Current educational strategies to integrate HIV care into primary medical care in Central America have traditionally targeted managers or higher-level officials, rather than local health care workers (HCWs). We developed a complementary online and on-site interactive training program to reach local HCWs at the primary care level in underserved communities.The training program targeted physicians, nurses, and community HCWs with limited access to traditional onsite training in Panama, Nicaragua, Dominican Republic, and Guatemala. The curriculum focused on principles of HIV care and health systems using a tutor-supported blended educational approach of an 8-week online component, a weeklong on-site problem-solving workshop, and individualized project-based interventions.Of 258 initially active participants, 225 (225/258 = 87.2%) successfully completed the online component and the top 200 were invited to the on-site workshop. Of those, 170 (170/200 = 85%) attended the on-site workshop. In total, 142 completed all three components, including the project phase. Quantitative and qualitative evaluation instruments included knowledge assessments, reflexive essays, and acceptability surveys. The mean pre and post-essay scores demonstrating understanding of social determinants, health system organization, and integration of HIV services were 70% and 87.5%, respectively, with an increase in knowledge of 17.2% (p<0.001). The mean pre- and post-test scores evaluating clinical knowledge were 70.9% and 90.3%, respectively, with an increase in knowledge of 19.4% (p<0.001). A survey of Likert scale and open-ended questions demonstrated overwhelming participant satisfaction with course content, structure, and effectiveness in improving their HIV-related knowledge and skills.This innovative curriculum utilized technology to target HCWs with limited access to educational resources. Participants benefited from technical skills acquired through the process, and could continue working within their underserved communities while participating in the online component and then implement interventions that successfully converted theoretical knowledge to action to improve integration of HIV care into primary care. Central America, which consists of Belize, Costa Rica, El Salvador, Guatemala, Honduras, Nicaragua, and Panama, has been particularly affected by the rapidly spreading HIV epidemic in Latin America. Four of the countries with the highest prevalence rates in Latin America are in Central America and 200,000 people live with HIV/AIDS in the region Although guidelines are developed and discussed between national authorities and HIV experts, many training activities target doctors at specialized care clinics or hospitals In order to develop an effective training program targeted directly to local HCWs in Central America, a government/academic partnership was formed between the Johns Hopkins Center for Clinical Global Health Education, Gorgas Memorial Institute in Panama, and the Pan American Health Organization (PAHO), with an evaluation component by the Epidemiological Research Center in Sexual and Reproductive Health in Guatemala (CIESAR by its name in Spanish) and funded by the US Department of Health and Human Services. Through this collaborative effort, we developed a novel 3-pronged blended educational strategy contextualizing theory into practical skills through an online distance learning component, followed by an on-site problem-solving workshop, and the development of intervention-action projects. The goal of this curriculum was to provide participants with the knowledge, skills, and attitudes necessary to improve the delivery of health services by integrating high-quality basic HIV and related infectious disease care with primary care. Our strategy was novel as it offered tutor-supported complementary online and on-site interactive components that translated knowledge into action and allowed us to specifically reach local HCWs in underserved communities with limited access to educational resources. In addition, the 8 week online component prepared students for the on-site training workshop by reinforcing fundamental concepts while avoiding prolonged absences from the workforce.The Ministries of Health of each of the four participating countries  were approached. The baseline evaluation and the planned training program components and processes were described to the local authorities and a formal written approval was obtained to conduct the program with participants from their countries. Additionally, the Ministries of Health provided formal written statements that approval by their national bioethics committees was not needed, as this was an educational activity and the evaluation activities posed no risk to participants. The Johns Hopkins Medicine Institutional Review Boards approved the analysis of the data from the training program (NA_00075375), which qualified for exemption as not human subjects research. The analysis used program evaluation activities to examine the overall effectiveness, feasibility, and acceptability of the educational program, and posed no risk to participants. Written consent was obtained for all surveys in the baseline evaluation, but was not required for the anonymously analyzed data from the course evaluation activities.A general needs assessment consisting of interviews with regional leaders, content experts, and community stakeholders, and a review of local and regional data from country reports CIESAR carried out a baseline evaluation of HCWs, administrators, and patients in the selected regions. This evaluation set out to determine Knowledge, Attitudes and Practices (KAP) regarding the integration of HIV and other infectious diseases into primary health care. Three different questionnaires were directed at i) physicians, nurses and health educators, ii) directors of selected health services facilities, and iii) health service users (outpatients) in the targeted areas. The questionnaires were validated and adjusted in each participating country with changes to adapt for local language.Following the results of the needs assessment, the training program was deployed in Guatemala, Nicaragua, the host country of Panama, and the Dominican Republic in the Caribbean. At the time of the development of the program, other countries were considered based on HIV prevalence but were ruled out due to either political instability at the time (Honduras) or language (Belize). The focus was on HCWs  who work directly in primary care in underserved areas with limited access to health services and limited access to traditional onsite training. Participants were nominated by the national health authorities of their countries following specific selection criteria: 1) operational staff located in the area selected, 2) permanent contract staff, 3) staff enthusiasm and commitment to participate, 4) potential to assist in replication of course, and 5) basic computer knowledge.The main goal of the training program entitled “Integration of HIV and other prevalent infectious disease care into primary medical care” was to strengthen the knowledge and skills of health workers in Latin America and the Caribbean to integrate HIV and other infectious disease care into primary care, with a special emphasis in PMTCT. A competency-based curriculum was developed aimed at not only improving knowledge, but also modifying attitudes and developing abilities that could be measured as outcomes of the course The competency-based curriculum for this training program focused on principles of HIV care and health systems using a novel blended educational strategy consisting of 150 hours of activities grouped in 3 components: an 8-week online component (Component 1), a weeklong on-site problem-solving workshop (Component 2), and individualized project-based interventions (Component 3) . The onlTwenty-six potential tutors working at academic institutions were nominated by national health authorities in each country to attend a five day workshop in Panama where they were trained in competency-based education. The workshop focused on educational methodologies, an overview of program topics, and operation of the program, while allowing evaluation and selection of the final 24 tutors for the program. The selected tutors were given a manual outlining their roles in the training program (Supporting Information S2), and were further trained during the on-site component to support the final project component. In total, the tutors received 65 hours of training. The selected tutors were a mix of doctors, nurses, and public health experts with extensive experience in education as professors or tutors in universities or administrators of academic programs, and several had experience in virtual education.Topic 1: Integration of HIV in the context of the renewal of primary health care focusing on general concepts of health politics, rights, and systems in the region and 22 lectures on Topic 2: Clinical management of HIV and other prevalent infectious diseases in primary care . The online component included weekly evaluations, assignments, and forums where participants could ask questions and interact with other participants. Lectures provided by multiple international experts in HIV and health care systems were prerecorded using Mediasite multimedia capturing software by Sonic Foundry and available on-demand for participants on the program website. In addition to the recordings, transcriptions of the lectures and handouts of the presentation slides were also available from the program site. To ensure access to the program materials regardless of internet connectivity, CD-ROMS with lecture material were delivered to all participants. In order to obtain baseline information on the participants who enrolled in the online component, a brief registration survey was conducted using SurveyMonkey The training program began with an 8 week online component consisting of 21 recorded lectures on The second component was an on-site problem-solving workshop in Panama. The 200 top-scoring participants from the online component were invited to participate in the on-site component. Participants spent five 8-hour days in workshops tailored to reinforce and apply concepts they had learned during the online component. These workshops included clinical cases, role playing and group work to outline the relationship between HCW and patient, the effective transfer of information, and changes in provider attitude that can reflect in patient care and potentially lead to changes in patient conduct  to identify barriers to care, identify and analyze the organization of the provision of care in her/his health center, and to propose changes to improve effective care. Clinical knowledge in the online component was tested using multiple-choice pre and post-tests for each module in Topic 2 . Two surveys were administered to obtain participant feedback: the first evaluated the online component exclusively, and the second evaluated the entire program at the completion of all three components. The feedback surveys used Likert scale from 1 to 5 (1 = strongly agree to 5 = strongly disagree), multiple choice, and open-ended questions to evaluate overall program satisfaction, acceptability of the online component, and perceived benefit from the training program. These surveys were completed using Survey Monkey Quantitative and qualitative evaluation instruments were used to evaluate student progress during the online component. Participants wrote reflexive essays at the beginning and end of the online component which qualitatively measured understanding of social determinants, health care system organization, and integration of HIV services. Additionally, participants had to write weekly small essays during Topic 1  region, and even lower rates in the selected countries  For the baseline evaluation, CIESAR completed a total of 1,735 interviews which were carried out across the four countries .The results of the three questionnaires in the baseline evaluation pointed out discrepancies in the perception of integration of HIV services. Results indicated that 33/44 (75%) of directors from all four countries reported that their health facility offered HIV tests for the general at risk population, whereas only 427/750 (57%) of health providers reported offering the test and 541/941 (57%) of outpatients reported having been tested. When looking at individual countries, Nicaragua showed the largest discrepancy with 105/113 (93%) of providers reporting that they offer HIV tests, but only 104/190 (55%) of outpatients reporting they have been tested. While 95% and 89% of directors and health service providers, respectively, affirmed that HIV testing was routinely offered during prenatal care in their health facilities, only 423/650 (65%) of the women reporting they were or had been pregnant stated they had been tested for HIV. This difference was statistically significant in each of the countries except for Panama.Of the 269 participants who were initially enrolled by the Ministries of Health, 258 became active participants by completing one or more of the initial activities . As the online component progressed, thirty-three participants were dropped due to inactivity, resulting in 225 (87.2%) participants that completed the online component. Only 2 of the 225 participants who completed the online component did not receive the required score (70%) to pass this component. Due to logistical restraints, only the top-scoring 200 participants were invited to the onsite workshop and 30 of those could not attend due to lack of government support from one of the countries. The training program concluded with the presentation of 152 project proposals in the 4 countries, some (10) from participants who were not able to attend the onsite workshop. In total, 142 participants completed all three components.At the initiation of the online component, 240/258 (93%) of the initially active participants completed an initial registration survey . The majAll available reflexive essay pairs  from the online component were evaluated by tutors using a predetermined checklist of criteria qualitatively measuring understanding of social determinants, health care system organization, and integration of HIV services. The mean pre-essay score was 70.3%  and the mean post-essay score was 87.5% . The mean difference between scores for paired pre- and post-essays was 17.2% .Clinical Management of HIV) evaluating clinical knowledge were analyzed for the 225 participants who completed the online component. The mean pre-test score was 70.9% , and the mean post-test score was 90.3% . The mean difference between scores for the paired pre- and post-test knowledge assessments was 19.4% .Scores on the pre- and post-tests from Topic 2  completed a feedback survey evaluating content, structure, tools, tutors, and evaluations of the online component. The participants highly rated structure of the online component including clarity of objectives and appropriate number of lectures (1.36), educational tools and materials including recordings of lectures, handouts, transcripts, and CD-ROMS (1.3), and utility of content for integrating HIV into primary care (1.1).Ratings for tutors were consistently agreeable with an average score of 1.28. Participants highly rated the tutors stating that they facilitated the general process (1.21) and the technical aspects (1.25) of the online component, and the overall process of learning (1.25). They also reported that the number of participants per tutor was adequate (1.59) and that the tutors responded to questions in a timely manner and with correct information (1.22). Overall, participants believed that their tutor was fundamental in completing the online component (1.22).While the majority of participants agreed that the online component prepared them for the onsite component, only one fifth (19%) believed that the online component alone would have been sufficient to acquire the knowledge to integrate HIV services. Most participants (85%) agreed that the on-site component and project development improved their ability to apply the theoretical knowledge acquired in the online component to their practice. Participants highly rated the online component (1.16), on-site component (1.15), and the development of project proposals (1.43).Overwhelmingly, the two most important factors that caused difficulties in completing the program were time commitment in fulfilling their normal responsibilities in the clinics while also completing the online component and availability/reliability of the internet connection. However, participants reported substantial improvement in computer literacy; self-reported ability to perform tasks in a computer with ease (excellent skill) doubled during the course of the training program . In openIn total, 152 participant-developed project proposals from the four countries were presented: 131 in three countries in national workshops and 21 to course coordinators and academic professionals through web-based conferencing. Projects included baseline knowledge surveys and needs assessments , capacity building of health workers, community education programs, reviews of clinic procedures and regulatory compliance, and evaluation of existing programs. Topics included preventing HIV mother-to-child transmission, sexual and reproductive health, family planning, stigma, testing and counseling, and adherence. Although there was a difference in complexity of the proposed projects from different countries, they were all small and focused in scope with an average budget of $8500, providing realistic approaches to health officials for achieving integration. Following the project presentations in the national workshops, the USAID put forth a proposal to fund one of the projects from Panama and the PAHO office in the Dominican Republic has set aside $12,000 to fund several projects in their country. Additionally, reports from a few participants in Guatemala stated that several projects had been implemented or were in the process of being implemented at the time of this publication.Internet-based education has become increasingly popular for medical education Achieving high retention rates is a goal of all educational programs, but in internet-based courses retention rates are typically low The lack of human connection in internet-based courses is a major factor leading to low retention rates Another potential factor in the high retention rate was the feasibility, acceptability, and applicability of the program which was highly valued by our participants. For most participants working in underserved communities with limited access to educational resources, taking time off work to attend training workshops outside of their communities was unrealistic, much like health care providers in general The program aimed to address the deficiencies in knowledge, lack of integration, and pervasive stigma identified in health providers at the primary level of care. We found that this intervention was highly effective in building both the knowledge and skills needed for the health care workers to integrate HIV into primary care. The majority of our participants felt that the online component alone would have been insufficient to acquire the knowledge, attitudes, and skills necessary to integrate HIV services, suggesting that the blended approach of online, onsite, and project components played a major role in making this program successful. As participants presented their projects, it was apparent that they not only acquired knowledge and skills that will help them in their present work, but they have learned how to search for answers to questions and to develop and research new ideas.Throughout the process of developing and implementing this training program, our multi-partner team learned several important lessons. Although technology was a useful tool to reach the most underserved areas, it was also a bigger barrier than expected For many participants, completion of this program was a personal achievement that empowered them to integrate HIV care into their practice and identify barriers to optimal care so that they may overcome them. As participants advanced through the program, they formed new learning communities among their peers locally and internationally that have the potential to sustain the momentum of integrating HIV services into primary care in the long term. Furthermore, partnering with local government, academic, and health ministry representatives throughout the program was also very important in providing participants a channel to make a difference in their communities. Particularly, the final presentation of their project proposals in front of these higher level officials, gave them the opportunity to present their work to people who have the resources to help them implement their projects. This requirement served as an additional learning experience and as an incentive to complete the program as they presented to potential funders.Development of Human Resource Competencies which used this training program as a model, the development of a self-paced version of the online component (available at www.ccghe.net), and the development of self-contained Learning Modules  aim to ensure long-term sustainability of this training program.Despite the success of the program and the positive feedback from the participants, there were limitations, and several elements could be improved. An introductory interactive module to familiarize participants with basic computer functions and how to navigate the course more effectively was available to participants but not required, and because of delays at the beginning of the course, few participants completed this introductory module. Future deployments of this training program should require this introductory module to assist in the smooth operation of the online component. Limitations to internet connectivity in these underserved areas also added undue stress for some participants. Potentially, a minimal amount of funding could be obtained to provide a central location where participants could access the internet; provide a small stipend for internet cafes or to purchase personal mobile hotspots, which several of our highly motivated participants did with their own funds; or the use of smart phones given that cell phone use and connectivity may be more common than internet. Additionally, future deployments should establish the infrastructure needed to finance the small-scale intervention projects which have the potential to contribute to national health programs by implementing local effective and feasible strategies to integrate HIV care into primary care This innovative curriculum utilized technology to target HCWs with limited access to educational resources. The novel 3-pronged blended educational strategy successfully impacted capacity to convert theoretical knowledge to action with its competency-based curriculum that achieved our goal of providing participants with the knowledge, skills, and attitudes necessary to improve the delivery of health services by integrating HIV care with primary care. Our program achieved a high retention rate with the support from tutors who provided the needed human connection that is usually lacking in distance education. Initially, technology proved to be a barrier but participants benefited from technical skills acquired through the process and they were able to continue working in their underserved communities while participating in the online component. They could then learn how to apply their newly acquired knowledge in problem-solving workshops, and propose practical interventions to improve integration of HIV care into primary care. More investment in training programs for underserved areas and future deployments of similar training programs in different countries could make a significant impact in improving HIV care and access to health care services by building the capacity of local HCWs.Supporting Information S1Alternative Language Abstract. Spanish(DOCX)Click here for additional data file.Supporting Information S2Manual for Tutors. This manual was created for the tutors to assist in their training and to describe their roles and responsibilities throughout the different phases of the program. This manual also serves as a detailed description of the program that others can use in order to replicate the program.(DOC)Click here for additional data file.Supporting Information S3Components and breakdown of the training program. Topics covered, length, and modality used for each component.(PDF)Click here for additional data file.Supporting Information S4Online Component Participant Feedback Survey. This survey was constructed and delivered using SurveyMonkey (PDF)Click here for additional data file.Supporting Information S5Overall Program Participant Feedback Survey. This survey was constructed and delivered using SurveyMonkey (PDF)Click here for additional data file.Supporting Information S6Learning Module 1 - HIV and the Social Dimension of Health. Each Learning Module includes a sample of 2 video conferences, a self-assessment (quiz), and complementary materials that were originally presented in the online component of the program. Learning Module 1 includes video conferences on “Epidemiology of HIV in Latin America” and “The Social Dimension of Health: The Social Determinants”.(RAR)Click here for additional data file.Supporting Information S7Learning Module 2 - HIV, health systems and programs. Each Learning Module includes a sample of 2 video conferences, a self-assessment (quiz), and complementary materials that were originally presented in the online component of the program. Learning Module 2 includes video conferences on “A Comprehensive and Integrated Approach” and “Basic Health Programs: Healthy Life Cycle”.(RAR)Click here for additional data file.Supporting Information S8Learning Module 3 - Managing Change for the Integration of HIV into Primary Care. Each Learning Module includes a sample of 2 video conferences, a self-assessment (quiz), and complementary materials that were originally presented in the online component of the program. Learning Module 3 includes video conferences on “Change Management for the Renewal of Primary Health Care” and “Community Participation: Intersectoriality”.(RAR)Click here for additional data file.Supporting Information S9Learning Module 4 - Integration of HIV into Primary Care. Each Learning Module includes a sample of 2 video conferences, a self-assessment (quiz), and complementary materials that were originally presented in the online component of the program. Learning Module 4 includes video conferences on “Models of Integration of HIV Services” and “HIV Prevention in Adolescents and Youth”.(RAR)Click here for additional data file.Supporting Information S10Learning Module 5 - Initial Management of HIV. Each Learning Module includes a sample of 2 video conferences, a self-assessment (quiz), and complementary materials that were originally presented in the online component of the program. Learning Module 5 includes video conferences on “Mechanisms of Transmission and Natural History of HIV Infection” and “HIV Counseling and Testing”.(RAR)Click here for additional data file.Supporting Information S11Learning Module 6 - HIV in pregnant women. Each Learning Module includes a sample of 2 video conferences, a self-assessment (quiz), and complementary materials that were originally presented in the online component of the program. Learning Module 6 includes video conferences on “HIV Testing in Prenatal Care” and “Management of Delivery and the Exposed Infant”.(RAR)Click here for additional data file.Supporting Information S12Learning Module 7 - Chronic Management of HIV. Each Learning Module includes a sample of 2 video conferences, a self-assessment (quiz), and complementary materials that were originally presented in the online component of the program. Learning Module 7 includes video conferences on “Tuberculosis and HIV Coinfection” and “Toxicity, Adherence, and Laboratory Monitoring”.(RAR)Click here for additional data file.Supporting Information S13Learning Object 8: Prevalent Infections and HIV. Each Learning Module includes a sample of 2 video conferences, a self-assessment (quiz), and complementary materials that were originally presented in the online component of the program. Learning Module 8 includes video conferences on “Hepatitis and HIV Coinfection” and “Parasites in Patients with HIV/AIDS”.(RAR)Click here for additional data file.


Increasing evidence indicates that tumor endothelial cells (TEC) differ from normal endothelial cells (NEC). Our previous reports also showed that TEC were different from NEC. For example, TEC have chromosomal abnormality and proangiogenic properties such as high motility and proliferative activity. However, the mechanism by which TEC acquire a specific character remains unclear. To investigate this mechanism, we focused on tumor-derived microvesicles (TMV). Recent studies have shown that TMV contain numerous types of bioactive molecules and affect normal stromal cells in the tumor microenvironment. However, most of the functional mechanisms of TMV remain unclear.Here we showed that TMV isolated from tumor cells were taken up by NEC through endocytosis. In addition, we found that TMV promoted random motility and tube formation through the activation of the phosphoinositide 3-kinase/Akt pathway in NEC. Moreover, the effects induced by TMV were inhibited by the endocytosis inhibitor dynasore. Our results indicate that TMV could confer proangiogenic properties to NEC partly via endocytosis.We for the first time showed that endocytosis of TMV contributes to tumor angiogenesis. These findings offer new insights into cancer therapies and the crosstalk between tumor and endothelial cells mediated by TMV in the tumor microenvironment. Tumor blood vessels have been recognized as an important target for cancer therapy since Folkman introduced the concept that tumor growth is dependent on angiogenesis. Tumor angiogenesis is necessary for solid tumor progression and metastasis Tumor blood vessels are different from their normal counterparts and have altered morphology, altered blood flow, enhanced leakiness, structural abnormalities in the basement membrane, and abnormal pericytes We reported that TEC are cytogenetically abnormal Recent studies report that crosstalk between tumor cells and stromal cells are important for tumor progression or acceleration of tumor malignancy by secreting microvesicles (MVs) In the present study, to investigate the contribution for the specific phenotype of TEC, we analyzed the effect of TMV on NEC and the mechanism of their uptake.2 and 95% air at 37°C. Human microvascular endothelial cells (HMVEC) were purchased  and cultured in microvascular endothelial growth medium . Human renal epithelial cells (hREC) were purchased  and cultured in renal epithelial cell growth medium .A375-SM, a super-metastatic human malignant melanoma cell line, was kindly gifted by Dr. Isaiah J. Fidler . The cells were cultured in Minimum Essential Medium  and supplemented with 10% fetal bovine serum. NEC were isolated according to the following method and cultured in a microvascular endothelial growth medium  in a humidified atmosphere of 5% COBandeiraea simplicifolia isolectin B4 (BS1-B4 lectin) and the expression of CD31, CD105, and CD144 indicated a high purity of isolated EC in flow cytometric analysis. Furthermore, RT-PCR revealed that NEC expressed CD31, CD105, CD144, VEGF receptor-1, and VEGF receptor-2 (VEGFR1 and VEGFR2), indicating that NEC possessed EC characteristics during culture. Isolated EC were negative for the monocyte marker CD11b and hematopoietic marker CD45 by RT-PCR. Template-free samples were used as the negative controls.NEC were isolated from mouse dermal tissue using a magnetic activated cell sorting system  with anti-CD31 antibody as previously described Total RNA was extracted using the RNeasy Micro Kit , and complementary DNA (cDNA) was synthesized using ReverTra-Plus  as described previously Rat anti-mouse CD31 antibody and fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD31 antibody was purchased from eBioscience . FITC-conjugated BS1-B4 lectin was purchased from Vector Laboratories Burlingame, CA, USA). PE-conjugated anti-mouse CD31 antibody, anti-mouse CD105 antibody, rat anti-mouse CD144 antibody, and normal rat IgG were purchased from BD Pharmingen . Anti-β-actin, total Akt, phospho (Ser473)-Akt, and anti-rabbit HRP-congujated secondary antibody were obtained from Cell Signaling Technology . LY294002 was obtained from Calbiochem  and dynasore from Santa Cruz Biotechnology .g for 1 h. Then, A375-SM cultured media was replaced minimum essential medium supplemented with ultracentrifuged 10% FBS. After incubation for 2 days, the conditioned medium was collected and centrifuged at 2,000 g for 15 min at 4°C to thoroughly remove cellular debris. The supernatant was centrifuged again at 110,000 g for 70 min at 4°C. The pellets were washed with phosphate-buffered saline, and after ultracentrifugation, were resuspended in phosphate-buffered saline. Protein levels were determined using the BCA assay from Pierce Chemical Co .TMV were obtained from A375-SM as previously described For surface phosphatidylserine detection, TMV were resuspended in PBS and incubated with annexin V-FITC  and anti-HLA-FITC for 30 min at room temperature. After washing to remove unbound reagents, size calibration of TMV was performed using microbeads  Next, TMV were analyzed on a BD FACS Aria II Flow Cytometer . Data were analyzed by Flow Jo .TMV were incubated on a coverslip at 37°C for 4 h. It was fixed with 2% glutaraldehyde in PBS. TMV were post fixed for 1 h with 1% osmium tetraoxide in PBS and were dehydrated in graded ethanol solutions of 50%, 70%, 95%, and 100% before being placed in an HCP-1 critical point drying machine . TMV were sputter-coated with platinum and palladium before being observed under an S4000 scanning electron microscope .4 solution, dehydrated with a graded series of ethanol and propylene oxide, and embedded in epoxy resin. Ultra-thin sections  were cut, stained with lead citrate, and examined using HITACHI H-7000 electron microscope .TEM assay was performed as described previously TMV were labeled using the PKH26 red fluorescent labeling Kit  according to the manufacturer's instructions. Labeled TMV (50 µg/ml) were incubated at 37°C for 1 and 24 h. For FACS analysis, the cells were washed twice and trypsinized. After washing, the cells were resuspended in PBS containing 2 mM EDTA and 2% FBS. Next, PKH26 fluorescence in the cells was analyzed by FACS.To determine PKH26-labeled TMV localization in NEC, the cells were fixed with 4% paraformaldehyde, washed, and stained with MFP488-phalloidin  and 4′,6-diamidino-2-phenylindole . PKH26-labeled TMV localization by ECs was determined using an FV10i confocal laser scanning microscope .Cells were plated on glass bottom dishes coated with poly-L-lysine . After overnight incubation, the medium was changed with or without dynasore (50 µM) and LY294002 (80 µM) for 30 min. Cell movements were monitored with or without TMV (50 µg/ml) under an FV10i microscope  at 10 min intervals. Velocity was determined by tracking the positions of cell nuclei using ImageJ . Migrated distance was integrated and divided by monitored time (12 h).5 cells per well with or without TMV (50 µg/ml), followed by incubation at 37°C for 12 h. Tube formation was observed using an inverted microscope and the experimental results were recorded at different times. The mean tube length was calculated using ImageJ.Diluted Matrigel  was transferred to a glass bottom dish and incubated at 37°C for 30 min to allow the matrix solution to solidify. Cells were harvested and resuspended in appropriate media and preincubated with or without dynasore (50 µM) and LY294002 (80 µM) for 30 min. Cells were then seeded at a density of 1×103VO4] for 10 min on ice, and the lysate was clarified by centrifugation. Samples were electrophoresed on 10% polyacrylamide gels and transferred to Immobilon-P membranes . The membranes were blocked for 60 min in TBS containing 5% skimmed dry milk and 0.1% Tween 20, and then incubated for overnight at 4°C with primary antibodies followed by incubation with HRP-conjugated secondly antibodies. Signals were developed using the ECL Western Blotting Detection Reagent  and detected using an LAS-4000 mini image analyzer .Western blot analysis was performed using antibodies specific to phosphorylated-Akt, total-Akt and HRP-conjugated secondary antibody as described previously t-test was used.Mean ±standard error (SE) values were compared using one-way ANOVA with Tukey-Kramer multiple comparison testing. When only two groups were compared, a two-sided Student's NEC were isolated from mouse dermal tissue using a magnetic cell sorting system as described above. Flow cytometric analysis revealed the binding of BS1-B4 lectin and the expression of CD31, CD105, and CD144 in NEC . RT-PCR It has not been established conclusively how TEC acquire their abnormality in a tumor microenvironment. There are several reports that MVs secreted from malignant tumor cells (tumor-derived MVs: TMV) affect surrounding normal cells TMV reportedly have a size of less than a few micrometers. Flow cytometric analysis of human TMV was performed to determine the size of TMV. Most isolated TMV were found to be around the forward and side scatter signal corresponding to 1-µm beads by flow cytometry, using micro beads as internal size standards .Scanning electron microscopy showed that TMV had a spheroid morphology less than 1 µm similar to flow cytometric assay , althougVarious reports have suggested that the surface of TMV express various cell membrane components, such as HLA originated from host cells Isolated TMV were analyzed by flow cytometry using annexin V, which binds to phosphatidylserine, and anti-human HLA. These results suggested that TMV expressed phosphatidylserine and HLA . FurtherA previous report indicates the possibility that protein and/or nucleic acids  are packaged into MVs and can be taken up by cells, acting as biomolecules After treatment with PKH26-labeled TMV for 1 and 24 h, NEC were analyzed for their fluorescent intensity using flow cytometry . The fluThen, we hypothesized that TMV uptake is dependent on endocytosis because many types of extracellular fluids and nutrients are taken up by this process. To confirm this hypothesis, we performed the TMV uptake assay with low temperature treatment. Since endocytosis is energy-dependent, low temperature treatment is a common method for inhibition of endocytosis Endocytosis requires a small GTPase dynamin, which pinches off endocytic vesicles from the cytomembrane These results suggested that TMV is taken up by NEC via endocytosis.We have shown that TEC have higher motility, which is important for angiogenesis, than NEC P<0.01). When NEC were treated with the endocytosis inhibitor dynasore, TMV could not enhance NEC motility  for 2 h, followed by stimulation with TMV. Akt phosphorylation was examined by western blotting. It was determined that LY294002 inhibited TMV-induced Akt phosphorylation , which sNext, to investigate the activation of the PI3K/Akt pathway by TMV, we used dynasore. After NEC were pretreated with dynasore for 30 min, TMV no longer induced Akt phosphorylation . These rIn order to analyze involvement of PI3K/Akt pathway in cell motility and tube formation enhanced by TMV, the PI3K inhibitor, LY294002 was used. TMV did not stimulate cell motility when NEC was treated with LY294002 . FurtherThe aim of this study was to investigate how TMV affect phenotypic changes in EC in a tumor microenvironment. This study yielded several results:TMV are taken up by NEC via endocytosis.TMV promote cell motility, tube formation, and Akt phosphoarylation in NEC.TMV endocytosis is required for induction of the proangiogenic phenotype in EC.In this study, we tried to isolate MV from normal cells as normal control. However, normal endothelial cells (HMVEC) and normal epithelial cells (hREC) secreted few amount of microvesicles compared to melanoma cells , consistWe previously reported that TEC had higher motility than NEC Although TMV are classified by size and several markers, their whole character have not been cleared, yet. In this study, isolated TMV had a wide range of size distribution observed by SEM and flow cytometry. TMV expressed HLA and β-actin, which are the markers for shedding microvesicle derived from tumor plasma membrane There have been a few studies about the uptake on TMV by normal cells Several reports have demonstrated that TMV can activate Akt phosphorylation There have also been several reports that TMV transfer bioactive molecules to recipient cells To investigate if TMV-induced motility of NEC is regulated in an autocrine manner, NEC was treated by TMV in bottom chamber of transwell. Migration assay was performed to analyze whether TMV-NEC can attract NEC from upper chamber in an autocrine manner or not . As a reRegarding with bioactive molecules in TMV, the possible bioactive molecules in TMV are phospholipids. Phosphatidylserine and sphingomyelin, which are the components of the membrane of TMV, activate Akt phosphorylation In this study, we for the first time demonstrated that TMV are taken up by NEC via endocytosis. TMV uptake confers proangiogenic properties on NEC. Inhibiting TMV endocytosis may lead to the development of a useful prevention strategy against tumor angiogenesis.Figure S1Tumor-conditioned medium activates NEC. A375-SM cells were cultured in MEM (0.5% FBS supplemented). After two days, conditioned medium was centrifuged at 2,000 g to remove cells and debris. Random motility, cell proliferation and apoptosis were analyzed in NEC with NEC conditioned medium (NEC CM) or tumor conditioned medium (tumor CM). (A) Random motility was measured by time-lapse observation. Mean velocities are presented ± SE . Tumor CM enhanced motility in NEC. (B) NEC proliferation was measured using MTS assay kit . Mean MTS activity are plotted (n = 4). Cell proliferation was stimulated by Tumor CM. (C) The cells were stained with the Annexin-V-FLUOS Staining kit  and were analyzed by flow cytometry . The percentages of dead cells decreased when NEC were treated by tumor CM.(TIF)Click here for additional data file.Figure S2Analysis of cytotoxicity of Dynasore. Cytotoxicity of dynasore was measured by apoptosis assay kit. NEC were treated with a various concentration of dynasore for 16 h. The cells were analyzed by flow cytometry. Dynasore was not cytotoxic to NEC at the concentration of 50 µM, that was used in our study.(TIF)Click here for additional data file.Figure S3Normal cells secrete small amounts of MV. A375-SM, HMVEC and hREC were seeded with 50% confluency. After two days, cells were trypsinized and total cell number were counted respectively and MV-derived from each cell lines were isolated as mentioned below. (A) Protein amount of each MV were measured by BCA protein assay kit. Then, secreted amount of MV per single cell were estimated. The amount of MV secreted by tumor and normal cell was analyzed. Under same conditions, the amounts of normal cell derived MV were little , compared with that of tumor cells . Secreted amount of MV per single cell (pg/cell) were presented. (B) MV numbers isolated from each conditioned medium was counted using flow cytometry. It was suggested that normal cell secreted little amount of MV. Counted numbers of MV in each conditioned medium are presented.(TIF)Click here for additional data file.Figure S4TMV-induced migration was not regulated in an autocrine manner. To discuss how TMV enhance motility in NEC, transwell assay  was performed according to manufacturer's protocol. (A) Briefly, NEC were seeded on bottom chamber (1×104 cells/cm2). After 4 h, adhered NEC were treated with or without TMV (50 µg/ml) in bottom chamber. Then, NEC were seeded on upper chamber (1×105 cells/ml). Twelve hours later, NEC were fixed with 10% formalin and stained with Mayer's Hematoxylin solution. (B) Migrated NEC numbers towards supernatant from NEC treated with or without TMV were counted . NEC did not migrate towards supernatant from TMV-treated NEC in bottom chamber.(TIF)Click here for additional data file.


International interest in pay-for-performance (P4P) initiatives to improve quality of health care is growing. Current programs vary in the methods of performance measurement, appraisal and reimbursement. One may assume that involvement of health care professionals in the goal setting and methods of quality measurement and subsequent payment schemes may enhance their commitment to and motivation for P4P programs and therefore the impact of these programs. We developed a P4P program in which the target users were involved in decisions about the P4P methods.For the development of the P4P program a framework was used which distinguished three main components: performance measurement, appraisal and reimbursement. Based on this framework design choices were discussed in two panels of target users using an adapted Delphi procedure. The target users were 65 general practices and two health insurance companies in the South of the Netherlands.th percentile of group performance. The incentive for clinical care was set twice as high as the one for practice management and patient experience. Quality scores were to be calculated separately for all three domains, and for both the quality level and the improvement of performance. The incentive for quality level was set thrice as high as the one for the improvement of performance. For reimbursement, quality scores were divided into seven levels. A practice with a quality score in the lowest group was not supposed to receive a bonus. The additional payment grew proportionally for each extra group. The bonus aimed at was on average 5% to 10% of the practice income.Performance measurement was linked to the Dutch accreditation program based on three domains . The general practice was chosen as unit of assessment. Relative standards were set at the 25Designing a P4P program for primary care with involvement of the target users gave us an insight into their motives, which can help others who need to discuss similar programs. The resulting program is in line with target users' views and assessments of relevance and applicability. This may enhance their commitment to the program as was indicated by the growing number of voluntary participants after a successfully performed field test during the procedure. The elements of our framework can be very helpful for others who are developing or evaluating a P4P program. International interest in pay-for-performance (P4P) initiatives to improve quality of health care is growing. Despite the proliferation of P4P programs, the evidence to support their use is still inconclusive ,2. One oAnother remarkable feature of current P4P programs is that they are mostly designed and implemented top-down by policy makers and managers . P4P proEvaluation of the involvement of target users in de decisions about the P4P program may contribute to the growing field of P4P research. One may assume that involvement of health care professionals in the goal setting and methods of quality measurement and subsequent payment schemes may enhance their commitment to and motivation for P4P programs and therefore the impact of these programs. Nevertheless, you have to reckon with conflicts of interest when involving target users. Therefore it is important to develop the P4P program in a systematic way, such as the Delphi procedure . The perWe searched the literature for relevant elements for our P4P program, to be discussed by the target users. Table performance indicators covered three domains, clinical care, practice management and patient experience, and were derived from the Dutch National Accreditation Program for general practices [The ractices . The tarractices , they coractices , which eappraisal and reimbursement elements and options to be discussed were derived from the literature [th percentile or 90th percentile) or a combination. Improvement can best be stimulated by feedback in a reachable range [The terature ,8,10,11.terature . The tarterature . For calterature . In ordele range , thus prreimbursement the options for the method of payment were either money, human resources, a sabbatical leave or a combination of these. For the size of the bonus we asked the target users whether an average bonus of 5000 to 10000 Euros (depending on practice size), which is on average 5-10% of the practice income, would be appropriate. In other P4P programs the size of the bonus varied between US$2 per patient and US$ 10000 per practice [Concerning the practice . The sizpractice ,24,25. TAn action research approach  was applIn each round a written questionnaire with the design options for the P4P program was sent to the target users two weeks before the planned meeting. In the questionnaire they were provided with background evidence on the options as described in the section 'The design options in the P4P framework' and they were asked to make a choice. Each meeting started with explaining the aim of the discussion and feedback on the results of the questionnaires. All design options were discussed, but for the performance indicators the project team decided not to discuss indicators with high consensus, defined as less than 30% or more than 70% in favour. At the end of each meeting the panel members completed the same questionnaire again. The decision rule for inclusion of clinical indicators was set at more than 70% in favour, and for the other design options a majority rule was applied.All panel meetings were held in the region in question to enhance participation. Payers and GPs attended the same discussion meetings which lasted 2 hours. The general practices in the first panel received 1500 Euros for participating in the panel as well as in the field test. Each GP in the second panel received 100 Euros for attending the meetings.The number of general practitioners and health insurance representatives that filled in the questionnaires and attended the meetings for the specific panels are presented in Figure The successive panel procedures and the field test resulted in a P4P program which is presented in Table clinical conditions to be assessed were mainly focused on chronic care, though GP care comprises much more. Especially communication skills were missed. Although patient experiences were to be assessed, some GPs stated that communication was not reflected enough in the indicators. The GPs also discussed the fact that choosing indicators would result in a certain focus that could distract them from the more general goal of quality improvement. Practices may concentrate their performance on the indicators from the P4P program. It was proposed that in the long-term a large set of indicators will be needed. Then the P4P program could have different sets for different years. Some GPs even suggested that the practice should not be aware of the existing set. For clinical care, GPs were convinced that the outcomes were a mixed result of patient and doctor's performance. It was therefore decided that the payment should be based on the process measures only, but the GPs would like to receive feedback on the outcome indicators as well. So, data for both process and outcome indicators were collected. Although the health insurance representatives stated they would leave the decisions on clinical care options to the GPs, they joined the discussion in the meetings on the prescription indicators. The prescription indicators were highly valued by the health insurance representatives. The GPs questioned these indicators which resulted in not including the acid suppressive drugs indicators in the program, and including indicators on prescribing antibiotics. Some items of practice management were excluded due to their estimated low correlation with the quality of practice management such as financial accounting. The EUROPEP instrument, consisting of 23 items measuring patient experience, was supplemented with four items regarding the possibility to ask for a longer consultation, accessibility by phone, getting an appointment with your own doctor and an accessible procedure for complaints. All selected quality indicators can be found in additional file The target users thought clinical care, practice management and patient experience to be appropriate domains for the P4P program, as well as the subjects within these domains  because clinical care is the major domain of quality of care. The data from the 22 practices in the field test showed that the quality scores should be calculated separately for each domain because otherwise the performance on clinical care would dominate the overall quality score. Having data available for two or more years made it possible to calculate improvement of performance as well. Panel 2 decided to reward quality level as well as improvement of performance in a ratio of 3:1 for the bonus payment in the next year. In that case practices with high scores would receive a bonus for delivering quality and practices with low scores would be stimulated to improve. In the following years two separate scores will be calculated for each practice; one on quality level and one on improvement of performance for the three domains clinical care, practice management and patient experience. The panel thought of the P4P program as a three years cycle in which practice management was only measured at the beginning of each cycle. The users preferred to have a detailed division in levels of quality scores to make small differences in quality visible and to make it easier to achieve next levels. The range of quality scores of all participating practices were therefore divided into seven equal levels (relative 'thresholds'). Practices that do not improve will be rated in level 0 (no bonus) and practices showing improvement will be rated in one of six levels with a differentiation in bonus accordingly. For feedback the target users preferred a benchmark with the 25th (minimum standard), 50th and the 75th percentile because that would give them a good overview and stimulate practices at the bottom as well as at the top. Risk adjustment for the process indicators was discussed. The target users preferred stratification, which is a comparison with a benchmark consisting of comparable practices instead of correction of their own data. However, stratification would require a large sample of practices, so we decided not to include this aspect in the experiment. Following the experiences with the field test, a two-step procedure was chosen by the GPs. Practices will receive feedback (indicator scores and benchmark) and the bonus after they have had the opportunity to respond to their feedback. The feedback was accompanied with clear information on the calculation procedure.The general practice was chosen as The discussion on the type of financial reward resulted in the conclusion: "Money is the best method, because money can buy you anything". The target users agreed on a bonus that was 5 to 10% of practice income with a minimum of 0 Euros and a maximum of 15000 Euros. None of the users indicated this amount was too high and half of them thought this was too low. However, agreement was reached with the argument that the proposed size of the bonus was in line with bonuses paid in trade and industry. A decline in the bonus for the years to follow had to do with the budget of the health insurance companies who rationalized it by arguing that the data collection was most labour intensive at the start of the project. According to the discussion on the appraisal we need two formulas to calculate the bonus, one on the quality level and one on quality improvement level. The quality improvement level can only be calculated after the first year. The formulas are:The exact bonus at baseline and in following years is presented in Table P4P proves to be a complex innovation and knowledge needs to be acquired over time . AssuminIn line with other P4P programs our target users selected performance indicators for clinical care, practice management and patient experience. It was not surprising that the chronic diseases were chosen for the program concerning the attention for these diseases and concerning the health care costs due to these diseases. However, our program seems to be more balanced compared to other programs with regard to the position of patient experiences in the program ,5-7. GPsMostly, P4P programs are designed and implemented top-down by policy makers and service managers . In our The target users had a realistic estimate of the required size of the bonus in order to achieve a quality stimulus. According to the target users a bonus of on average 5-10% of practice income was considered to be appropriate. The target users were aware of the risk of gaming when the incentive is too high ,24,25. OThe target users opted for relative P4P standards. Until now, programs mainly base their incentives on absolute performance standards ,5,6,8,28The strength of our study lies in its developmental process, assuming a greater probability of acceptance of the program and subsequent quality improvement. Involving the target users resulted in good discussions and consensus about the design options. The field test was performed successfully as part of the procedure. Many practices participated in the field test as well as in the panels, which resulted in a reasonably balanced P4P program.This study has some limitations. First, due to time constraints patients were not included in the design of the P4P program. However, they had been previously involved in discussions about the objectives of the Dutch accreditation program, which was part of the initial framework of our program. Second, there is a drop in the number of participants in panel 2. A possible explanation is that the subjects we discussed in panel 2 were more restricted and detailed and therefore less attractive than those in panel 1. Third, practices could voluntarily register for this experiment, which may have resulted in overrepresentation of early adopters of a P4P program. It is important that the early majority as well as later on the late majority support the P4P program. To involve them in design choices that are acceptable and applicable is still a challenge.A strength of the design choices is the involvement of the target users. The behavioral change of the P4P program is therefore grounded in extrinsic (reimbursement) as well as intrinsic motivation. To stimulate the motivation further the feedback will be discussed within the practice supported by a facilitator.The performance measures do not cover all aspects of general practice. Just stimulating the incentivized parts of the performance can result in a possible decline in quality of care of the non-incentivized aspect . By discGPs have decided that the outcome indicators on clinical care will not be incentivized, and the health insurance companies agreed. We have to study the effect of this decision on the outcome indicators. By incentivizing the process indicators an indirect effect is expected on the reported outcome measures, but that still has to be proven.As in other P4P programs the focus of the clinical performance measures is on the chronic conditions. Policy makers show a lot of interest in the performance on these conditions, resulting in several improvement projects. This might have an effect on our baselines measures in which case the room for improvement decreases. In our effect study we will take into account the baseline measures to get more insight into this problem.The relative thresholds might evoke calculating behavior, that is if no one improves the bonus will still be dived. The question is which practice will take this risk. This design choice introduces a prisoner's dilemma with unclear results. Although, based on the involvement of the participating GPs in quality of care we would assume that in this group the urge of improvement is larger. The sustainability of the relative thresholds can become tensed in a broader probably less involved group of GPs.The health insurance companies decided together with the GPs on the available budget for the bonus. However, after the first bonus was allocated, the health insurance companies started the discussion on the bonus again. They suggested that the data collection was much easier in the following year, and therefore the bonus could be reduced. This lead to a lot of turbulence among the participating GPs. No consensus was reached, but the practices were still willing to participate. This is a demonstration of the inequity in the relation between payers and GPs that is hard to cover with a consensus procedure.The sustainability of the P4P program is also stressed if the performance measures stay the same each cycle, because the indicators scores increase due to the P4P program to a certain optimum. This phenomenon has been described with the UK-QOF data [By performing a procedure to involve target users in designing a P4P program for general practice, a detailed framework to define design choices was established. This framework as well as the insight into motives for design choices of the target users can be helpful for others who are developing or evaluating a P4P program. The resulting design resembled the P4P programs from other countries, but ours was also in line with target users' views and assessments of relevance and applicability. As already shown by the growing number of voluntary participants during the study, this may enhance general practitioner's commitment to the program.The authors declare that they have no competing interests.KK, the main investigator, carried out this study. She analyzed the questionnaires, organized the panel meetings and drafted this manuscript. JB, the project leader, was involved in all aspects of the study. AJ and RG participated in discussions about the reporting. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2296/13/25/prepubThe indicator set of the P4P program.Click here for file


In 2009 an estimated 5.3 million people in the United States were afflicted with Alzheimer's disease, a degenerative form of dementia. The impact of this disease is not limited to the patient but also has significant impact on the lives and health of their family caregivers. The Resources for Enhancing Alzheimer's Caregiver Health (REACH II) program was developed and tested in clinical studies. The REACH II program is now being delivered by community agencies in several locations. This study examines the impact of the REACH II program on caregiver lives and health in a city in north Texas.Family caregivers of Alzheimer's patients were assessed using an instrument covering the multi-item domains of Caregiver Burden, Depression, Self-Care, and Social Support upon enrollment in the program and at the completion of the 6 month intervention. The domain scores were analyzed using a multivariate paired t-test and Bonferroni confidence interval for the differences in pre- and post-service domain scores.A total of 494 families were enrolled in the program during the period January 1, 2011 through June 30, 2012. Of these families 177 completed the 6 month program and have pre – and post service domain scores. The median age for the caregivers was 62 years. The domain scores for Depression and Caregiver Burden demonstrated statistically significant improvements upon program completion.The REACH II intervention was successfully implemented by a community agency with comparable impacts to those of the clinical trial warranting wider scale implementation. In 2009 an estimated 5.3 million people in the United States were afflicted with Alzheimer's Disease (AD), a degenerative form of dementia which begins with memory loss and leads to disorientation, impaired judgment, behavioral changes, and difficulties in speaking, walking, and swallowing in later stages. As Americans age, the number of persons with this condition are anticipated to rise to 11 to 16 million by 2050 In order to alleviate this burden on Alzheimer 's disease caregivers and maintain their ability to care for the patient, several interventions have been developed and tested Millions of adults in the US are afflicted with Alzheimer's disease (AD) type dementia and majority of them continue to live in the community. According to Schubert et al. Providing care to elder family members can at times come at a great cost to the caregiver. Caregivers of AD patients invest significant time, energy, and money, and in many cases over a long period of time, which involves exhausting tasks leading to high levels of burn-out symptoms The Resources for Enhancing Alzheimer's Caregiver Health (REACH) study was funded by the National Institute on Aging and the National Institute of Nursing Research as a multisite research program. It aimed to test REACH as an effective caregiver intervention, and it was performed in 2 phases. The first phase, REACH I, tested different interventions at 6 U.S. sites , in order to test various psychosocial interventions and their impact on the health and wellbeing of family caregivers of persons with AD dementia REACH II was conducted as a multi-ethnic, multi-site, randomized clinical trial (intervention vs. control group) to reduce burden and depression of caregivers of AD patients The United Way of Tarrant County contracted with the Alzheimer's Association to implement the REACH II program as a component of support services to Alzheimer families. The Alzheimer's Association Caregiver Education and Counseling REACH II staff consisted of two dementia care specialists (counselors), who received training in the REACH II program implementation. The counselors were trained during a two day workshop offered by a co-investigator of the original REACH development and implementation team. The workshop included training on the content of the resource book, role playing, and discussion of proper responses to various situations raised by caregivers.The REACH II counselors assessed the caregivers at home and followed up in-person and by telephone counseling, covering topics including home safety, feelings and stress management, behavioral skills training, and provided additional resources in conjunction with material from caregiver notebooks provided by the REACH II program which were tailored to specific needs of the family. The resource notebook was used when the counselors visited or contacted the caregivers by phone. The notebook was composed of sections covering each of the areas addressing the quality of life indicators. The sections included pictures and information with particular attention to the needs identified by the caregivers as their priorities. The REACH II staff made modifications in the delivery of REACH II services to accommodate program implementation in the community setting. One example was that in order to resolve communication barriers with families by changing terminology in interactions substituted “dementia care specialists” for “counselors”. A professionally printed caregiver notebook in Spanish was developed based on a former program in Pittsburgh, PA, to enable program delivery to Hispanic families.Referral sources to the program included a 24/7 telephone helpline, support groups, case managers, the Aging & Disabilities Resource Center (ARDC), partnering agencies, and home health agencies were utilized to recruit program participants.A questionnaire developed by the REACH II study was administered to the caregivers of Alzheimer's patients who received REACH II services at the beginning of services and was repeated at the time when services were completed. The intake process was developed and implemented which entailed an initial telephone contact, the first home visit and assessment, followed by additional home visits and or phone calls based on the caregiver needs as established by the assessment tool.Families included in this analysis were served by the program during the period January 1, 2011 through June 30, 2012. A total of 494 families were served during this period. For caregivers whose loved ones passed away prior to the completion of the 6-month REACH program the relevant parts of the Quality ofLife questionnaire were completed and renamed bereavement battery. This led to improved data capture of services delivered by mainly including the depression and self-care sections.The variables recorded for each client include demographic characteristics , start and end dates of service, reason for service termination, and pre- and post-service scores for the four domains – Depression, Caregiver Burden, Self-Care, and Social Support. The domains scores were obtained through a sixteen item risk appraisal instrument developed for the REACH II program administered upon intake to and completion of the program We first summarized the demographic characteristics of the study sample. We also classified the clients by their reasons for service termination. As not everyone completed the program, for each demographic category, we found the percentage that completed the program. Finally we compared the pre- and post-service domain scores for clients who had both of these scores available For formal tests on comparison of pre- and post-service scores, we used paired t-test for each domain. As the four domains are related, we also carried out multivariate paired t-test to test if there are differences between pre- and post-service scores over all domains. As this overall test showed significance, we followed it up by 95% Bonferroni confidence intervals (CIs) for differences between pre and post-service scores for each domain. If a CI for difference does not include the null value of 0, then it gives evidence that the difference between pre- and post-service scores for that domain is statistically significant.The study participants did not sign a consent letter per se. The participants were recipients of program services being evaluated by the researchers. As such they signed a form given permission for their data to be shared with the Dallas Fort Worth Hospital Council Foundation. This Foundation matches their data with hospital data and provided a de-identified file to the evaluators. This procedure was approved by the University of North Texas Health Science Center (UNTHSC) Institutional Review Board. The study was also approved by the UNTHSC Institutional Review Board.We had a total of 494 clients in the sample. The demographic characteristics are shown in th to 75th percentiles of the distribution of scores and the connected lines show the full range of scores for the domain. As shown in this figure the Pre-Service scores were similar among the clients who terminated for the various reasons. Only for the Self Care domain were the scores of those who completed the program higher than those who terminated services. Also of note is that the Caregiver Burden score was highest for those clients who were placed in nursing homes.n varies slightly from one domain to other as some clients chose not to respond to some questions in one or more domain. In that case, the total score for that domain for that client cannot be determined. All of the domains showed some improvement between the pre and post scores.th to 75th percentiles within the box, and the full range of scores in the connected lines. They are intended to show the distribution of scores for each domain.The boxplots of Pre and Post Service scores in Almost 10 million family members, friends, and neighbors provided care to Alzheimer's patients in 2005 without payment The Resources for Enhancing Alzheimer's Caregiver Health (REACH) was developed through a study funded by the National Institute of Aging and the National Institute of Nursing Research from which the REACH intervention was selected as an effective intervention to assist caregivers of Alzheimer's disease Our study has demonstrated that similar proportions  of caregivers with their Alzheimer's patients complete the program or terminated the program due to the death of the Alzheimer's patient in similar proportions. White caregivers had a lower proportion (31%) completing or terminating the program due to death of the Alzheimer's patient than did Black (44%) or Hispanic (47%) caregivers. This finding is consistent with the earlier REACH II clinical implementation for Hispanics but in our study the proportion of Black caregivers completing was higher than for Whites whereas the opposite was true in the original study Of the four caregiver domains which were measured in our study, significant improvements were found for Depression and Caregiver Burden. The domain scores for Self Care and Social Support demonstrated improvement but the changes were not statistically significant. These findings are consistent with the original clinical REACH II study Limitations of this study are related to the “real world” implementation which provided the REACH II services by a community provider  with funding from the regional United Way organization. All caregivers who enrolled in the program received the REACH II intervention with no randomization to a control group. We believe that withholding of services could not be ethically justified due to the finding from the original implementation that caregivers significantly benefited from the services when compared to a control group. Delivery of services by a community organization through local funding was not sufficient to support a large array of measurement instruments and no “post” measures were available from caregivers who voluntarily or involuntarily terminated from the program or moved out of the service area.Earlier studies of the REACH II intervention have established that this program can improve the quality of life of caregivers of Alzheimer's patients Through the United Way of Tarrant County support for a small scale evaluation of multiple programs funded as part of their Healthy Aging and Independent Living initiative, we were able to establish that the REACH II intervention could be successfully implemented in a community setting with outcomes comparable to the original REACH II clinically based study. These findings support a wider scale implementation of the REACH II intervention in community settings to help address the growing demands on family and community caregivers of Alzheimer's patients in the coming decades as the U.S. population continues to age and acquire this condition. Financial support for these services will continue to be a challenge as resources available at the community level are constrained. Policymakers should consider the value of these services as part of their efforts to keep elderly dementia patients in their homes and communities.


MtsslWizard is a computer program, which operates as a plugin for the PyMOL molecular graphics system. MtsslWizard estimates distances between spin labels on proteins quickly with user-configurable options through a simple graphical interface. In default mode, the program searches for ensembles of possible MTSSL conformations that do not clash with a static model of the protein. Once conformations are assigned, distance distributions between two or more ensembles are calculated, displayed, and can be exported to other software. The program’s use is evaluated in a number of challenging test cases and its strengths and weaknesses evaluated. The benefits of the program are its accuracy and simplicity.The online version of this article (doi:10.1007/s00723-012-0314-0) contains supplementary material, which is available to authorized users. Distance determination by pulsed electron–electron double resonance (PELDOR) [or double electron–electron resonance (DEER)] spectroscopy has become a popular tool to determine exact distances in bio-macromolecules , 28, 29.An intrinsic limitation of SDSL is due to the widely used methanethiosulfonate spin label (MTSSL ), which Different approaches have been used to deal with this problem. The uncertainty can be allowed for by adding ±14 Å to any distance restraints . This ahttp://www.pymol.org) molecular graphics program . PyMOL is widely used for the generation of figures of macromolecules in publications and its source code is available free of charge (http://sourceforge.net/projects/pymol/). Our program has a graphical user interface (GUI) that is fully integrated into PyMOL.We present here a computer program, mtsslWizard that allows the user to attach MTSSL labels to selected positions of a protein by pointing and clicking with a computer mouse. The program is designed as a plugin for the PyMOL , calculation times on the order of 2–10 min may occur. Wherever the protein is a symmetric multimer, only one calculation per unique site is required; the distribution is easily transferred to other symmetry related positions by simply clicking in “Copy and Move” mode of the program.Wherever the label is attached close to a hydrophobic surface pocket, conformations that place the label inside the pocket might be particularly stable. Such a fixed conformation would give a very different distance distribution than models assuming a flexible spin label. Identifying such a pocket would be key to correctly interpreting PELDOR data and avoiding “identifying” a conformational change where none occurred. As a possible solution to this problem, mtsslWizard sums the number of atoms that reside outside the vdW cutoff of a non-clashing conformation but are still within a 4.5 Å sphere around the same label conformation. We then use the number of these contacts as a surrogate for fit to the protein surface. The conformation with the highest number of such contacts is flagged as a “snuggly fit”, as are all those conformations that have at least 75% of this maximum value . The “snuggly fit” algorithm is only activated when the program runs with the thoroughness set to “painstaking” since otherwise the conformational space is not sampled well enough for this feature to work properly. We would like to stress that the “snuggly fit” algorithm merely flags up possibilities for the scientist to further investigate.MtsslWizard was initially designed as a tool to assist in the selection of labeling sites for PELDOR experiments. The program gives the user an immediate idea if a planed labeling site is too occluded for the attachment of MTSSL and if the distance between two planed labeling positions will fall into the PELDOR window of 15–80 Å. Furthermore, we found that the program is very useful to interpret PELDOR data if two or more models for a protein structure are available. In this case, a comparison of the experimental distance distributions with the in silico data can often help to decide which of the available models shows the best agreement with the experimental data.Almost by definition, SDSL is performed on solvent-accessible surface residues; however, the local environment of the protein structure can change due to the introduction of the spin label. A good example for this is the spin-labeled Spa15 structure which shows some significant conformational changes compared to the wild-type structure , 32. It Once two or more sites of a protein have been labeled in silico, the mtsslWizard is switched into “Distance” mode and all distances between a pair of label ensembles are determined (N–N distances between nitroxide groups), displayed and written out to a file if desired Fig. c. For a 2 histone [r>) and the most probable/peak distances rpk are listed. The reason for the usage of the mean distance is that the value for the mean distance is more stable in DEER analysis-derived distance distributions than the peak distance or the asymmetry of the distribution, especially in the case of broad distributions [r> = 1.2 Å, σ(Δr) = 3.0 Å) estimate of the PELDOR experimental distance than the Cβ–Cβ distance  = 3.5 Å) and performs on par with the MMM program [r> = −0.9 Å, σ(Δr) = 3.3 Å). Here, <Δr> is the mean value of the difference between in silico and experimentally derived distance and σ(Δr) is its standard deviation. Thus, mtsslWizard is “safe” to use, it is not worse than MMM, the current gold standard on average. Bowman et al. [r) = 3.5 Å that is not much worse than any of the simulation approaches. Thus, as a rule of thumb, 6.5 Å can be added to any Cβ–Cβ distance to get a good idea of which experimental distance is to be expected between a pair of MTSSL spin labels. However, the benefit of simulation programs is the calculation of distributions that can be compared to experimental data.We tested mtsslWizard against a dataset of 52 published PELDOR and CW-EPR-derived distances of double spin-labeled T4 lysozyme (T4L) and H3–H4) histone . The proibutions . The res–H4 histo program   and in 298 K mode. In 175 K mode we found that MMM often produces very sharp but spiky distance distributions, which can be difficult to interpret and that are usually not found in PELDOR experiments. At 298 K the MMM distributions are much more smooth and rather wide, which means that most of the MTSSL rotamers that MMM predicts for a certain site are assumed to be populated at this temperature . In essence, this comes close to the mtsslWizard strategy of sampling the whole conformational space of the label at this site and calculating an average distance. Furthermore, Fig. In 2009, Endeward et al.  analyzed1 and Chi2 of the predicted ensemble are close to the experimental values of −60 and 180° indicating that in this case the protein environment drives the label into this conformation In the Wza mutant Q335C the label is attached to the bottom of a relatively large hydrophilic crevice in the molecular surface . Since mtsslWizard is based on the “tether-in-a-cone” approach , 17, 18 Wherever the spin label is not disordered but adopts a fixed or restricted set of conformations due to interaction with the protein, only a fraction of the possible conformations that mtsslWizard finds will match the observed data. Additional parameters such as the surface complementarity score (“snuggly fit”) have been introduced to improve the predictions (example 3: Spa15). We also found that it is often helpful to interpret mtsslWizard results in the light of room temperature CW spectra because the latter contain experimental information about the flexibility of the spin label and would thus hint to whether a label is indeed trapped in a pocket or pointing into the solvent.MtsslWizard is a simple to use and freely available program that allows users to generate plausible for MTSSL labels attached to a protein, to measure distances between labels and indeed to vary parameters to allow superficially implausible models. We have argued above that there are sound reasons  not to assume each side chain of an amino acid is positioned correctly .As we would predict our approach produces similar results to MMM in most cases, any benefit in such cases would derive only from it being a PyMOL plugin, rather than MATLAB. Importantly, the integration of the program into a freely available, sophisticated and powerful molecular graphics program like PyMOL makes it easy for users to critically evaluate mtsslWizard results. We believe the advantage of our program occurs when PELDOR data does not match the simple model. Incorrectly interpreting disagreement between PELDOR data and a distance model in functional terms must be avoided. Determining crystal structures to resolve such anomalies is definitive but may not be possible in each case. Our approach allows the scientist to explore whether the measured distances are plausible from a range of models. As we show the program does a very good job for a number of test cases where there are unusual positions of the spin label, we stress the program is intended to provide a basis for further analysis and experimentation. By eliminating alternative explanations for discrepancy between model and data, scientists will be able to confidently identify conformational changes.htp://www.pymolwiki.org/index.php/mtsslWizard.The source code of mtsslWizard is freely available at Supplementary Figure 1green) and MMM in 175 K  and 298 K (blue) mode. If available experimental distributions were overlaid in red. The T4L distributions were taken from [ Superposition of distance predictions by mtsslWizard Below is the link to the electronic supplementary material.


P < 0.05), whereas there were no significant differences in ADC values between bulging and herniated discs (P > 0.05). Moreover, statistically significant relationship was found in the mean ADC values of NP between “non-bulging/herniated and non-degenerated discs” and “non-bulging/herniated degenerated discs” as well as herniated discs (P < 0.05). Linear regression analysis between ADC value and disc level revealed an inverse correlation (r = -0.18). The ADC map of the NP is a potentially useful tool for the quantitative assessment of componential and molecular alterations accompanied with lumbar disc abnormalities.The aim of the present study was to compare the relationship of morphologically defined non-bulging/herniated, bulging and herniated intervertebral lumbar discs with quantitative apparent diffusion coefficient (ADC). Thirty-two healthy volunteers and 28 patients with back pain or sciatica were examined by MRI. All intervertebral lumbar discs from L1 to S1 were classified according to morphological abnormality and degenerated grades. The ADC values of nucleus pulposus (NP) were measured and recorded. The significant differences about mean ADC values of NP were found between non-bulging/herniated discs and bulging discs as well as herniated discs ( The group included 32 healthy volunteers (16 men and 16 women) without symptoms of back pain or sciatica and the median age was 38 years (age range 18-73 years). Others included 28 patients (12 men and 16 women) with back pain or sciatica and the median age was 47 years (range 20-74 years). Individuals were excluded in the presence of diabetes mellitus, major systemic diseases, serious illnesses , back surgery, spinal fractures, or diagnosed osteoporosis. This prospective study was approved by the Institutional Review Board in the First Hospital of Medical School, Xi'an Jiaotong University and all participants provided written informed consent before enrollment.2 value by diurnal variation in the intervertebral discsLumbar spine imaging was achieved on a whole-body 1.5T clinical scanner . Each MR scanning in this study was performed in the afternoon in order to diminish possible influence on ADC and T2-weighted images (T2WI) fast spin echo (FSE) sequence in the sagittal and axial orientation. The scanning parameters were as follows: TR/TE, 3,500/1,200 ms; thickness, 4.0 mm; field of view (FOV), 250 mm; matrix, 512×512; number of signal acquisitions (NSA), 4; turbo factor, 17; number of sagittal sections, 11; number of axial sections in each disc, 3. Moreover, sagittal T1-weighted imaging was performed by FSE sequence with TR/TE, 400/14 ms; thickness, 4.0 mm; FOV, 256 mm; matrix, 512×512; NSA, 2; turbo factor, 3; number of sections, 11. The sagittal DWIs of the lumbar spine were obtained by a single-shot SE echo-planar imaging sequence (DWEPI) with Syn-spine coil in three orthogonal directions and then an average ADC was calculated from the three ADC directions. A b-value of 500 s/mm2 was chosen as a suitable compromise for diffusion-weighted imaging of intervertebral discs in this study. Other parameters were as follows: TR/TE of 3,000/81 ms, thickness of 6.0 mm, FOV of 250 mm, matrix of 512×512 and NSA of 8. The total scan time was 3 min 34 s for 3 slices passing through the lumbar center.Conventional lumbar MRIs were acquired with a 4-channel cervical-thoracic-lumbar spinal (Syn-spine) coil by TAll discs were classified morphologically on MRI images by two experienced radiologists with over 10 years' experience in MR imaging of the spine. They divided all discs into three groups: non-bulging/herniation discs group, disc bulging group and disc herniation group (protrusion or extrusion) according to the degree of disc extension beyond the interspace (DEBIT). 1) Non-bulging/herniation discs indicates no disc extension beyond the DEBIT; 2) Bulge refers to circumferential, symmetric DEBIT (around the end plates); 3) Herniation includes two types: Protrusion indicates focal or asymmetric DEBIT into the canal, and the base against the parent disc broader than any other diameter of the protrusion. Extrusion means focal, obvious DEBIT, and the base against the parent disc narrower than any diameter of the extruding material itself, or no connection between displaced disc material and parent disc2WI compared with that of normal intervertebral discs in the same individual; 3) loss of distinctness of the intranuclear cleft compared with that of normal discs in the same individual. Discs without three features were classified as non-degenerated discs. Moreover, discs with only one of these findings were defined as mildly degenerated discs, while those with at least two findings were defined as severely degenerated discsFor evaluation of disc degeneration, radiologists also classified all discs into the following categories: non-degenerated, mildly-degenerated and severely-degenerated grades according to the absence or presence of three findings: 1) loss of disc height; 2) reduction in signal intensity on TThe two radiologists worked independently and without knowledge of the clinical information and findings on ADC map in three consecutive weeks. If disagreements occurred, they reviewed the images together and reached a consensus for analysis.2WI).All discs were divided into three groups described above. The column displayed degenerative grades of discs according to three findings  For discs that the boundary between the NP and annulus fibrosus (AF) was not distinguishable, the irregular or regular ROIs were selected by the operator according to the expected location of the inner portionAll data were transferred into the imaging workstation . A radiologist performed the region of interest (ROI) in central discs from L1 to S1, including NP: 1) For non-degenerated and some of degenerated discs, an elliptical ROI, on the middle section of sagittal T2WI with corresponding sagittal ADC map was acquired from a 30-year-old asymptomatic volunteer. The lumbar discs were graded according to the sagittal T2WI as follows: L1-L2, L2-L3 as mildly degenerated, L3-L4 and L4-L5 as non-degenerated and L5-S1 as severely degenerated. The elliptic ROIs with red dotted line were manually placed at the center of the lumbar discs including the NP. The irregular ROIs with red dotted line for L5-S1 were selected by the operator according to the expected location of the NP. Then, the ROIs were copied to the ADC map at the same level.The typical sagittal Tr). The degree of correlation was evaluated by r absolute value as follows: r > 0.7 was defined as strong; 0.5 < r ≤ 0.7 as moderate; r ≤ 0.5 as weak. Statistical significance was set at P < 0.01. One-way ANOVA and post hoc Games-Howell tests were used for the comparison of the mean ADC values of NP in non-bulging/herniated discs, bulging and herniated discs. In addition, we also evaluated the relationship between the mean ADC values of NP and degenerative grades according to the three groups: non-bulging/herniated and non-degenerated group, non-bulging/herniated and degenerated group, and herniated group. A P value of less than 0.05 was considered significant. In this study, statistical analyses were performed by using SPSS for Windows version 13.0 .The variations of ADC values of NP in lumbar intervertebral discs along with anatomic levels were assessed by Spearman correlation coefficient . A total of 295 discs were successfully measured on ADC map. Five discs were excluded because three discs were obscured due to susceptibility artifacts at L3-S1 in one person, and two discs severely degenerated at L5-S1 had so narrow space that precluded the ROIs. The percentages of discs in the different classifications showed that most non-bulging/herniated discs were present in the non-degenerated disc group; nevertheless, most abnormal discs were seen in the mildly degenerated and severely degenerated groups ×10−3 mm2/s for bulging discs, and (1.83±0.32)×10−3 mm2/s for herniated discs, respectively. There was a significant relationship in the mean ADC values of NP between non-bulging/herniated discs and bulging discs, between non-bulging/herniated discs and herniated discs ×10−3 mm2/s for the non-bulging/herniated and non-degenerated group, (1.81±0.29)×10−3 mm2/s for the non-bulging/herniated and degenerated group, and (1.83±0.42)×10−3 mm2/s for herniated discs. There was a marked statistical difference in the mean ADC values of NP between the “non-bulging/herniated and non-degenerated group” and herniated group as well as the “non-bulging/herniated and degenerated group” ×10r = -0.18, P < 0.01). In addition, the ADC value of non-bulging/herniated discs was markedly higher than bulging and degenerated discs, and revealed statistically significant differences (P < 0.05). Further results depicted statistically significant differences in the ADC values of the NP between non-bulging/herniated and non-degenerated group and herniated group, between non-bulging/herniated and degenerated group and herniated group (P < 0.05).In this study, the ADC values of NP were measured in subjects with different categorizations including disc abnormalities and disc degeneration. It confirmed the previous report that most non-bulging/herniated discs were at the cephaled level, and most of the bulging and herniated discs were localized at the caudal levelThe lumbar intervertebral disc is the largest avascular structure in the body. Nutrients are dependent on diffusion of fluid either from the vertebral bodies across cartilaginous endplate or through the AF from the surrounding capillaries to reach the NP, and metabolic waste products of discs are removed from the tissue by the reverse routeet al.In this investigation, most bulging and herniated lumbar discs were at the caudal level, while most of the non-bulging/herniated intervertebral discs were located at L1-L2 and L2-L3 (P > 0.05, The intervertebral lumbar disc matrix consists of a complex network of macromolecules whose composition varies in different regions of the discADC map has shown the potential to quantitatively evaluate degeneration of disc molecular composition and structural integrity because ADC values are sensitive to water content and arrangement of collagen network structure2WI and copy to the same slice of ADC map.Nevertheless, there were some limitations of ADC map. First, echo planar imaging sequence is highly sensitive to artifacts, especially to motion artifacts that include gastrointestinal and respiratory movement. In order to minimize and control the possible effect of these artifacts, we not only required every participant to maintain quiet respiration, but also compressed all the subjects in the lower abdomen with abdominal bandage. Second, flow artifact caused by the cerebrospinal fluid often results in confusion between NP and AF and it is easy to cause a measurement bias. To minimize the bias, we placed ROI in the middle slice of sagittal Tin vivo, ADC value is a potentially quantitative tool to assess componential and molecular alterations accompanied with disc abnormalities. Additionally, it may estimate therapeutic effect in which standard MRI does not show any change even though clinical symptoms may improve or deteriorate.As a noninvasive method of measuring the diffusion of water within tissue