Public health practitioners and researchers for many years have been attempting to understand more clearly the links between social conditions and the health of populations. Until recently, most public health professionals in English-speaking countries were unaware that their colleagues in Latin America had developed an entire field of inquiry and practice devoted to making these links more clearly understood. The Latin American Social Medicine (LASM) database finally bridges this previous gap. brings important information, originally known mostly within professional networks located in Latin American countries to public health professionals worldwide via the Internet. The LASM database uses Spanish, Portuguese, and English language trilingual, structured abstracts to summarize classic and contemporary works.This public health informatics case study describes the key features of a unique information resource intended to improve access to LASM literature and to augment understanding about the social determinants of health. This case study includes both quantitative and qualitative evaluation data. Currently the LASM database at The University of New Mexico This database provides helpful information for public health professionals on the social determinants of health and expands access to LASM. Public health practitioners have long recognized the connections between patients' socioeconomic conditions and their health -8. Yet tAmerican Journal of Public Health that focused on LASM [Until recently, however, most of the knowledge base in this discipline has remained largely unknown outside Latin America. Language barriers and disincentives to distribute this information more widely are two major reasons for this lack of awareness. Some readers might have first learned about Latin American social medicine (LASM) through recent critical reviews  or throu on LASM ,11.LASM traces its historic origins to European researchers such as Rudolf Virchow and the belief systems of indigenous cultures. Both the European and indigenous sources of current social medicine practices emphasized the importance of linking social conditions to health status. Contemporary social medicine in Latin America continues to emphasize these linkages between social conditions and the health of populations. Social medicine professionals participate in a wide array of settings in Latin America and represent diverse specialties. Their integration into healthcare systems has varied by era and country .Innovative approaches to disseminating work in LASM have become increasingly available due to Internet technology. The project "Enhanced Access for Latin American Social Medicine" at The University of New Mexico with funding from the U.S. National Library of Medicine seeks to make information on the connections between social conditions and health problems available to a wide audience. The project has sought to bridge the prior information gap primarily through delivering structured abstracts of social medicine publications in Spanish, English, and Portuguese via the Internet on the LASM database beginning in 2001. Other goals of this project include: publishing full text social medicine electronic journals on behalf of medical societies in Latin America; and, maintaining a repository for key classic and contemporary social medicine publications.Structured abstracts are posted in three languages in the LASM database at The University of New Mexico for both the classic and contemporary social medicine literatures. The first phase of this project involved preparing and posting the structured abstracts of 25 landmark books, 50 book chapters, and 100 journal articles from the classic social medicine literature in Spanish, Portuguese, and English.A peer selection committee identified and agreed upon the specific selections of classic books, book chapters, and journal articles to be abstracted for this project. Table The contemporary literature summarized in the LASM database has been drawn primarily from the 12 journals currently or previously published in Latin America. Table Saúde em Debate (Brazil) and SaluCo Bulletin (Cuba), are available from the host website. The host website also posts structured abstracts of important articles from these full text journals.As a pilot, the project has made two social medicine journals available in electronic full text format. Several issues of these journals, The LASM database encompasses several broad themes within LASM. Subject areas include the history, theories, methodologies, and organizational dimensions of social medicine. Other subjects pertain to institutional analysis, social/critical epidemiology, and strategic planning. Table The LASM database is a web application developed using the Cold Fusion application server. The data are stored in a Microsoft SQLServer relational database. The data sources are the original Latin American publications, which are summarized in Spanish, Portuguese, and English languages in structured abstract format. The English-language structured abstracts are quality checked by the principal investigator, who reviews the abstracts for substantive content, and then by the librarian investigator who reviews the structured abstracts for final quality assurance purposes. Each record contains fields for author, title , place of publication, publisher and structured abstracts in each language. Records contain volume, number, and pagination when applicable. All records contain terms the from Medical Subject Headings (MeSH) system, a controlled vocabulary developed and maintained by the National Library of Medicine. Users can search the database on controlled fields of author, title, and MeSH terms in any of the three available languages. Full text abstract searching is also available. In addition to the abstract searching facilities, the LASM database can be browsed alphabetically by title. The browsing interface offers a convenient way to become familiar with the extent and diversity of the LASM literature.From January 2002 through December 2003, a total of 17, 853 visits were made to the website hosting the LASM database. The largest numbers of visits in descending rank order originated from Brazil, Mexico, Argentina, Spain, and Colombia. A preliminary, qualitative evaluation has been favorable. Following completion of this project, we will conduct and publish a comprehensive summative evaluation. A total of 250 structured abstracts in Spanish, Portuguese, and English had been posted to the LASM database as of June 30, 2004. The host website presents more detailed information about this project, as well as the structured abstracts themselves.The LASM database comprises a dynamic and searchable web application containing structured abstracts in English, Portuguese, and Spanish. It is designed using industry standard web application design principles, but its content is unique to the LASM domain.A database searching design and utility problem unique to the LASM database, and other web applications like it, relates to the problem of web-based multilingual searching. Widely available search engines cannot preprocess a multilingual language translation of search terms (e.g. retrieving results containing the Spanish word "pública" for the English search term "public"). Additionally, search engines do not recognize that the unaccented "publica" (as it might be entered by an English speaker as a search string) might be the same word as the Spanish accented "pública" and therefore will not return the user's expected search result. Key combinations and modifiers that are used to create special and accented characters in word processing programs like Microsoft Word often do not work in browser based search and form fields. Our primary instruction to users for constructing search terms containing special and accented characters  suggests that they use a separate text editor that accepts keyboard modifiers to construct special and accented characters to build the search term and then copy and paste the completed search string into the field. On our search pages we also list common special and accented characters in Spanish and Portuguese that users can copy and paste into browser based search fields.Although there are no other alternatives for entering special and accented characters into browser form fields, both methodologies are somewhat cumbersome. Therefore, to improve the searching utility of the LASM database we also preprocess entered search strings to allow the search engine to perform selected character substitution on the entered search string and attempt to solve the accented character problem from the search engine side. All search strings are first passed through a regular expression routine, which substitutes single character search engine wildcards for the following characters: A, E, I, O, U, a, e, i, o, u, N, C, n, c. This effectively removes potential special and accented character misspellings. To return to the previous example, a user might enter the search string "publica" ("public" in English) in a search field as an attempt to find article titles that contain the Spanish or Portuguese word "pública". Properly spelled, "Pública" uses the accented character "ú" rather than "u" (ASCII 163 rather than ASCII 117). Unfortunately, current search engines are not intelligent enough to infer that "pública" is a match for the search string "publica". Therefore, the literal search for publica (unaccented) will not return any search results that contain "pública" although there are hundreds of instances of the word "pública" in the LASM database.In the case of "publica", the preprocessed search string that is finally submitted to the search engine is "p*bl***". The search engine will return all seven-letter word matches that contain the letters p, b, and l in the first, third, and fourth positions respectively. The net effect of this technique is to under-specify the search result. That is, the search engine may possibly return records that contain other words that happen coincidentally to match the submitted search string. On the other hand, the returned result set can be guaranteed to contain the desired search result.Problems created from under-specifying the search are limited based on experience gained from using this technique. The positional constraints of submitted characters within the search string generally are restrictive enough to prevent most problems. Specifically, within a limited domain search surface like the LASM database, the likelihood of the occurrence of most alternative word matches is very low.The character substitution methodology described here is not perfect, and many other alternative strategies for addressing the multilingual search problem have been explored by LASM technical staff. Most of the alternative strategies considered, however, involved much higher costs in terms of acquiring or developing specialized search engine capabilities or much higher abstract preparation costs. Therefore, we chose the character substitution strategy as a compromise between implementation cost and search utility.Internet technology via websites and web browsers has created numerous opportunities for public health colleagues to inform one another and to collaborate across wide geographic space. The LASM database clearly demonstrates the efficacy of the Internet for communicating its informative structured abstracts posted in the Spanish, Portuguese, and English languages. This database provides useful information that would otherwise be unavailable for public health professionals on the social determinants of health. Furthermore, it expands access to LASM through its inclusion of both the classic and contemporary literature.. As the data reported above indicate, the LASM database already has been accessed steadily since its initial small-scale publicity began in 2002. We hope that public health readers will utilize the LASM database to improve their research, teaching, and practice.Anyone with access to the World Wide Web and a web browser can access all structured abstracts in the LASM database Neither the authors nor any support personnel possess competing interests related to the LASM database or to this article's publication.JE conceived of and wrote the initial version of this article, served as an early collaborator on and helped design the project, was responsible as an investigator for acquiring journals for the Latin American social medicine collection, provided quality assurance editing on structured abstracts, played a major role in the formative evaluation of the project, and coordinated all revisions to this article. HW initiated and designed the project, served as principal investigator, obtained funding, translated and edited abstracts in the English-language, and edited this manuscript. HSB served as co-principal Investigator, helped design this project, obtained funding, and led the effort for the formative evaluation of this project. JT helped administer the project and edited this manuscript. CI coordinated the project, wrote Spanish-language structured abstracts, and provided reference materials for this article. KW and JT developed all programming aspects of the website including the search strategies, managed the web interface and underlying database and contributed the text for portions of this article.The pre-publication history for this paper can be accessed here:


Daily energy expenditure consists of three components: basal metabolic rate, diet-induced thermogenesis and the energy cost of physical activity. Here, data on diet-induced thermogenesis are reviewed in relation to measuring conditions and characteristics of the diet.Measuring conditions include nutritional status of the subject, physical activity and duration of the observation. Diet characteristics are energy content and macronutrient composition.Most studies measure diet-induced thermogenesis as the increase in energy expenditure above basal metabolic rate. Generally, the hierarchy in macronutrient oxidation in the postprandial state is reflected similarly in diet-induced thermogenesis, with the sequence alcohol, protein, carbohydrate, and fat. A mixed diet consumed at energy balance results in a diet induced energy expenditure of 5 to 15 % of daily energy expenditure. Values are higher at a relatively high protein and alcohol consumption and lower at a high fat consumption. Protein induced thermogenesis has an important effect on satiety.In conclusion, the main determinants of diet-induced thermogenesis are the energy content and the protein- and alcohol fraction of the diet. Protein plays a key role in body weight regulation through satiety related to diet-induced thermogenesis. Diet induced thermogenesis (DIT) can be defined as the increase in energy expenditure above basal fasting level divided by the energy content of the food ingested and is commonly expressed as a percentage. It is, with basal metabolic rate and activity induced thermogenesis, one of the three components of daily energy expenditure. Although DIT is the smallest component, it could play a role in the development and/or maintenance of obesity. De Jonge and Bray  evaluateMethodological issues include: was the baseline appropriate, what was the energy content and nutrient composition of the test food consumed, what was the duration of the postprandial measurement period, and what was the error associated with the calculation of DIT from the measured energy expenditure. Weststrate et al  investigHere, the focus is on DIT as a function of the energy content and nutrient composition of the test food consumed and the duration of the postprandial measurement period in adult subjects with a normal bodyweight. The review is based on literature published over the last 15 years.The experimental design of most studies on DIT is a measurement of resting energy expenditure before and after a test meal, with a ventilated hood system. The observation is started after an overnight fast, where subjects are refrained from eating after the last meal at 20.00 h at the latest. Thus, with observations starting between 08.00 and 09.00 h the next morning, the fasting interval is at least 12 h. Postprandial measurements are made for several hours where subjects have to remain stationery, most often in a supine position, for the duration of the measurements. In some studies, measurements are 30 min with 15 min intervals allowing i.e. for sanitary activities.The use of a respiration chamber to measure DIT has the advantage of reproducing more physiological conditions over a longer period of time while regular meals are consumed throughout the day ,6. The DStudies on DIT were selected from Medline. Studies were selected when information was presented on energy intake, diet composition with respect to carbohydrate, protein fat and alcohol of the test food, duration of the postprandial measurement, and DIT.Reported intra-individual variability in DIT, determined with ventilated-hood systems, is 6 to 30% ,8. ReporThe mean pattern of DIT throughout the day is presented in figure Fifteen studies on DIT with information on energy intake, on diet composition and on the postprandial measurement period were selected from literature Table . Five stFor a comparison of DIT between studies as a function of the nutrient composition of the test food consumed, the energy content of the test food was divided by the length of the measurement interval after food consumption and expressed in MJ/h. Only three of the 22 studies presented in table The main determinant of DIT is the energy content of the food, followed by the protein fraction of the food. The thermic effect of alcohol is similar to the thermic effect of protein.Diet induced thermogenesis is related to the stimulation of energy-requiring processes during the post-prandial period. The intestinal absorption of nutrients, the initial steps of their metabolism and the storage of the absorbed but not immediately oxidized nutrients . As suchTheoretically, based on the amount of ATP required for the initial steps of metabolism and storage, the DIT is different for each nutrient. Reported DIT values for separate nutrients are 0 to 3% for fat, 5 to 10% for carbohydrate, 20 to 30% for protein , and 10 Of the studies presented in table The higher DIT value of alcohol and protein compared with carbohydrate and fat has implications for the effect of these nutrients on energy balance. However, the main effect on energy balance does not seem to be primarily linked to the lower bioavailability of alcohol-and protein energy than that of fat and carbohydrate. Alcohol energy is largely additive to the normal diet but does not seem to affect energy balance positively . ProteinAlcohol forms a significant component of many diets and it supplements rather than displaces daily energy intake. Alcohol consumption as an aperitif has even been shown to result in a higher subsequent intake with no intake compensation afterwards . Yet, alThe main effect of protein on energy balance is thought to be DIT related satiety. Satiety scores were higher during meals with a high-protein/high-carbohydrate diet, as well as over 24 h, than with a high-fat diet . The obsIn conclusion, the main determinants of diet-induced thermogenesis are the energy content and the protein-and alcohol fraction of the diet. Protein plays a key role in body weight regulation through satiety related to diet-induced thermogenesis.


In view of clinical observations and laboratory results that support a central role of the spleen in idiopathic thrombocytopenic purpura (ITP) pathophysiology, we studied the effect of splenectomy on type-1 and type-2 cytokine gene expression in an adult ITP case, refractory to conservative treatment.6/L within 10 days after the operation. Two consecutive blood samples were obtained from the patient, 3 and 7 months after the splenectomy for the purposes of this study. A control group consisted of 11 healthy adults. Peripheral blood mononuclear cells were prepared from each blood sample and cultured in vitro for 8 h with the addition of the mitogens phorbol myristate acetate and ionomycin. Total cellular RNA extracted from 106 cells was submitted to semiquantitave reverse transcriptase-polymerase chain reaction (RT-PCR) for the amplification of IL-2, IFN-γ, IL-4, IL-5, and IL-10 metagraphs. The PCR products were run on ethidium-stained agarose gels, photographed and quantified by densitometry.The patient was subjected to splenectomy 9 months after the diagnosis with complete response, attaining platelet counts over 150 × 106/L to 265 × 106/L. The change of type-2 cytokine expression  was slight and the Th2 activity (IL-4+IL-5) remained largely unchanged. The Th1/Th2 ratio, that reflects the pathogenic disease-specific T-cell immune deviation, was accordingly reduced 7 months post-splenectomy (Th1/Th2 = 1.3) compared to 3 months (Th1/Th2 = 3.5).A steep decrease of type-1 cytokine expression  and their calculated sum expressing Th1 activity was observed at 7 months post-splenectomy compared to 3 months post-splenectomy, in parallel with a rise of platelet count from 190 × 10The reduction of the Th1/Th2 cytokine ratio that was observed over time after splenectomy was accompanied by full clinical remission. Nevertheless, the persistence of a type-1 polarization, even after several months following spleen removal, is suggestive of a more basic abnormality of the immune function in these patients. Adult autoimmune thrombocytopenic purpura (ITP) is a chronic acquired organ-specific autoimmune thrombocytopenic syndrome . The lowThe spleen is considered to be the primary site of the autoimmune response where initiation, maintenance and regulation of the autoimmune attack take place. The spleen is the site of autoreactive T- and B-cell interaction and activation, and autoreactive anti-platelet antibody production . Platelein vitro cultures [+CD4+, CD3+CD8+, CD3+HLADR+, and CD3+CD25+ cells increases significantly in ITP patients refractory to splenectomy [Splenectomy is followed by reduction of autoantibody peripheral blood titre ,13. Splecultures . The percultures . The numenectomy . Changesenectomy .Splenectomy is the most clinically effective therapeutic intervention in ITP patients, resulting in complete remission in two thirds of the patients with more than 60% maintaining the therapeutic effect in the long term ,18. IrreGiven the important immunoregulatory role of the spleen, we looked at the effects of splenectomy on immune activation and immune deviation indices in the peripheral blood of an ITP patient after splenectomy in association with peripheral platelet counts.6/L). Following clinical exclusion of causes of secondary thrombocytopenia [A 42 year old woman presented in October 1999 in Patras University Hospital (PUH) with lower limb purpura and low platelet count . Informed consent was obtained from the patient. PUH abides by the Helsinki declaration on ethical principles for medical research involving human subjects.in vitro for 8 h with the addition of 20 ng/ml phorbol myristate acetate (PMA) and 1 μM ionomycin .Peripheral blood mononuclear cells (PBMC) were prepared from each blood sample by centrifugation over a Ficoll-Paque gradient . The cells were cultured 6 cells was submitted to semiquantitative RT-PCR for the amplification of IL-2, IFN-γ, IL-4, IL-5, and IL-10 metagraphs [Total cellular RNA extracted from 10tagraphs . Primerstagraphs .6/L to 265 × 106/L. Regarding type-2 cytokine gene expression, IL-4 increased, IL-5 decreased, and IL-10 remained unchanged, whereas the change in Th2 activity (IL-4 units plus IL-5 units) was slight (Figure A sharp decrease in the expression of the type-1 cytokines IL-2 and IFN-γ and their calculated sum expressing Th1 activity was observed at 7 months after splenectomy compared to 3 months after splenectomy (Figure The above results show that in this patient type-1 polarization persists after removal of the spleen and attainment of clinical remission. This may mean that the spleen is not exclusively responsible for the coordination or the maintenance of the pathological immune response in this patient, provided that no accessory splenic tissue exists. Other disease centres may control the autoimmune reaction as well, such as the liver or the bone marrow. Alternatively, it is possible that ITP is the manifestation of a general immune system malfunction that pre-existed before the development of thrombocytopenia and persists after removal of what seems to be the effector of a manifestation of an autoimmune proclivity.Unfortunately, a pre-splenectomy sample was not available for analysis. As a result, no conclusions can be drawn about the effect of splenectomy on the direction of change of Th1 activity. Based on phenotypic studies showing increased presence of T lymphocytes with activated phenotype after splenectomy in ITP patients , it is pThe clinical remission may be due to the removal of a major platelet destruction site, although the underlying immune activity that drives the destruction may remain unaffected. Complete remission does not mean that increased platelet destruction has stopped after splenectomy. Platelet life span may still be shortened in this patient and/or her normal platelet count may be even higher than what was achieved after splenectomy.The pathological immune activity seems to decrease over time after splenectomy, as reflected by the lower Th1/Th2 ratio that is indicative of the degree of immune polarization. This may be explained by reduced stimulation of the immune system by activated spleen reticuloendothelial cells that present platelet antigens to T helper lymphocytes. In this way, it may be hypothesized that removal of one vital component of the self-attacking immune process can break the vicious circle that culminates in even greater immune activation, polarization, and platelet destruction. Removal of a major site of autoimmune activity may have abrogated recruitment of naïve T-cells. As a result, overall autoimmune activity wears off, as existing activated Th1 effector cells perish leaving behind a much smaller population of peripheral memory cells that retain the initial Th phenotype.Another consideration that stems from the results of this case study is that immune polarization and immune deviation of the pathological response depend more on upregulation of type-1 mediators rather than on suppression of type-2 cytokines, or that type-2 response is inadequate to control excess type-1 response in active disease.Clinical improvement after splenectomy is associated with reduced but not normalized immune activation and polarization in the patient studied. However, the spleen seems not to be absolutely necessary for the maintenance of the autoimmune reactivity.The authors declare that they have no competing interests.FPP prepared the case report, performed the experiments and drafted the manuscript. AM conceived of the study, participated in its design and coordination and co-wrote the manuscript. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


A healthcare provider's recommendation to undergo screening has been shown to be one of the strongest predictors of completing a colorectal cancer (CRC) screening test. We sought to determine the relationship between the general quality of self-rated patient-provider communication and the completion of CRC screening.A formative study using qualitative data from focus groups and quantitative data from a cross-sectional survey of church members about the quality of their communication with their healthcare provider, their CRC risk knowledge, and whether they had completed CRC screening tests. Focus group participants were a convenience sample of African American church members. Participants for the survey were recruited by telephone from membership lists of 12 African American churches located in rural counties of North Carolina to participate in the WATCH (Wellness for African Americans Through Churches) Project.Focus Groups. Six focus groups (n = 45) were conducted prior to the baseline survey. Discussions focused on CRC knowledge, and perceived barriers/motivators to CRC screening. A theme that emerged during each groups' discussion about CRC screening was the quality of the participants' communication with their health care provider. Survey. Among the 397 participants over age 50, 31% reported CRC screening within the recommended guidelines. Participants who self-rated their communication as good were more likely to have been screened (36%) within the recommended guidelines than were participants with poor communication (17%) . Participants who had adequate CRC knowledge completed CRC screening at a higher rate than those with inadequate knowledge (p = 0.011). The percentage of participants with CRC screening in the recommended guidelines, stratified by communication and knowledge group were: 42% for good communication/adequate knowledge; 27% for good communication/inadequate knowledge; 29% for poor communication/adequate knowledge; and 5% for poor communication/inadequate knowledge.Participants who rated their patient-provider communication as good were more likely to have completed CRC screening tests than those reporting poor communication. Among participants reporting good communication, knowledge about colorectal cancer was also associated with test completion. Interventions to improve patient-provider communication may be important to increase low rates of CRC screening test completion among African Americans. Colorectal cancer (CRC) screening among average risk adults 50 years and older can decrease the incidence and mortality rates for CRC -7. NatioA healthcare provider's recommendation to undergo screening has been shown to be one of the strongest predictors of completing a CRC screening test -17, and . The WATCH Project was a church-based colorectal cancer prevention study designed to increase fruit and vegetable consumption, reduce fat intake, increase moderate physical activity, and increase CRC screening among church members. In this study of patient-provider communication and CRC screening, we used data from two different sources: 1) focus groups of African American men and women, and 2) surveys of church members. The Institutional Review Board from the University of North Carolina at Chapel Hill approved this study.Data for this study were obtained as part of a larger study, the WATCH (Wellness for African Americans Through Churches) Project As part of the formative data collection in early 1998, we conducted six focus groups of African American adults. There were three focus groups of African American adult men and three of African American adult women. The focus groups comprised of a convenience sample drawn from members of African American churches located in central North Carolina. The focus groups were conducted in the churches, lasted approximately 60 minutes, were tape recorded, transcribed, and reviewed for accuracy. A $15 incentive and refreshments were provided for the participants. The focus groups were led by sex-matched African American moderators and explored issues associated with colon cancer, CRC screening (barriers and motivators), nutrition, and physical activity. The information from the focus groups was used to develop the questionnaires and to help guide the intervention strategies used in the culturally appropriate colon cancer prevention program.The second source of participants for the survey study was the baseline intervention sample for the larger WATCH project. The telephone survey collected information focused on general health status, nutrition, physical activity, patient-provider communication, CRC risk knowledge and screening behaviors. The baseline survey was completed, on average, in approximately 40 minutes and was administered by trained interviewers prior to the intervention. The baseline telephone surveys were conducted between October 1998 and October 1999.In this study, a history of CRC screening was defined as the self-report of undergoing a fecal occult blood test (FOBT), flexible sigmoidoscopy, or colonoscopy within the recommended time period. Participants were asked whether they had each of the CRC screening tests, and if they responded yes, the participants were asked when they had their last test. The items in the survey included a brief explanation of each screening test. Items were described as follows: FOBT, "which is stool slides"; sigmoidoscopy, "which is a tube inserted in the rectum to look at the colon and the bowel"; and colonoscopy, "which is a tube inserted in the rectum to look at the entire intestine, usually given in a hospital or specialist's office." Responses included, "less than 1 year"; "1–2 years"; "3–5 years"; or "more than 5 years."CRC screening was considered to be within the recommended time period based on an algorithm using ACS guidelines considering which test and how recent the test was performed (e.g. a person who reported having FOBT within the past year was considered within the recommended time period). Participants were considered to have been screened within the recommended time period if they had a FOBT within the preceding year, and sigmoidscopy within the preceding 5 years. Although current CRC screening guidelines recommend colonoscopy every 10 years for average-risk adults, we examined colonoscopy use in the past 5 years because of the limitations of the survey instrument. Self-report of CRC screening behavior has been demonstrated to be a reliable endpoint for intervention trials .Analyses of data from this study included factor analysis, analysis of variance, and logistic regression and were conducted using SPSS, version 10.1. Logistic regression analyses were performed to evaluate whether the level of perceived patient-provider communication was significantly related to CRC screening behavior in this population. Sociodemographic variables were identified as potential covariates if there was plausible theoretical or empirical evidence that the variable might be associated with the communication variable or with CRC screening. Variables that were significantly associated with communication level (p < 0.05) were retained and tested as covariates in the logistic models. Only the sex of the participant, receiving healthcare at a doctor's office versus a clinic/emergency room, and knowledge of CRC risk were significantly associated with communication and only these three covariates were entered into the initial logistic model. Variables in the model were evaluated by the Wald test and interpreted using odds ratios and confidence intervals. The overall fit of each model was evaluated using the Hosmer-Lemeshow Goodness of Fit test and by examining classification tables .Several major themes associated with CRC emerged from the personal experiences expressed about the medical community during the focus groups. One of the themes discussed by the participants in each focus group was patient-provider communication. Selected comments by focus group members about CRC and patient-provider communication are listed in Table Originally, 2480 names were obtained from the 12 church rosters located in five rural counties in North Carolina. Many members were ineligible  or we were unable to contact them by telephone, and 239 members who declined to participate in the WATCH Project. There were 850 church members who participated in the WATCH Project and completed the baseline survey. The adjusted response rate was 66% using a calculation method, suggested by the Council of American Survey Organizations (CASRO) , that acThe participants in this study were the 397 church members who participated in the WATCH Project and were 50 years and older. The characteristics of the 397 participants are shown in Table Factor analysis of the five communication items was performed from the baseline survey responses and two factors were identified; one with three items and the other with two items. The second factor was dropped because it had only two items and did not add reliability to the scale. The three communication items about shared decision-making and patient satisfaction demonstrated good reliability  and were summed to calculate a communication score. The communication score was used to categorize the participants into three groups: good, fair, and poor communication with providers. Participants were categorized as having "good" communication if they perceived receiving enough information from their provider, being involved in medical decisions, and thinking that their provider understood their health needs almost all the time or always. Participants who rated all three items 'sometimes', 'rarely', or 'never' scored "poor" on the communication scale, and individuals who rated the items with a mix of the above listed responses were assigned to the "fair" group.In terms of quality of communication, 75% 298/397) responded positively to all 3 questions and were considered have "good" communication; 10% (42/397) responded positively to none of the 3 questions and were considered to have "poor" communication; and 13% (50/397) had fair results. Participants in the good communication group were more likely to be female (p = 0.031), and were more likely to receive their healthcare at a doctor's office versus a clinic/emergency room/health department (p = 0.028). None of the other sociodemographic factors listed in Table 97 responOnly 45% (175/389) of the participants reported that their providers had recommended CRC screening, and just 31% (120/389) of all participants reported being screened within the recommended time interval. Of the individuals who reported being screened, 65% (78/120) stated that their doctor had recommended CRC screening, compared with 36% (97/269) of those who did not report screening.Knowledge of CRC was assessed using seven items Table  with a mKnowledge about CRC was considered adequate (knowledge score > = 4) for 57% (228/397) and inadequate for 43% (197/397). Participants with adequate CRC knowledge were more likely to have completed a CRC screening test within the recommended time period compared to those with inadequate CRC knowledge (21% vs. 10%). Adequate knowledge was associated with a higher level of education (p < 0.001), a higher level of income (p < 0.001), having health insurance (p < 0.001), and having Medicare/ Medicaid as one's health insurance (p < 0.001).Results of the logistic regression analyses are shown in Table CRC screening within recommended guidelines by perceived communication and knowledge is listed in Table Our study found that participants had higher rates of CRC screening when their self-rated communication with their healthcare provider was classified as perceived as more positive. In addition, we found that participants who self-rated their communication as good and who had adequate CRC knowledge completed recent CRC screening at higher rates than those with good communication and inadequate knowledge. In addition, screening rates are higher with both good communication and adequate CRC knowledge than when only one factor is present. These findings suggest that both good patient-provider communication and CRC knowledge are important for CRC screening. Because of the cross-sectional nature of our study, we cannot determine causality, and it is possible that CRC knowledge improves by going through the screening process.Improving CRC screening rates in the African American population may require strategies that address both improving physician-patient communication skills and increasing CRC knowledge. Good patient-provider communication is fundamental to a patient's perceived quality and satisfaction with their healthcare. Better communication, including the use of shared decision making, is associated with trust between patients and providers . HoweverThe association between the lack of physician recommendations for cancer screening tests and low patient utilization of those tests has been documented in previous investigations ,19,27-30The results of our study and other previously published work ,35-39 suAlthough the results from our cross-sectional study have implications for developing colorectal cancer prevention programs, it must be recognized that prospective longitudinal studies are needed that specifically address the effect of patient-provider communication on CRC screening. Because of its cross-sectional design, we cannot infer causal relationships. In addition, since all participants were recruited from 12 churches, there may be some clustering of exposures and outcomes by physician that may affect the results; we did not measure this effect. Patient-provider communication was measured by using only three self-reported communication items, which may not fully capture the full extent of the patient-provider relationship. In addition, we do not know whether our participants had racially discordant or concordant physicians, and we did not have information regarding the physician's beliefs or practices about CRC screening or the physicians' assessment of the quality of communication with the patients. The outcome measure of having undergone CRC screening was self-reported, and these results were not validated. Finally, because of the relatively small number of participants and the use of convenience sampling, results from this study may not be generalizable.The findings from this cross-sectional study suggest that not only do patients need to be informed about their CRC risk and the importance of screening tests but that having good communication with their healthcare provider may also important. The burden of having good communication in the patient-provider relationship is the responsibility of both individuals. Both the patient and the provider should strive to improve their communication skills so that patients who want to participate in the decision about how to be screened for colorectal cancer may do so effectively.Because culturally distinct factors may contribute to poor communication and mistrust for African-Americans, specific new strategies need to be developed . BeliefsThe author(s) declare that they have no competing interests.M.L.K. participated in the conception of the study, the analyses, the interpretation of the results, writing the first draft and revising the manuscript. A.S.J. participated in the conception of the study, performed the statistical analyses, participated in the interpretation of the results, and the editing of the manuscript. M.P.P. participated in the interpretation of the results and the writing of the manuscript. M.H., E.J., and V.O. assisted with the qualitative analysis and surveys. M.K.C. participated in the conception of the study, the statistical analyses, interpretation of the results, and the writing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Essential hypertension is a common, polygenic, complex disorder resulting from interaction of several genes with each other and with environmental factors such as obesity, dietary salt intake, and alcohol consumption. Since the underlying genetic pathways remain elusive, currently most studies focus on the genes coding for proteins that regulate blood pressure as their physiological role makes them prime suspects.The present study examines how polymorphisms of the insertion/deletion (I/D) ACE and M235T AGT genes account for presence and severity of hypertension, and embeds the data in a meta-analysis of relevant studies.The I/D polymorphisms of the ACE and M235T polymorphisms of the AGT genes were determined by RFLP (restriction fragment length polymorphism) and restriction analysis in 638 hypertensive patients and 720 normotensive local blood donors in Weisswasser, Germany. Severity of hypertension was estimated by the number of antihypertensive drugs used.No difference was observed in the allele frequencies and genotype distributions of ACE gene polymorphisms between the two groups, whereas AGT TT homozygotes were more frequent in controls . This became significant (p = 0.035) in women only. AGT TT genotype was associated with a 48% decrease in the risk of having hypertension , and this risk decreased more significantly in women . The meta-analysis showed a pooled odds ratio for hypertension of 1.21  in Caucasians. No correlation was found between severity of hypertension and a specific genotype.The ACE I/D polymorphism does not contribute to the presence and severity of essential hypertension, while the AGT M235T TT genotype confers a significantly decreased risk for the development of hypertension in the population studied here. This contrasts to the findings of meta-analyses, whereby the T allele is associated with increased risk for hypertension. Essential hypertension is a common, polygenic, complex disorder resulting from interaction of several genes with each other and with environmental factors such as obesity, dietary salt intake, and alcohol consumption. Since the underlying genetic pathways remain elusive, currentThe Renin-Angiotensin System (RAS) has a central role in regulating blood pressure and sodium homeaostasis. Genes encoding components of RAS, including angiotensinogen (AGT), angiotensin-converting enzyme (ACE), angiotensinogen II type-1 receptor (AGTR1), and renin, have been extensively investigated as genetic determinants of essential hypertension . PolymorIn 1992, the M235T AGT TT polymorphism was first reported to be associated with hypertension . This fiThe inconsistent results might be explained in part by the genetic and environmental heterogeneity among different ethnic groups . On the The present study investigates the relationship between variants of the I/D ACE gene and M235T AGT gene, and the presence and severity of essential hypertension in a large homogeneous German population. The effect of a combination of ACE and AGT gene polymorphisms on hypertension was also examined.The design of the study followed the guidelines proposed by Cooper et al , and the2 or dialysis), 15 were due to diabetic nephropathy, 5 to chronic pyelonephritis, 1 to chronic glomerulonephritis, 1 light chain deposits, 1 polycystic kidney disease, and 14 of unknown cause (no biopsy obtained). The severity of hypertension was estimated based on the number of antihypertensive medications used, a surrogate marker for the severity of hypertension[This cross-sectional study comprised a total of 1358 individuals from Weisswasser, a county town of 25,000 in Saxony, Germany. After giving informed consent, 720 normotensive subjects were selected from local blood donors and 638 hypertensive patients from the local renal care center. All hypertensive patients included in the study had been diagnosed as suffering from primary hypertension by the attending consultants on first contact with the clinic. Hypertensives were defined as those who received at least one antihypertensive medication. At the time of blood sampling, 34.2% were diabetic , and 65.1% were suffering from kidney disease. Of the 37 patients with K/DOQI stage 5  and restriction analysis were used to determine the frequencies of the I/D polymorphisms of the ACE gene, and homo-/ heterozygoty of the M235T AGT gene,21. ACE AGT M235T polymorphism was studied by first amplifying a 104 bp long fragment of the AGT gene using the following primer sequences: sense- 5'CCGTTTGTGCAGGGCCTGGCTCTCT3', and antisense: 5'CAGGGTGCTGTCCACACTGGACCCC3'.Tth 111I.The M -> T point mutation at position 235 creates a detection site for the restriction enzyme t test for continuous data and Pearson's χ2 test for categorical data. Allele frequencies were calculated with the gene-counting method. χ2 test was used for assessment of the Hardy-Weinberg equilibrium for the distribution of genotypes. Odds ratios were calculated with a 95% confidence interval. A P < .05 was considered significant.Statistical analysis was carried out using SPSS personal computer statistical package . Demographic characteristics were compared by 2 test using P-value < 0.05. The Mantel-Haenszel odds ratios were calculated by applying both fixed effect model and random effect model in case of heterogeneity.A meta-analysis was performed using Review Manager 4.2 (The Cochrane Collaboration) to further examine the association of the AGT M235T gene polymorphisms with essential hypertension in Caucasians. A systematic literature search in PubMed Medline for articles published between April 2002 and June 2004 was carried out using the following MESH-headings: "angiotensinogen/genetics", "hypertension/genetics", "blood pressure/genetics", and "adult". The search was limited to articles published in English and studies on Caucasian human subjects. Only 2 studies were left after strict examination according to the exclusion criteria listed in . A totalDemographic data are summarized in Table AGT M/T genotyping was successful in 637 hypertensives and 720 normotensives, and ACE I/D genotype was analyzed in 636 hypertensive and 719 control subjects. The roles of age and gender in the association between hypertension and ACE and AGT gene polymorphisms were examined by comparing the effects of ACE and AGT genotypes for hypertension in men and women, young and elderly subjects respectively. "Young" was defined as "age < 50 years old", and "elderly" was characterized as "age ≥ 50 years old".No differences were observed in ACE allele and genotype frequency distribution between hypertensives and controls with respect to gender and age, and no deviations from Hardy-Weinberg equilibrium were observed in any of subgroups P > 0.1). ACE genotypes I/I, I/D and D/D were of almost identical frequency within both groups  compared with those with MM genotype Figure . The stuThe effect of eight combinations  on hypertension was examined. No statistically significant association was observed between any combination above and hypertension in all subjects combined. Nonetheless, in women, both genotypes of TT, DD/ID and TT, II/ID were significantly associated with lower prevalence of hypertension  and a more significant association was found in women , compared to men. This is in stark contrast to findings from previous studies, including two German datasets [AGT 235 T-allele and/or TT genotype significantly increased the risk for essential hypertension in Caucasians: odds ratio T vs. M was 1.20 (95% CI: 1.11 to 1.29) [Surprisingly, the cross-sectional study presented here shows a higher prevalence of the T/T M235T AGT gene in the control group compared to the hypertensive group. AGT TT genotype was associated with a datasets ,32, and datasets ,22,47, wto 1.29) , odds rato 1.29)  includedto 1.29) AGT T allele among normotensives was higher than that among hypertensives (0.36 vs. 0.33). This was inconsistent with one previous meta-analysis in Caucasians, which showed that among controls, the mean allele frequency for the AGT T allele was 0.41 (95% CI: 0.34 to 0.48), and among cases, increased to 0.45 (95% CI: 0.38 to 0.52) [AGT T allele among hypertensives (0.33) was outside the lower border of the 95% confidence interval (0.38 to 0.52) reported in [Although no significant difference was observed in AGT T allele frequency distribution between hypertensives and controls with respect to age and gender, the frequency of the to 0.52) . In the orted in . This maThe AGT genotype distribution is not in Hardy-Weinberg equilibrium (P < 0.001) while no deviations from Hardy-Weinberg equilibrium are observed on the ACE genotype distribution in the same population. This may be explained by a shift toward a higher frequency of MT individuals (62.4%) instead of TT individuals (3.7%) in this specific population.The study population presented here contains a large proportion (65%) of patients with renal disease. While selection of participants based on patient records excluded those patients that had symptoms suggesting the diagnosis of secondary hypertension at first contact, the possibility remains that at least a part of the study population suffers from renal rather than essential hypertension. It should be noted, however, that the majority of studies included in the presented meta-analysis does not give specific information regarding renal function of hypertensives, and the largest study  is populAnother possible explanation is that the AGT T allele frequency may decrease with age, which was reported from the United Arab Emirates . The preA number of studies -54 examiThe effect of a combination of RAAS genes' polymorphisms on blood pressure has been investigated in the participants to the Olivetti Heart Study , in whicAlthough in most surveys the prevalence of hypertension appears to be equal in women and men , sex-speIn addition to the factor declared above, the result may be influenced by the study design and the composition of the sample population. The study design itself may influence the results. As mentioned above, this study was a cross-sectional study where subjects were assessed at a single time, and at that time most of the controls were younger than 50 years old. Animal studies have shown that hypertension genes may be activated for only certain periods during the life history of an organism . Hence, The population can be described as static population with a mixed Germanic-Slavonic background. Due to the location  and political and historical setting , the population may be assumed as homogeneous. Several recent studies -61 have In the meantime, it should be noted that the present study was carried out on an unusually large population . Given Despite the limitations mentioned, this cross-sectional study does not support the notion that the ACE I/D polymorphism contributes to the prevalence and severity of essential hypertension. However, the M235T TT genotype of AGT gene was detected to confer a significantly decreased risk for the prevalence of hypertension in women from this particular population. Despite the large sample size, the present study fails to revise the odds ratio in a meta-analysis of a total of 25 studies on the association between the AGT M235T polymorphisms and hypertension in caucasians. This observation may reflect a very specific local inheritance pattern of the AGT genotypes. If this holds true, studies aiming at drug development based on genomic traits must be scrutinized rigorously as therapeutic recommendations may be valid for selected subpopulations only.The author(s) declare that they have no competing interests.M.N. designed and initiated the study, analyzed the samples and wrote part of the manuscript. A-L.Z. and M.L. did the statistical analysis. A.M. devised the concept for meta-analysis and wrote the manuscript. L.P. created the figures. M.N. and A.M. should be considered as joint co- authors.The pre-publication history for this paper can be accessed here:


Transport of fatty acids within the cytosol of adipocytes and their subsequent assimilation into lipid droplets has been thoroughly investigated; however, the mechanism by which fatty acids are transported across the plasma membrane from the extracellular environment remains unclear. Since triacylglycerol-rich lipoproteins represent an abundant source of fatty acids for adipocyte utilization, we have investigated the expression levels of cell surface lipoprotein receptors and their functional contributions toward intracellular lipid accumulation; these include very low density lipoprotein receptor (VLDL-R), low density lipoprotein receptor-related protein (LRP), and heparan sulfate proteoglycans (HSPG).We found that expression of these three lipoprotein receptors increased 5-fold, 2-fold, and 2.5-fold, respectively, during adipocyte differentiation. The major proteoglycans expressed by mature adipocytes are of high molecular weight (>500 kD) and contain both heparan and chondroitin sulfate moieties. Using ligand binding antagonists, we observed that HSPG, rather than VLDL-R or LRP, play a primary role in the uptake of DiI-lableled apoE-VLDL by mature adipocytes. In addition, inhibitors of HSPG maturation resulted in a significant reduction (>85%) in intracellular lipid accumulation.These results suggest that cell surface HSPG is required for fatty acid transport across the plasma membrane of adipocytes. The adipocyte plays a central role in overall metabolic regulation serving as a storage depot for fatty acids and as an endocrine cell to regulate energy utilization and feeding behavior ,2. The mExtracellular fatty acids that are available for adipocyte uptake are either 1) associated with circulating albumin, 2) hydrolyzed from triacylglycerol-rich lipoprotein particles by lipoprotein lipase, or 3) in the form of VLDL particles which can be directly internalized by adipocyte lipoprotein receptors. In the circulation, VLDL represents the major source of fatty acids for peripheral tissues in the form of triacylglycerols and provides a concentrated source of esterified fatty acids. It is interesting that in light of the well studied processes of cytosolic transport and assimilation of free fatty acids into triacylglycerol-rich storage droplets, the mechanism of transport of fatty acids across the adipocyte plasma membrane remains controversial. Two mechanisms, which are not mutually exclusive, have been proposed: one involves passive diffusion across the plasma membrane ,7, the oIn vivo studies corroborate these observations as uptake of long-chain fatty acids is impaired in CD36 knock-out mice [Several cell surface proteins are expressed by adipocytes which potentially contribute to receptor-mediated uptake of extracellular fatty acids; these include CD36, fatty acid transport protein-1 (FATP1), very low density lipoprotein receptor (VLDL-R), low density lipoprotein receptor-related protein (LRP), and heparan sulfate proteoglycans (HSPG). CD36 is a cell surface glycoprotein that binds long-chain fatty acids with high affinity and demonstrates a subcellular distribution that is consistent with a role in fatty acid transport across the plasma membrane -23. Follout mice  or when out mice . Althougout mice . FATP1 iout mice . In adipout mice . Similarout mice ,28.Studies using a VLDL-R knock-out animal model have suggested a role for this receptor in the accumulation of fatty acids by adipose tissue; mice lacking VLDL-R demonstrate only modest weight gain and a reduction in adipose tissue mass when placed on a high-fat diet . VLDL-R To better understand the process of fatty acid transport across the plasma membrane of adipocytes, we have examined the expression levels and functional contributions of lipoprotein receptors, VLDL-R, LRP and HSPG toward intracellular lipid accumulation. Our findings suggest that cell surface HSPG play an essential role in fatty acid uptake and intracellular lipid accumulation in adipocytes.in vitro model for adipocyte differentiation [Since members of the LDL receptor family are known to play a predominant role in the uptake of lipoproteins, we chose to examine the protein expression levels of VLDL-R and LRP during adipocyte differentiation using 3T3-L1 cells , which antiation ,43. 3T3-ntiation  and LRP ntiation .125I-RAP at 4°C  and p-nitrophenyl-β-D-xylopyranoside (pNP-Xyl) are reagents that can serve as alternative acceptors within cells for heparan sulfate moieties and are thus able to compete for heparan sulfate chain addition to proteoglycan core proteins ,52. The To determine if HSPG are involved in intracellular lipid accumulation by mature adipocytes, we assessed cytosolic lipid droplet formation in induced 3T3-L1 cells following treatment with either 4-MUmb or pNP-Xyl. Again 3T3-L1 pre-adipocytes were incubated with 4-MUmb or pNP-Xyl concurrently with the addition of differentiation reagents and intracellular lipid accumulation was assessed by Oil Red O staining. Upon Oil Red staining, untreated mature adipocytes demonstrated large intracellular droplets; the characteristic morphologic feature of cytosolic lipid accumulation Fig. . TreatmeIntracellular lipid accumulation is a hallmark event of adipocyte development and the major factor in adipocyte hypertrophy. This prompted us to investigate the molecular mechanism by which adipocytes take up extracellular lipid components. In the present study, we provide new information on the expression levels and functional activities of certain cell surface receptors in mature adipocytes that are known to play primary roles in lipoprotein processing and clearance. These receptors have been extensively studied in liver, the major organ for dietary lipoprotein clearance , howeverThese findings offer a novel observation that cell surface HSPG appear to be essential for lipid accumulation and lipid droplet formation in adipocytes. However, this observation also raises the question as to the exact role of HSPG in the transport of fatty acids across the adipocyte plasma membrane. Based on the results presented in this study and those reported elsewhere, we have drawn a testable model to define how HSPG contribute to intracellular lipid accumulation by adipocytes Fig. . HSPG arThe identity of this HSPG is currently unknown; however, its large relative molecular mass and composition containing both heparan and chondroitin sulfates are consistent with the structural properties of the syndecan ,61 and pThe availability of circulating triacylglycerol-rich lipoproteins for adipocyte utilization and storage relies on transport of these particles across the endothelial barrier. Transcytosis of albumin across endothelium has been shown to occur by a vesicular transport pathway -70 and iHow HSPG might coordinate its activity with CD36 or FATP1 for fatty acid uptake or possibly with members of the LDL receptor family for assisted internalization is under current investigation. Presently, our results provide novel findings indicating that cell surface HSPG activity is necessary for lipid accumulation by adipocytes. It is anticipated that these observations will aid in our understanding of the complex mechanism leading to adipose hypertrophy and obesity, and provide a novel avenue to explore for targeted reduction of intracellular lipid accumulation35SO4 were purchased from MP Biomedicals . p-nitrophenyl-β-D-xylopyranoside and 4-methylumbelliferyl-β-D-xylopyranoside were from Calbiochem . Heparin, heparinase I, chondroitinase ABC, p-nitrophenylbutyrate, lipoprotein lipase, and Oil Red O were purchased from Sigma-Aldrich. Optiprep was from Greiner Bio-One . DiI  was purchased from Molecular Probes . Apolipoprotein E was obtained from Calbiochem. RAP-GST fusion protein was purified as previously described [Anti-LRP polyclonal antibody was raised against an 18 amino acid peptide from the cytoplasmic tail of human LRP  and antiescribed . Tissue 2 and passaged twice weekly. To differentiate 3T3-L1 cells into adipocytes, cells were incubated with 100 ng/ml dexamethasone, 100 μg/ml 3-isobutyl-1-methylxanthine, and 1 μg/ml insulin for 4 days, followed by 1 μg/ml insulin for an additional 4–8 days.3T3-L1 cells were obtained from American Type Culture Collection  and grown in Dulbecco's modified Eagle's medium (DMEM)  supplemented with 10% (v/v) fetal calf serum , 1 mM sodium pyruvate, 100 μg/ml streptomycin sulfate, and 100 units/ml penicillin. Cells were cultured at 37°C with 5% COCell lysates were prepared with 20 mM Tris pH 7.4, 150 mM NaCl (TBS) containing 1% (v/v) Triton-X100. For leptin, culture supernatants were concentrated by mixing with 3 volumes ice cold acetone, incubating at -20°C for 1 h, followed by centrifugation at 10,000 × g at 4°C for 15 min. Protein pellets were resuspended with SDS-PAGE sample buffer supplemented with 2% (v/v) β-mercaptoethanol. Proteins were then separated by SDS-PAGE and transferred to Immobilon-P  using a wet tank transfer system . Membranes were blocked with TBS, 0.1% (v/v) Tween-20, 5% (w/v) non-fat dry milk for 20 minutes at 23°C and incubated with the indicated antibody for 2 h at 23°C. Membranes were washed three times (10 min each) with TBS, 0.1% (v/v) Tween-20, and bound antibodies were detected with species-specific HRP-conjugated secondary antibodies  followed by chemiluminescence detection according to the manufacturer's instructions . Images were captured using a Syngene GeneGnome system equipped with a Peltier-cooled 16-bit CCD camera and saturation detection. Densitometric analysis was performed using Scion Image, version 4.0.2.125I as previously described [125I-RAP-GST (500 ng/ml) diluted into 20 mM Hepes, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 1% (w/v) bovine serum albumin (buffer A) at 4°C for 3 h in the presence or absence of a 50-fold molar excess of unlabeled RAP-GST. Unbound 125I-RAP-GST was removed by rinsing cells three times with cold buffer A after which cells with bound ligand were solubilized with 0.1 N NaOH. Solubilized proteins were added to EcoLume  and subjected to scintillation counting . Results were normalized to total cellular protein . Specificity was determined as the difference between total binding (without competition) and non-specific binding (non-competable) [125I-labeled ligand. All data points represent averages of duplicates or triplicates with standard errors of <5%.RAP-GST was purified  and labeescribed . Differepetable) . The act2 for 3 h with 500 ng/ml 125I-RAP-GST diluted into DMEM containing 1% (w/v) bovine serum albumin in the presence or absence of a 50-fold molar excess of unlabeled RAP-GST. Media was then removed and processed for trichloroacetic acid (TCA) precipitation [Differentiated 3T3-L1 cells were incubated at 37°C/5% COpitation . TCA-sol2 35SO4 for 20 h at 37°C/5% CO2. Cells were detached by incubating with phosphate buffered saline containing 5 mM EDTA for 20 min at 23°C. Following centrifugation to pellet cells , they were lysed by incubation with solubilization buffer  for 1 h at 4°C. Insoluble material was removed by centrifugation  and supernatant was batch adsorbed onto 0.5 ml Macro-Prep DEAE Support (BioRad) (pre-washed with solubilization buffer) with gentle agitation for 1 h at 4°C. DEAE resin was applied to a 1.0 cm × 5.0 cm column and washed with 10 column volumes solubilization buffer followed by 10 column volumes 100 mM Tris, pH 7.5, 150 mM NaCl. Bound proteins were eluted with 100 mM Tris, pH 7.5, 1.0 M NaCl and 120 μl fractions were collected. Fifteen μl from each fraction was mixed with 3 ml EcoLume and subjected to scintillation counting. Peak fractions from DEAE enrichment of proteoglycans obtained from labeled differentiated adipocytes  or chondroitinase ABC (5 units/ml) or both for 20 h at 37°C. The material was applied to a 1.0 cm × 12 cm Sephacryl S-200 HR  gel filtration column and eluted with 20 mM Tris, pH 7.4, 150 mM NaCl. 350 μl fractions were collected of which 200 μl was mixed with 3 ml EcoLume and analyzed by scintillation counting.3T3-L1 pre-adipocytes and differentiated adipocytes grown in 100 mm tissue culture dishes were incubated with sulfate-free DME containing 10% (v/v) fetal calf serum and 125 μCi/ml NaNew Zealand White rabbits were placed on a high-fat chow diet (10% peanut oil/1% cholesterol) for a minimum of 4 d, blood was drawn into in 1 mM EDTA and centrifuged at 2000 × g, 15 min to remove cells. Chylomicrons were floated by centrifuging plasma at 100,000 × g for 10 min and removed by pipetting. Plasma was then mixed with OptiPrep™  and centrifuged at 350,000 × g for 3 h (SW55Ti rotor) with slow acceleration and deceleration. VLDL particles (density of 1.006 g/ml) were removed from the top of the gradient by pipetting. Purified VLDL was analyzed by SDS-PAGE and Coomassie R staining to confirm the presence of apoB100 (515 kD) and apoE (35 kD). Animal protocol (#2032) was approved by the University of New Mexico, Health Sciences Center Laboratory Animal Care and Use Committee. For DiI labeling, a working stock of 3 mg/ml was made in dimethylsulfoxide and 0.15 mg was slowly added to 1.67 mg VLDL (in 1.9 ml) with vortexing to rapidly mix. The mixture was then wrapped in foil and incubated for 8 h at 37°C. Unbound DiI was removed from DiI-labeled VLDL by OptiPrep gradient centrifugation as described above.3T3-L1 cells were plated on glass coverslips and incubated with differentiation reagents as described above. After conversion to mature adipocytes, cells were rinsed twice with DMEM and incubated with DiI-VLDL (4 μg/ml) and apolipoprotein E (3 μg/ml) diluted into DMEM in the presence or absence of either heparin (500 μg/ml) or RAP-GST (50 μg/ml). After 3 h at 37°C, cells were rinsed with phosphate buffered saline, fixed with 1.5% (w/v) paraformaldehyde for 30 min, and mounted in Gelvatol  containing 1 mg/ml p-phenylenediamine. Cells were observed with a Zeiss Axioskop microscope equipped for epifluorescence. Images were capture with a Hamamatsu digital/video camera and AxioVision software.Treated and non-treated 3T3-L1 cells (as indicated in figure legends) were cultured overnight in serum-free DMEM without phenol red. Media was removed and combined with an equal volume of 100 mM sodium phosphate buffer, pH 7.2, 150 mM NaCl, 0.5% (v/v) Triton-X100. p-nitrophenyl butyrate in acetonitrile was added to a final concentration of 0.5 mM and absorbance at 400 nm was recorded every 10 s for 5 min using a Genesys UV Spectrophotometer. A standard curve for LPL activity was generated by plotting absorbance values obtained with varying concentration of purified LPL (from bovine milk). Quantitation of LPL activity in the individual media samples was determined from the standard curve.Cells were fixed with 10% (v/v) formaldehyde in phosphate buffered saline for 1 h at 23°C, rinsed twice with water, then stained for 2 h at 23°C with 0.1% (w/v) Oil Red O in 75% (v/v) isopropanol, followed by rinsing twice with water to remove unincorporated dye. Stained cells were viewed and photographed using a Zeiss AxioVert microscope with phase contrast optics and Hamamatsu digital/video camera. For quantitation, cells were dried for 2 h at 37°C, followed by incubation with 100% isopropanol for 15 m at 23°C to extract bound dye. Solubilized dye was then quantitated by spectrophotometry in a BioRad Model 680 Microplate Reader by measuring absorbance at 510 nm. Statistical significance was determined by performing a paired t-test.ApoE = apolipoprotein EHSPG = heparan sulfate proteoglycanVLDL = very low density lipoproteinVLDL-R = VLDL receptorLRP = low density lipoprotein receptor-related proteinSyn-1 = Syndecan-1FATP1 = fatty acid transport protein-1RAP = receptor associated proteinLPL = lipoprotein lipaseDiI = 1,1'-dictadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate4-MUmb = 4-methylumbelliferyl-β-D-xylopyranosidepNP-Xyl = p-nitrophenyl-β-D-xylopyranosidebFGF = basic fibroblast growth factorLCW carried out the majority of studies and drafted the manuscript. SC performed the xyloside titration study and DN performed the LPL assays. RAO provided the original conceptual framework for the study, carried out pilot experiments, participated in the experimental design and finalized the manuscript for submission. All authors read and approved the final version.


The fusion protein RUNX1-CBFA2T1 associated with t-positive acute myeloid leukaemia is a potent inhibitor of haematopoetic differentiation. The role of RUNX1-CBFA2T1 in leukaemic cell proliferation is less clear. We examined the consequences of siRNA-mediated RUNX1-CBFA2T1 depletion regarding proliferation and clonogenicity of t-positive cell lines.The t-positive cell line Kasumi-1 was electroporated with RUNX1-CBFA2T1 or control siRNAs followed by analysis of proliferation, colony formation, cell cycle distribution, apoptosis and senescence.Electroporation of Kasumi-1 cells with RUNX1-CBFA2T1 siRNAs, but not with control siRNAs, resulted in RUNX1-CBFA2T1 suppression which lasted for at least 5 days. A single electroporation with RUNX1-CBFA2T1 siRNA severely diminished the clonogenicity of Kasumi-1 cells. Prolonged RUNX1-CBFA2T1 depletion inhibited proliferation in suspension culture and G1-S transition during the cell cycle, diminished the number of apoptotic cells, but induced cellular senescence. The addition of haematopoetic growth factors could not rescue RUNX1-CBFA2T1-depleted cells from senescence, and could only partially restore their clonogenicity.RUNX1-CBFA2T1 supports the proliferation and expansion of t-positive leukaemic cells by preventing cellular senescence. These findings suggest a central role of RUNX1-CBFA2T1 in the maintenance of the leukaemia. Therefore, RUNX1-CBFA2T1 is a promising and leukaemia-specific target for molecularly defined therapeutic approaches. The chromosomal translocation t8;21) , which is associated with 10–15% of all cases of acute myeloid leukaemia, fuses the DNA binding domain of the transcription factor RUNX1  to the almost complete open reading frame of CBFA2T1  -propansulfonat (CHAPS), 1% (w/w) Dithiothreitol) and treated for 30 min on ice with 2.5 u/ml Benzonase . Total (10–20 μg) and nuclear lysates (10 μg) were analyzed as described . Band in-  as suggested by the manufacturer. Real time PCRs were performed using Sybr-Green Mix , 300 nM primers and 20% v/v diluted RT reaction mix using standard conditions on a 7000 Sequence detection system . The primer sequences for STAT1 (5'-CAT CAC ATT CAC ATG GGT GGA (forward primer) and 5'-GGT TCA ACC GCA TGG AAG TC (reverse primer)), for CD34 (5'-TCC AGA AAC GGC CAT TCA G (forward primer) and 5'-CCC ACC TAG CCG AGT CAC AA (reverse primer)), for G-CSF (5'-CCC ACC TTG GAC ACA CTG C (forward primer) and 5'-AGT TCT TCC ATC TGC TGC CAG (reverse primer)) and for GAPDH (5'-GAA GGT GAA GGT CGG AGT C (forward primer) and 5'-GAA GAT GGT GAT GGG ATT TC (reverse primer)) were designed with Primer-Express software .Total RNA was isolated using the RNeasy Kit . Reverse transcription was performed with random hexamers using MMLV-RT, RNase HTo determine proliferation rates and doubling times, cells were counted using trypan blue. For single cell expansion assays, 24 h after electroporation cells were diluted to a density of 10 cells/ml and plated in 100 μl aliquots into 96 well tissue culture plates. Colony formation assays were performed 24 h after electroporation with a density of 5,000 cells/ml in semisolid medium . Colonies were counted 14 days after plating. The cytokine concentrations were 20 ng/ml for G-CSF and GM-CSF in the cell cycle and senescence assays, and 10 ng/ml for all growth factors in the colony formation assays.6 cells were suspended in 200 μl citrate buffer  followed by the addition of 800 μl staining solution  NonIdet P40, 500 μM EDTA) and 10 μl boiled RNase A (10 g/l). Cells were kept for 30 minutes on ice and analyzed by flow cytometry . Data analysis was performed with FCSPress 1.3 .Cell cycle analysis was performed as described . Four da for data analysis.For quantification of apoptotic cells, cells were stained with FITC-labeled annexin V as suggested by the supplier . CD34 expression was monitored by staining with anti-human CD34-FITC . Analysis was performed by flow cytometry  using FCSPress 1.3 Electroporation of t-positive cells with the RUNX1-CBFA2T1 siRNA siAGF1, but not with the mismatch control siRNA siAGF6, resulted in a three- to fourfold reduction of RUNX1-CBFA2T1 fusion protein in both Kasumi-1 cells fig. . This fiTransfection with siRNAs may induce an interferon response leading to non-specific effects on cellular processes such as proliferation or differentiation . Since SIn comparison to mock-transfected cells, electroporation with RUNX1-CBFA2T1 siRNAs resulted in a ten- to twenty fold decreased clonogenicity in both single cell expansion (data not shown) and colony formation assays -negative myeloid leukaemia cell lines MV4-11, NB4, HL60, U937 and K562 was not affected upon electroporation with RUNX1-CBFA2T1 siRNA (data not shown), which argues against a general, unspecific effect of the siRNAs used in this study on leukaemic clonogenicity. Therefore, the application of RUNX1-CBFA2T1 siRNAs is sufficient to inhibit the clonogenic growth of Kasumi-1 cells, indicating that RUNX1-CBFA2T1 is essential for the clonogenicity of these leukaemic cells.To examine the effects of RUNX1-CBFA2T1 on the progenitor status of t-positive leukaemic cells, we analyzed the CD34 expression levels in siRNA-treated Kasumi-1 cells. When compared to mock-transfected cells, depletion of RUNX1-CBFA2T1 caused a twofold decrease in CD34 surface expression 7 days after electroporation . Two or more consecutive electroporations with RUNX1-CBFA2T1 siRNA caused a reduction of the amount of S phase cells by 50% and a 30% reduction in G2/M phase cells, with a corresponding increase of the G1/G0 fraction by 20% fig. . TreatmeKip1), a general inhibitor of Cyclin-CDK complexes and, thereby, of cell cycle progression. Two electroporations with RUNX1-CBFA2T1 siRNA caused a twofold increase in CDKN1B protein levels in RUNX1-CBFA2T1-depleted cells, but not in control cells (fig. The changes in the cell cycle distribution are associated with changed expression levels of CDKN1B (p27lls fig. . TherefoA possible reason for the observed inhibition of proliferation is the induction of apoptosis upon suppression of RUNX1-CBFA2T1. We addressed this possibility by examining the amount of apoptotic cells by annexin V staining and of hypodiploid (subG1) cells by flow cytometry. Electroporation of Kasumi-1 cells together with the RUNX1-CBFA2T1 siRNA siAGF1 neither increased the amount of hypodiploid cells nor the amount of annexin V positive cells when compared to control cells. Instead, after siAGF1 treatment we observed a slight but reproducible decrease in the amount of apoptotic cells in both assays. This decrease was more pronounced after two consecutive electroporations with siRNAs. Repeating the siAGF1 electroporation after 4 days resulted in a threefold decrease of the amount of annexin V stained cells fig.  indicatiThe observed G1 arrest upon RUNX1-CBFA2T1 depletion could be either reversible or irreversible. An irreversible G1 arrest is a hallmark of cellular senescence. To address the question of the nature of the G1 arrest, and to analyze a possible influence of RUNX1-CBFA2T1 on cellular senescence, we stained siRNA- and mock-treated cells for senescence-associated β-galactosidase activity. After two or more electroporations with RUNX1-CBFA2T1 siRNA, but not with control siRNA, a significant fraction of the Kasumi-1 cells stain positive for β-galactosidase fig. . Cell coA possible mechanism of how RUNX1-CBFA2T1 prevents cell cycle arrest and senescence is the autocrine production of growth factors. Kasumi-1 cells express, for instance, G-CSF fig. , but notDepletion of RUNX1-CBFA2T1 for 16 days reduced the fraction of S phase cells twofold compared to control cells. This decrease is independent of G-CSF or GM-CSF fig. . When anFinally, we examined the effects of several haematopoetic growth factors on colony formation of siRNA-treated Kasumi-1 cells. Supplementing the semisolid medium with the corresponding growth factors increased colony numbers for both control cells and RUNX1-CBFA2T1-depleted cells fig. . G-CSF eTaken together, the addition of G-CSF or GM-CSF delays, but does not prevent the G1 arrest in RUNX1-CBFA2T1-depleted cell. Furthermore, senescence is not inhibited by these growth factors. In line with these findings, neither of these growth factors completely restored the impaired clonogenicity of such cells. These results indicate that neither G-CSF nor GM-CSF can rescue leukaemic cells suffering of siRNA-mediated RUNX1-CBFA2T1 depletion.3 receptor [Arf [ex vivo and in vivo [The leukaemic fusion protein RUNX1-CBFA2T1 inhibits haematopoetic differentiation by repressing gene expression via recruitment of histone deacetylases -7, and breceptor -11. In areceptor ,46, in stor [Arf . The not in vivo ,21. In aWe show that RUNX1-CBFA2T1 also supports proliferation, CD34 expression and colony formation in the t-positive leukaemic cell line Kasumi-1. Thus, RUNX1-CBFA2T1 functions are similar in HSCs and in Kasumi-1. In the latter, support of proliferation is not caused by an inhibition of cell death. Instead, as observed with many other cell types ,46, RUNXKip1 (CDKN1B) during RUNX1-CBFA2T1 depletion are in line with the recently demonstrated central role of this CDK inhibitor in the establishment of senescence [The reduced proliferation of Kasumi-1 cells upon RUNX1-CBFA2T1 depletion is paralleled by a cell cycle arrest in G1. Particularly from a therapeutic point of view, it is important to know, whether this G1 block will be relieved by recovering RUNX1-CBFA2T1 expression, or by growth factors, or whether this arrest is irreversible. We observe that RUNX1-CBFA2T1 depleted cells show enhanced levels of senescence-associated β-galactosidase activity, a hallmark of cellular senescence. Therefore, the inhibition of G1 to S transition is associated by the induction of senescence indicating that this G1 arrest is irreversible. Interestingly, the elevated levels of p27nescence ,50.Arf [Arf and of its human homologue, p14Arf [Furthermore, RUNX1 was shown to induce senescence in murine embryonic fibroblasts, probably by inducing p19Arf . Since R, p14Arf , an incr3 [An important point to consider is a possible relation between the effects of RUNX1-CBFA2T1 on clonogenicity, proliferation and senescence, and its anti-differentiation activity. For instance, increased β-galactosidase activity may also be associated with late monocytic differentiation . Moreove3 . However3 , and datin vivo might interfere with the development and maintenance of leukaemia. However, a remaining possibility might be that the stroma environment with its complex mixture of growth factors and cell-cell interactions may be able to rescue RUNX1-CBFA2T1-depleted cells. We are currently investigating this question both in cell culture and in vivo.The haematopoetic growth factors G-CSF and GM-CSF were shown to support clonogenicity and proliferation in Kasumi-1 cells . HoweverA major point of concern for siRNA applications is the specificity of the corresponding siRNA . ReasonsIn summary, we show that RUNX1-CBFA2T1 does not only inhibit the myeloid differentiation , but is HPV is an employee of Alnylam Europe AG, which also provided some of the siRNAs used in this study.NM performed the proliferation, senescence and cell cycle assays. BD analyzed the clonogenicity of growth-factor treated Kasumi-1 cells and of t negative cell lines. HR performed the immunoblot analyses and was involved in the analysis of clonogenicity and apoptosis. CC examined the expression levels of CD34 and of STAT1. HPV, AG, GH and AN participated in the coordination of the study. JK and OH conceived and designed the experiments. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


By aligning probe sequences with expressed sequence tags (ESTs) in NHP, inter-species conserved (ISC) probesets, which have two or more probes complementary to ESTs in NHP, were identified on human GeneChip® platforms. The utility of human GeneChips® for the assessment of NHP expression patterns can be effectively evaluated by analyzing the hybridization behaviour of ISC probesets. Appropriate normalization methods were identified that further improve the reliability of human GeneChips® for interspecies (human vs NHP) comparisons.While researchers have utilized versions of the Affymetrix human GeneChip® platforms  were identified for both monkey and chimpanzee. Expression data was generated from peripheral blood mononuclear cells (PBMCs) of 12 human and 8 monkey (Indian origin Rhesus macaque) samples using the Focus GeneChip®. Analysis of both qualitative detection calls and quantitative signal intensities showed that intra-species reproducibility (human vs. human or monkey vs. monkey) was much higher than interspecies reproducibility (human vs. monkey). ISC probesets exhibited higher interspecies reproducibility than the overall expressed probesets. Importantly, appropriate normalization methods could be leveraged to greatly improve interspecies correlations. The correlation coefficients between human (average of 12 samples) and monkey (average of 8 Rhesus macaque samples) are 0.725, 0.821 and 0.893 for MAS5.0 (Microarray Suite version 5.0), dChip and RMA (Robust Multi-chip Average) normalization method, respectively.ISC probesets in each of the seven Affymetrix GeneChip® platforms to assess the expression profiles of NHP for intra-species studies. Caution must be taken for interspecies studies since unsuitable probesets will result in spurious differentially regulated genes between human and NHP. RMA normalization method and ISC probesets are recommended for interspecies studies.It is feasible to use Affymetrix human GeneChip An underlying assumption in such studies is that transcripts of humans and NHP are highly conserved, and probe sequences designed to detect human genes will detect their orthologs in NHP samples. It is estimated that chimpanzees (Pan troglodytes) and humans shared 98.77 % DNA similarity [® for NHP expression analysis.Microarray studies on non human primates (NHP) have been used to address viral pathogenesis ,2, neuromilarity  comparedmilarity ,12, and ® platforms for NHP expression profiling have robustly addressed the quantitative aspects of cross platform performance. Vahey and colleagues [® and demonstrated that there was no significant difference in the dynamic range of the raw fluorescence distribution for equivalent amounts of human cRNA and macaque cRNA hybridized to the chip. Chismar and colleagues [® platform and compared the expression patterns of humans with that of the rhesus macaque. They concluded that the percentage of 'present' calls observed in the transcriptome of macaque brain is lower than that of human brain, and that this is especially true for genes with lower signal intensity. Caceres and colleagues [® platforms for the study of expression profiles in NHP is to employ a sequence analysis approach.Few published studies employing human GeneChiplleagues  used thelleagues  used thelleagues  used the® platforms to assess expression patterns in NHP samples by: a) identifying ISC probesets based on sequence analysis; b) assessing intra (within NHP species)- and interspecies (between NHP and human samples) reproducibility of GeneChip® data; and c) applying appropriate normalization methods to improve interspecies reproducibility.In this study, we address the power of human GeneChip® hybridizes with the transcriptome of a NHP, there are three possible outcomes: 1) it hybridizes with the ortholog of the NHP; 2) it cross-hybridizes with a non-ortholog transcript, or 3) it fails to hybridize due to sequence divergence. In Affymetrix GeneChip® system, a probeset is composed of 11–20 probes and each probe is a 25-mer oligo. We identified probes on the human GeneChip that are complementary to ESTs in NHP postulating that these probes would hybridize most optimally with the transcripts of NHP. We defined a probeset as an ISC probeset if it had at least two complementary probes. The rationale used to define the criterion that defines an ISC probeset is described in the methods section. The procedure used to generate ISC probesets is shown in Figure ® platforms  were generated for both monkey and chimpanzee. Detailed information about each ISC probeset such as probe sequence, GenBank accession and the position and degree of matching is provided in the supplemental materials (Macaca mulatta) than for chimpanzee (Pan troglodytes). This is not because monkey EST sequences are more similar to human sequences than chimpanzee EST sequences, but because we have a much greater amount of EST sequences available for monkey. At the time of writing this manuscript, there were 33,474 monkey ESTs available, while there were only 6,943 ESTs available for chimpanzee. As the number of defined ESTs will increase in the future, additional ISC probesets could be identified for both monkey and chimpanzee using this method.When a probe sequence on the human GeneChipaterials . Table 1®s, there are three probesets  that target the gene CXCR4 at different positions in its transcript. In order to address this redundancy issue, we converted the number of probesets into the number of unique UniGene clusters based on the GeneChip® annotation file provided by Affymetrix Website [® and the U133Plus2.0 GeneChip® have the lowest and highest frequency of redundant probesets for a given gene, respectively.It is not uncommon, especially in the U133Plus2.0 platform, that multiple probesets target the same gene. For example, in the U133A and the U133 Plus 2.0 GeneChip Website . While a® from Affymetrix and covers the human genome most extensively. Figure The U133Plus2.0 is the most current version of human GeneChip® data observed in intra- and interspecies samples. The intra-species reproducibility is displayed in Figure ® for NHP intra-species studies.The qualitative detection call (present / absent) output from MAS5.0 was the initial approach used to examine the reproducibility of GeneChip® platforms are used to compare the expression pattern of humans with those of NHPs, care must be taken in the interpretation of data. If we consider a probeset as being expressed when 50% or more of replicates have present calls, then 3445 (2059+1386) and 2321 (2059+262) probesets are expressed in the PBMC fraction of humans and monkeys, respectively  was created. Probesets that are not expressed in human PBMCs were excluded in this analysis. Pair-wise correlation coefficients were calculated for all 20 samples . The correlation coefficients were visualized using heat spectrum graphs where colors ranging from red to white correspond to correlation coefficients of 0.5 to 1.0, respectively. In figure To assess the intra- and interspecies reproducibility of GeneChip® data variation [® data at the probe level using a non-linear algorithm while MAS5.0 normalizes data at probeset level using linear scaling. Sequence divergence usually leads to one or very few probes in a probeset being problematic while the majority of probes in that probeset may still work reasonably well. If the variation generated from these problematic probes were normalized, the interspecies reproducibility should improve. Figure Different normalization methods have been shown to significantly affect GeneChipariation -17. We cariation -16 and d® expression data as applied to the derivation of meaningful expression profile data from NHP. The utility of the human Affymetrix GeneChip® for the assessment of expression profiles in NHP depends on the experimental design and on the approach to data normalization and analysis. Our observations suggest that: 1) it is feasible to use the human GeneChip® in the evaluation of expression profiles of NHP samples for intra-species comparisons; 2) use of ISC probesets and RMA normalization are recommended for interspecies studies; and 3) with the increasing amount of ESTs of NHP, additional ISC probesets (and perfect probesets) will be identified in the near future.This paper presents a comprehensive analysis of probe sequences and GeneChipMacaca mulatta) and chimpanzee (Pan troglodytes) were downloaded from NCBI website [Affymetrix GeneChip probe sequences were downloaded from Affymetrix website . The EST website .® probe sequences and monkey /chimpanzee EST sequences. The length of a probe sequence is always 25 nucleotides while the number of probes in a probeset varies from 11 to 20 depending on GeneChip® platforms  in comparison with an RT-PCR experiment, the primer length is equivalent to our probe length, and two primers (one forward and one backward) usually generate a unique sequence in a whole genome; 3) a probe sequence on the Affymetrix GeneChip® is a well designed sequence with a single probe hybridizing with a unique transcript in whole transcriptome; and 4) since the EST sequences in NHP are very limited so far, most of them do not cover whole transcript such that a false negative could be generated if we require all the probes in a probeset being complementary to known ESTs. In order to convert probeset IDs to UniGene IDs and map them onto chromosomes, probeset annotation files were downloaded from Affymetrix website [Stand alone BALST program was downloaded from NCBI website . Perl sc website . No animRhesus macaque) was collected and peripheral blood mononuclear cells (PBMCs) were separated by Histopaque-Ficoll (Sigma) gradient centrifugation. RNA preparation, Hybridization, staining and scanning of the GeneChip® was carried out as described by Vahey et al. [®. Signal values and detection calls (present or absent) for all samples were determined by using MAS5.0 . Signal values were scaled to the default target signal intensity of 500). A matrix of detection calls  and a matrix of signal intensities for all samples across all probesets were constructed. A gene must exhibit 50% or more of 'present' calls in all samples to be considered 'expressed'. In this study, an expressed probeset in human is a probeset that has 6 or more present calls among 12 human samples. Similarly, an expressed probeset in monkey means there were 4 or more present calls among 8 monkey samples. The signal intensities output from MAS5.0 were log2 transformed. Model-based normalization was performed using dChip version 1.3 [rma function in the package was used at its default setting, that is, 'RMA' background correction, 'quantile normalization', 'PM only model' and 'median polish summarization'. By default, the signal intensities were already log2 transformed.Briefly, peripheral blood from healthy human and NHP  and set color scales between 0.5 (red) and 1.0 (white).Intra- and interspecies correlation coefficients of signal intensities were calculated by built in function 'n 1.9.0. . VisualiNHP: non human primateEST: expressed sequence tagISC: inter-species conservedRMA: robust multi-chip averageMAS5.0: Microarray suite version 5.0ZW developed the original hypotheses, performed the bioinformatics analyses to test them and drafted the manuscript. MV provided critical input on design and execution of the laboratory experiments and with ZW interpreted the data sets and revised the manuscript. ML conducted all aspects of the animal handling including the harvest of well characterized primary samples. MN and AA are technical staff who extracted the nucleic acid and performed the laboratory portions of the microarray experiments. All authors read and approved the final manuscript.Macaca mulatta) in GeneChip® platforms Plus2.0, U133A and U133B, respectively. Note: All the four additional files are multiple-sheets MS excel files. Within each worksheet of a file, the rows are ISC probesets, the columns are probeset IDs, positions of a probe (x and y coordinates in the chip), GenBank accessions of a matching EST, probe sequence, matched EST sequence and position, BLAST e- values and matching identities, in that order.There are three worksheets in this file. Worksheet 1, 2 and 3 are ISC probesets for monkey (Click here for fileMacaca mulatta) in GeneChip® platforms Focus, FL, U95Av2 and U95B, respectively. Note: All the four additional files are multiple-sheets MS excel files. Within each worksheet of a file, the rows are ISC probesets, the columns are probeset IDs, positions of a probe (x and y coordinates in the chip), GenBank accessions of a matching EST, probe sequence, matched EST sequence and position, BLAST e- values and matching identities, in that order.There are four worksheets in this file. Worksheet 1, 2, 3 and 4 are ISC probesets for monkey (Click here for filePan troglodytes) in GeneChip® platforms Plus2.0, U133A and U133B, respectively. Note: All the four additional files are multiple-sheets MS excel files. Within each worksheet of a file, the rows are ISC probesets, the columns are probeset IDs, positions of a probe (x and y coordinates in the chip), GenBank accessions of a matching EST, probe sequence, matched EST sequence and position, BLAST e- values and matching identities, in that order.There are three worksheets in this file. Worksheet 1, 2 and 3 are ISC probesets for chimpanzee (Click here for filePan troglodytes) in GeneChip® platforms Focus, FL, U95Av2 and U95B, respectively. Note: All the four additional files are multiple-sheets MS excel files. Within each worksheet of a file, the rows are ISC probesets, the columns are probeset IDs, positions of a probe (x and y coordinates in the chip), GenBank accessions of a matching EST, probe sequence, matched EST sequence and position, BLAST e- values and matching identities, in that order.There are four worksheets in this file. Worksheet 1, 2, 3 and 4 are ISC probesets for chimpanzee (Click here for file