Eight volunteers  served as subjects with informed consent in the experiment, which was approved by the Ethical Committee of Helsinki University Central Hospital. The subjects participated in an MEG measurement and in a subsequent behavioral listening experiment. All subjects had also participated in the previous MEG study .The stimulus set comprised bursts of uniformly distributed white noise with four different bandwidths presented from one of five frontal horizontal directions . The stimuli used in the previous experiment  were useDuring the MEG experiment, the subject sat in a reclining chair under instruction not to pay attention to the auditory stimuli and to concentrate on watching a self-selected silent video. The stimuli were then presented through a custom-made wide band tubephone sound system  with a 306-channel whole-head MEG device . Before the acquisition, the dimensions of the skull and the location of the head position indicator (HPI) coils were measured using a 3-D digitizer, and HPI coil positions were determined prior to the presentation of each stimulus block. Horizontal and vertical eye movement sensors were used for removing epochs with eye movement artefacts defined as absolute displacements exceeding 150 μV. Over 150 responses for each stimulus type were collected for each subject and averaged in order to cancel out the cortical activity not time-locked to stimulus presentation. Averaged responses were baseline-corrected with respect to a 100-ms pre-stimulus baseline and band-pass filtered at 1–20 Hz.x-axis passing through the preauricular points (positive to the right), the y-axis passing through the nasion, and the z-axis as the vector cross product of the x- and y-unit vectors. The average goodness-of-fit of ECDs for all stimuli was 91%, with the minimal accepted goodness-of-fit being 60%. The left-hemispheric data for one subject was discarded due to too low goodness-of-fit values. In addition to the ECD properties  the dynamic range of the N1m response was investigated for each subject and stimulus bandwidth by subtracting the mean N1m amplitude for ipsilateral hemifield stimuli from its contralateral counterpart. In the left hemisphere, the mean of the N1m amplitudes evoked by the left-hemifield  stimulation was subtracted from the mean of the N1m amplitudes evoked by the right-hemifield  stimulation, while in the right hemisphere, the mean N1m amplitude evoked by right-hemifield stimulation was subtracted from the mean N1m amplitude evoked by left-hemifield stimulation. The procedure used to calculate the N1m dynamic range replicated the procedure in the previous experiment [Auditory N1m responses, defined as the peak of the negative deflection in the individual event-related response beginning at around 100 ms, were characterized for each subject through equivalent current dipoles (ECDs) . Separatperiment  and is bperiment ,15,16).M-way repeated measures analyses of variance (ANOVAs) and Newman-Keuls post-hoc comparisons.Differences according to stimulus type in the N1m amplitude, latency, location and dynamic range as well as in accuracy in the listening experiment were statistically investigated with The author(s) declares that there are no competing interestsPA, PM and HT designed the experimental setup of the study, and PA prepared the auditory stimuli. SL and LL acquired the data. SL performed the data and statistical analyses and prepared the manuscript. All authors participated in the writing process, and have approved the final version of the manuscript.