In Tanzania, many people seek malaria treatment from retail drug sellers. The National Malaria Control Program identified the accredited drug dispensing outlet (ADDO) program as a private sector mechanism to supplement the distribution of subsidized artemisinin-based combination therapies (ACTs) from public facilities and increase access to the first-line antimalarial in rural and underserved areas. The ADDO program strengthens private sector pharmaceutical services by improving regulatory and supervisory support, dispenser training, and record keeping practices.The government's pilot program made subsidized ACTs available through ADDOs in 10 districts in the Morogoro and Ruvuma regions, covering about 2.9 million people. The program established a supply of subsidized ACTs, created a price system with a cost recovery plan, developed a plan to distribute the subsidized products to the ADDOs, trained dispensers, and strengthened the adverse drug reactions reporting system. As part of the evaluation, 448 ADDO dispensers brought their records to central locations for analysis, representing nearly 70% of ADDOs operating in the two regions. ADDO drug register data were available from July 2007-June 2008 for Morogoro and from July 2007-September 2008 for Ruvuma. This intervention was implemented from 2007-2008.During the pilot, over 300,000 people received treatment for malaria at the 448 ADDOs. The percentage of ADDOs that dispensed at least one course of ACT rose from 26.2% during July-September 2007 to 72.6% during April-June 2008. The number of malaria patients treated with ACTs gradually increased after the start of the pilot, while the use of non-ACT antimalarials declined; ACTs went from 3% of all antimalarials sold in July 2007 to 26% in June 2008. District-specific data showed substantial variation among the districts in ACT uptake through ADDOs, ranging from ACTs representing 10% of all antimalarial sales in Kilombero to 47% in Morogoro Rural.The intervention increased access to affordable ACTs for underserved populations. Indications are that antimalarial monotherapies are being "crowded out" of the market. Importantly, the transition to ACTs has been accomplished in an environment where the safety and efficacy of the drugs and the quality of services are being monitored and regulated. This paper presents a description of the pilot program implementation, results of the program evaluation, and a discussion of the challenges and recommendations that will be used to guide rollout of subsidized ACT in ADDOs in the rest of Tanzania and possibly in other countries. Private sector drug outlets, including drug shops, general shops, and market vendors, are major sources of medicines in sub-Saharan Africa. A review of studies from some countries found that around half of caregivers initially sought medicines for the treatment of common childhood illnesses from private drug sellers .duka la dawa baridi (DLDB) are the most common source of medicines in the private sector [Historically, in Tanzania, retail drug shops commonly known as e sector . Becausee sector ; howevere sector . Most DLe sector ,5.Duka la Dawa Muhimu in Kiswahili. The goal of the ADDO program is to improve access to affordable, quality medicines and pharmaceutical services in rural and peri-urban areas where there are few or no registered pharmacies. The accreditation process for ADDOs takes a holistic approach, with a focus on training and changing behavior and expectations of those who use, own, regulate, and work in the drug shops.In 2002, the Ministry of Health and Social Welfare and TFDA worked with Management Sciences for Health's (MSH) Strategies for Enhancing Access to Medicines Program, with funding from the Bill & Melinda Gates Foundation, to pilot an innovative private sector drug seller program to transform the DLDB into government-accredited drug dispensing outlets (ADDO)--th January 2009) at which time ADDO coverage was expected to be complete  and technical support from MSH's Rational Pharmaceutical Management Plus (RPM Plus) Program, TFDA and NMCP began a pilot program to make subsidized ACTs available through ADDOs in 10 districts in the Morogoro and Ruvuma regions. At that time, the products . The pilThe results of the review will provide government stakeholders with evidence needed to guide policies related to increasing access to affordable and effective antimalarials through the private sector. Specific policy and implementation issues include ACT packaging and pricing, supply logistics, regulatory oversight and supervision, training, monitoring drug safety, and behavior change and communication. In addition, the data on the subsidization of preferred treatment will provide policy makers with key information on the effectiveness of that approach.The pilot program took place in five districts of the Morogoro region and five districts of Ruvuma region Figure . The burThe policy and regulatory environment in Tanzania allowed the private sector to deliver subsidized ACTs. The NMCP had already identified the ADDO program as a potential mechanism to supplement public sector distribution of subsidized ACTs and increase access in rural and underserved areas . To allo®. This decision allowed those visiting either ADDOs or public clinics to receive the same dosage instructions and public health messages; however, NMCP was concerned that using the exact same packaging might result in significant product diversion from public facilities to ADDOs. To reduce this likelihood, a tamper-proof sticker was used on subsidized packages sold in ADDOs, thereby, making it easier for TFDA inspectors to differentiate the two  for other patients. This cost excludes the cost for laboratory tests for malaria diagnosis, which when indicated, adds TSH 300-500 (USD 0.23-0.38) to the patient's cost. Anecdotal evidence suggests that facilities adhere to this price and provide free access to children under five. It was determined, however, that for this pilot, TSH 300 for the course of therapy was too low for ADDO owners to recover operational costs and produce a large enough profit margin to provide an incentive for them to stock the product. We examined drug shop clients' ability to pay based on the sales of other pharmaceuticals sold at ADDOs and reviewed research examining willingness and ability to pay for ACTs . ADDO owTo procure the supply of ACTs for the pilot program, PMI negotiated with Novartis, who agreed to supply the product to be distributed through ADDOs at the same price as the public sector receives. Procurement for ADDOs followed the same procedure as procurement in the public sector, where the consignee fills out and submits ACT order submission forms to the World Health Organization. For the pilot program, ACTs were procured in two consignments, which provided an opportunity to track the first consignment's sales before procuring the second consignment. In addition, because ACTs have a relatively short two-year shelf life, this ensured that the product in the second consignment would have close to the full useful life available. The program negotiated with the TFDA to obtain a waiver of the 2% importation fee that TFDA charges for all pharmaceutical imports.To determine quantities of ACTs needed for ADDOs for the pilot program, we used the morbidity method of quantification because no historical data was available on ACT sales in ADDOs. Therefore, we reviewed dispensing registers from a sample of 78 ADDOs to determine the number and age of customers who sought antimalarial treatment during the preceding 12-month period in Ruvuma and Morogoro regions. Interviews with owners and dispensers of selected ADDOs provided additional information. We also reviewed the Southern Highland Pharmacy's database in Songea town, which has a comprehensive record of ADDOs in Ruvuma region that have purchased from them since 2003. Estimates took into account the transmission patterns of malaria in the two regions and the age-specific dosage categories for ACTs, which were based on the NMCP criteria for estimating ACT needs for public health facilities. RPM Plus estimated that 661,393 courses of treatment would be needed to supply the approximately 650 ADDOs in the target districts for the first year.The plan to distribute ACTs to ADDOs was developed based on the existing private sector supply chain in Tanzania, where private pharmaceutical wholesalers are the main source of pharmaceuticals. Pyramid Pharma Ltd, based in Dar es Salaam, was chosen as the ACT distributor for ADDOs based on the following criteria: adequate and secure storage facility, readiness to track ACT inventory, reliable transportation to regional sub-distributors, high-quality record keeping, local availability of a wholesale unit in Ruvuma region, and previous working experience with major public and private sector clients. Pyramid Pharma was given responsibility to receive ACT consignments upon arrival in the country and to clear, store, and distribute the drugs to the two regional distributors.Pyramid Pharma identified Southern Highland Pharmacy as its subsidiary distributor for Ruvuma region and Marhaba Pharmaceuticals Ltd for Morogoro region. These regional distributors were already the main suppliers of ADDOs; 89% of all ADDOs in Ruvuma regularly procured their pharmaceutical supplies from Southern Highland pharmacy . To ensuGenerally, the regulation of ACT distribution through ADDOs capitalizes on the existing ADDO regulatory systems. Regulatory oversight and support by district- and ward-level inspectors help ensure that ADDO dispensers are held accountable for selling ACTs at the fixed price and for maintaining other ADDO standards. Supervision, focusing on education and mentoring of ADDO owners and dispensers, helps strengthen and maintain the quality of care and is vital to program sustainability. RPM Plus and TFDA trained and mentored council health management team supervisors at the district level to conduct quarterly supervisory visits with ADDO dispensers with the goal that the council team would eventually assume all supervisory responsibilities for ADDO dispensers, including their adherence to the ACT policy. Specifically, supervisors are supposed to ensure that ADDOs have ACT management tools on hand , provide technical support related to ACT dispensing, and evaluate dispensers' knowledge and skills. The main challenge with supervision is that the council health management teams also monitor public health facilities, but do not receive additional resources to cover transport costs and time needed to monitor ADDOs as well.The use of ADDO drug registers to record all sales information helps monitor and quantify ACT sales. In addition, quarterly report forms were created for ADDO dispensers to collate information on the number and age of malaria patients treated and product losses due to drug expiry. ADDOs are required to submit these forms quarterly to the regional distributor.Comprehensive training of both dispensers and owners is already a key requirement for the ADDO accreditation process. In addition to the six-week ADDO dispenser training program that had already taken place in Morogoro and Ruvuma regions prior to the ACT pilot program, RPM Plus and TFDA conducted an additional two-day training course for ADDO dispensers on the new malaria treatment policy. This training included the correct usage of ACTs, inventory management, and reporting on drug consumption, patient-related data, and ADRs. A total of 1,363 ADDO dispensers from 10 districts attended the training.As the availability of ACTs increases in the private sector through ADDOs, it is important to ensure that systems are in place to monitor and promote safe and appropriate use. TFDA already has an established passive (voluntary) ADR reporting system for medicines dispensed in public health facilities. To facilitate reporting by ADDOs, TFDA approved a simplified ADR reporting form for ACTs, inclusion of ADR reporting in ADDO dispenser training, and integration of ADDOs' ADR data into the district ADR reporting process. RPM Plus worked with the TFDA and council health management teams to link ADDOs with this reporting system. The ADDO dispenser training on ACT policy change addressed ADR issues to increase dispensers' awareness of the importance of drug safety monitoring and to instruct them on proper reporting using ADR forms. Dispensers were also instructed and encouraged on how to discuss drug safety issues with their clients. At the time of this evaluation, 20 ADDOs had reported on customers' "minor complaints" (skin rash and upset stomach) to the district authorities. Although these were nonspecific and might or might not have been related to taking ACTs, these early results indicated an increased awareness of the ADR issue.Behavior change and communication (BCC) activities were deemed a critical part of this intervention. During the assessment and planning stages of the pilot, RPM Plus consulted with NMCP to map out BCC activities related to malaria and to link this pilot program to ongoing BCC work to leverage resources and ensure that a consistent message was being delivered on ACT policy.The Communication and Malaria Initiative in Tanzania (COMMIT), which is led by the Johns Hopkins University's Center for Communication Programs, expanded their existing BCC activities related to the ACT policy in Morogoro and Ruvuma regions to include the availability of ACTs in ADDOs. COMMIT incorporated the availability of ACTs in ADDOs in road shows in the two regions and planned further strategies, including radio broadcasting and the dissemination of printed materials such as stickers, flyers, and t-shirts. However, a delay in the COMMIT BCC activities related to ADDOs meant that the activities did not coincide with start of ACT distribution, as planned, but instead started nine months later.Additionally, the Ifakara Health and Research Development Center's ACCESS program, which works on malaria projects in two Morogoro districts (Kilombero and Ulanga), has been using a BCC and social marketing approach that promotes ACTs as the treatment of choice for malaria symptoms, refers patients to health centers to obtain treatment, and emphasizes completing the full dosage for effective treatment .Approximately one year after the introduction of subsidized ALu in ADDOs, we planned this evaluation of the pilot program. One year allowed enough time to account for fluctuation in ACT consumption due to malaria seasonal patterns and to identify program barriers and identify recommendations to guide future delivery of subsidized ACTs in Tanzania's private sector. This program evaluation focused on processes in designing private sector subsidized ACT delivery, assessing the quantities of antimalarials that ADDOs dispensed, and the efficacy of regulatory and reporting systems related to ACTs. In addition, the evaluation sought to document challenges and lesson learned in launching an ACT delivery initiative through the ADDO program.One of the major advantages of the ADDO program is the improvement in record keeping, which is an accreditation requirement. Owners are required to keep records of all purchases and dispensing transactions for the list of products authorized for sale. To overcome logistical challenges and reduce the costs associated with collecting data from each individual ADDO, we asked ADDO dispensers to bring their drug registers, ACT purchase reports, and stock cards to central locations close to their communities. Most locations (primarily primary schools or ward offices) were in peri-urban areas with a few in villages. ADDO dispensers spent half a day in the meeting and were able to travel back to their shop. Prior to the meeting, the district team sent information such as which records to bring by calling or text messaging owners. The dispensers did not receive an allowance for participating, but were provided refreshments.Four hundred forty-eight ADDOs from 10 districts participated in this data collection exercise, 298 in Morogoro region and 150 in Ruvuma region, representing nearly 70% of ADDOs in operation in the two regions. ADDO drug register data were available from July 2007-June 2008 for Morogoro region and from July 2007-September 2008 for Ruvuma region. The drug register review was used to extract data on the number and age of customers who were sold treatment for uncomplicated malaria for each month of the period, including the type of antimalarial dispensed. The evaluation team also reviewed ACT purchase reports that had been submitted to the regional distributors. Triangulation of various records allowed us to verify and crosscheck discrepancies and accuracy of quantities purchased and dispensed during any period. Additionally, a questionnaire was administered to the ADDO dispensers to determine what type of ACTs shops were stocking and the reasons why. We also asked dispensers about the availability of ACT tools in their shops, including quarterly report forms, standard operating procedures for ACT purchasing, price indicator sheets, and dosage indicator sheets.The data from the ADDO drug registers suffers from limitations that are common with routine records in developing countries; however, this intervention involved working with district teams and ADDO owners to strengthen supportive supervision and the quality of data recording related to ACTs in the ADDOs. Regular teams visited ADDOs and worked with owners to ensure completeness, accuracy, and timely reporting of ACTs dispensed.The participation rate among eligible ADDOs was around 70%, which was an accomplishment given the logistical challenges . Follow-up with those who did not participate indicated that they did not receive the information about the evaluation workshop from the district health office, which was confirmed through government sources; however, other reasons may have contributed and may have biased the results. No particular geographic areas lacked participants; no-shows were fairly evenly distributed. In addition, problems still exist with ACT sales not being recorded in the drug register; however, observations from previous supervision visits suggests that this issue is not limited to the ACT program, but rather is related to overall underreporting of medicine sales to avoid taxes. This under-reporting, however, may bias overall results.all existing ADDOs in the two regions of Morogoro and Ruvuma to provide information on feasibility and implementation before large-scale nationwide rollout. One of the key requirements was that the implementation needed to be designed as a routine activity to reflect the reality on the ground and to assess its feasibility rather than a strictly controlled design using a naive region. From routine supervision and monitoring visits, we had documented that no ACTs were available in ADDOs prior to the intervention. Because the price for unsubsidized Coartem had been USD 8-10, none of the ADDOs could afford to stock it because customers could not afford to pay.The distribution of subsidized ACTs though ADDOs was piloted in As mentioned, results are based on data from 448 ADDOs--or about 70% of the total number of ADDOs in the 10 target districts. Over the pilot program implementation period (July 2007-September 2008), the 448 ADDOs dispensed antimalarial treatment to over 300,000 customers. Around 30% of malaria treatments were for children under five. Table The percentage of ADDOs that dispensed at least one course of ACT rose from 26.2% during July-September 2007 to 72.6% during April-June 2008. For the five districts in Ruvuma that reported data for July-September 2008, this increased to 81.5%. At the time of the evaluation, dispensers reported that 68% of ADDOs in Morogoro and 78% of ADDOs in Ruvuma were stocking at least one of the four types of ACTs . The main reasons they cited for ADDOs not stocking ACTs included the far distance to the regional distributor (92%), the high sales price of ACTs (55%), and the ADDOs' limited capital resources (52%). Slow uptake in ADDOs (as reported by dispensers) was also associated with patient perceptions that the ACT dosing schedule was difficult (30%) and the availability of free ACTs at public health facilities (10%). Only 4% of dispensers cited unavailability of ACTs at the regional distributor as a reason for not stocking. Dispensers also demonstrated a high level of compliance with ACT documentation requirements; over 80% of ADDO dispensers reported using drug registers, ACT quarterly report forms, ACT price indicator sheets, ACT dose indicator sheets, and referral forms. Evaluation teams verified all the records presented by ADDO dispensers.The ADDO data indicated that the number of customers seeking malaria treatment with ACTs gradually increased after the start of the pilot program, while the use of non-ACT antimalarials declined Figure . In the District-specific data showed substantial variation among the districts in ACT uptake through ADDOs, ranging from ACTs representing 10% of all antimalarial sales in Kilombero to 47% in Morogoro Rural Figure .This paper attempts to provide policy makers and program managers with concrete results from a year-long pilot program to guide their policy, regulatory, and implementation decisions for scaling up private sector delivery of ACTs through ADDOs. Although ADDOs have been operating in Tanzania on a limited scale since 2003, the use of subsidies in the private sector to promote the sales of a particular treatment--in this case, ACTs, is new; therefore, this data is an important contribution to the government's decisions in relation to developing a strategy to increase access to ACTs.As the results of this evaluation show, with extensive collaboration by numerous stakeholders a sustainable and reliable system to increase access to ACTs through the private sector has been established in two regions of Tanzania, using legally recognized accredited drug shops, the existing private sector pharmaceutical distribution system, trained drug dispensers, and local supervisory and regulatory oversight. Additional steps in program implementation involved procuring subsidized ACT products, determining packaging and pricing, training dispensers in the new ACT policy, and establishing an ADR monitoring system. Preliminary results showed that the intervention increased access to affordable ACTs for rural and underserved populations. In addition, early signs showed that antimalarial monotherapies not recommended by NMCP are being "crowded out" of the market. Importantly, this transition has been accomplished in an environment where the safety of the drugs and the quality of services are being monitored and regulated. While the results of the pilot program evaluation are encouraging, we have identified a number of challenges and opportunities for improvement.One year after initiation of the pilot, 70% of ADDOs that had reported data  for this pilot program were stocking ACT products. This figure exceeded 75% in 6 of the 10 districts. In the other four districts , the primary reason dispensers said that ADDOs did not stock ACTs was the long travel distance to the regional distributor for re-supply. This suggests that the current distribution system for ACT products, based on having one regional distributor in each region, is inadequate to meet the needs of ADDOs with limited capital or remote locations. For example, in Tunduru district, only 35% of ADDOs stocked ACTs at the time of data collection, and 80% of those dispensers cited distance to the distributor as the reason. In addition, a number of ADDOs reported having inadequate capital to procure enough ACTs to last until their next purchase, which is affected by long distances and poor road conditions.The best solution for distribution chain problems is to ensure that more certified wholesalers at the district level are available to serve ADDOs. Establishing certified wholesalers is a relatively slow and difficult process; for example, TFDA requires ADDO restricted wholesalers are to be supervised by a pharmacist, who is seldom available in rural areas. An interim option is to revisit the current requirement and consider allowing lower pharmaceutical cadres to supervise select ADDOs to serve as an ACT distributor to more remote ADDOs. Both strategies would help ensure that ADDO owners have a shorter distance to travel to obtain ACTs, lower travel costs, and greater ability to refill their stock. However, this option has regulatory implications that TFDA needs to consider carefully.While the pilot program demonstrated a generally successful mechanism to increase the availability of ACTs in the private sector, the sales of ACT products comprised only 26% of overall antimalarial sales in the 10 districts. The two main reasons cited by ADDO dispensers for the poorer than expected sales included the high prices of ACTs and the patient's perceptions of the drug. In addition, although our results show an increase in ACT sales at ADDOs, we cannot determine if this increase indicates an overall increase in ACT use in the community or merely a shift in access from one ACT source to ADDOs.Two studies have examined Tanzanian caregivers' willingness and ability to pay for ACTs for a sick child: one found a median level of willingness to pay of TSH 600 (USD 0.46) and the other found a median willingness to pay of TSH 650 (USD 0.5) ,15. ThisThe subsidized ACTs currently available in Tanzania have a more complicated dosing schedule than other common antimalarials, requiring two doses per day for three days versus a single dose for SP. Depending on the patient's age, one treatment comprises between 6- 24 pills. It is not known to what extent the relative inconvenience of the dosing schedule or other factors related to the appearance of the drugs or the packaging deters individuals from purchasing ACTs in ADDOs. Patient compliance to the proper ACT regimen purchased from ADDOs is also unknown. Further research examining the social, economic, and cultural factors affecting malaria treatment decision making would contribute to the design of effective BCC messages and marketing strategies for the new ACT policy.During the pilot program monitoring, supervisors noted that the profit margin for ADDO business owners for the adult course of therapy of subsidized ACT was significantly less (28-44%) than that for SP (61-164%)  in two districts of Tanzania, observed more rapid uptake of ACTs through DLDB than we observed in this study. One reason cited in the CHAI study was a more extensive and focused social marketing campaign that involved advertising the availability of ACTs in drug shops through numerous media outlets . The COMThe importance of drug seller training is well recognized and has been a vital component of most interventions aimed at improving the quality of services provided by drug shops and other medicine sellers ; howeverThe regulatory and supervisory systems inherent in the ADDO program and then adapted to monitor ACT dispensing make this intervention stand apart from other interventions to provide subsidized ACTs in private drug shops. While implementing these systems seems slow and requires significant financial and human resources, they help ensure that the population receives quality products and services and that public health goals are met.NMCP had initially raised ACT leakage as a concern before the program began; however, during the pilot program, no leakage of ACTs destined for the public sector to the ADDOs was identified. To prevent leakage from the public sector to ADDOs or from ADDOs to DLDB, a number of safeguards were put in place: (1) ACTs meant for ADDOs included a special ADDO sticker figure ; (2) ADDAnother concern was the potential risk associated with indiscriminant use of the drugs, which can accelerate the development of drug resistance and increase the risk of ADRs . This piAt the time of writing this paper Tanzania was among the countries selected to implement the Affordable Medicines Facility-malaria (AMFm). The AMFm, a price subsidy mechanism to expand access to affordable ACTs, is managed by the Global Fund to Fight AIDS, Tuberculosis and Malaria and has been described elsewhere ,20. The This evaluation found that the availability of management tools related to ACT dispensing in ADDOs was high. But while the majority of ADDOs had drug registers, ACT quarterly report forms, ACT price and dose indicator sheets, and referral forms, the evaluation suggested the need to improve dispensers' use of these tools. For instance, comparing the sales of ACTs recorded in drug registers with the purchasing slips from regional distributors indicated that dispensers were only recording approximately 60% of ACT sales. In addition, up to 70% of all malaria medicines sold in ADDOs are in response to written prescriptions from health facilities presented by patients, but in many instances, the sales of ACT from these prescriptions were not recorded in the drug registers. Using SP as an example, appropriate record keeping could indicate the percentage of SP sales that resulted from appropriate prescriptions to pregnant women and what percentage of sales was inappropriate. Strengthening supervision to ADDOs and emphasizing the importance of record keeping will continue to be important to ADDO program sustainability.As Tanzania expands access to effective antimalarial medicines, particularly in rural areas, ADDOs provide the mechanism to increase access in the private sector while ensuring quality of services and products to safeguard the public's health. This pilot program offered critical lessons and identified key challenges that need to be addressed for a successful and sustainable national scale-up. Market forces (price) alone will not increase access. Specific policy recommendations to assure the success of this strategy include: expanding the distribution system by allowing selected ADDOs in remote locations to resell products to other ADDOs until enough ADDO-restricted wholesalers are available to meet ACT as well as other ADDO product source needs; developing a comprehensive BCC strategy targeting ACTs in ADDOs; and developing policies that provide the districts with the capacity to supervise and inspect ADDOs to ensure quality of dispensing services and record keeping for accountability and monitoring. In addition, TFDA needs to continue to address the issue of under-reporting of sales of prescription-only medicines in the ADDO drug registers. TFDA should also explore opportunities offered by new technologies, such as use of cell phones in data collection and transmission.ADDO owners' participation in the pilot, the first of its kind to deliver subsidized products in Tanzania, was critical. The program provided incentives in terms of an adequate profit margin to cover owner operational costs and small profit. But a majority of ADDO owners and dispensers participated because they felt this program enhanced their reputation in the community.An intervention such as the ADDO program provides a platform to integrate interventions such as increasing access to ACT; however, such program requires substantial human and financial resources, high stakeholder participation, and time. Other countries planning to use the private sector drug sellers to increase access to ACTs can use such interventions as an initial step toward more comprehensively addressing the overall performance of the private sector health system. A drug seller intervention of limited scope can introduce ACT distribution with minimal dispenser training; however, the right path for a broader and more sustainable program should be based on innovative uses of the existing regulatory system, policy change, business incentives, capacity building, product quality monitoring, and consumer awareness and education.The authors declare they have no competing interests other than employment that includes activities related to the ADDO program.ER substantially contributed to the project design, data analysis and interpretation, and drafting and revision of the manuscript. BM helped analyze and interpret the data and drafted the early manuscript. JL collected the data, participated in data analysis and interpretation, and helped draft the manuscript. RM participated in data collection and analysis and drafting of the manuscript. WM participated in the design and implementation of the project and data collection and analysis. ME critically reviewed and revised the manuscript and contributed intellectual content. MG helped draft and review the manuscript. ES and BK participated in project design, data collection coordination, and helped draft the manuscript. SK participated in data collection and interpretation and helped draft the manuscript. All authors read and approved the final manuscript.


New strategies to overcome complications of cardiovascular diseases are needed. Since it has been demonstrated that atherosclerosis is an inflammatory disease, modulation of the immune system may be a promising approach. Previously, it was suggested that antibodies may confer protective effects on the development of atherosclerosis. In this study, we hypothesised that passive immunization with anti-oxLDL IgM antibodies specific for hypochlorite (HOCl) may be athero-protective in mice.−/− mice was induced by a perivascular collar placement around the right carotid artery in combination with feeding a high-fat diet. Subsequently, the mice were treated every six days with 500 µg Moab A7S8, non-relevant IgM or with PBS and the carotid arteries and aortic roots were studied for atherosclerosis. Passive immunization with this Moab A7S8 resulted in a significant reduced plaque volume formation in LDLr−/− mice when compared with PBS treatment (P = 0.002 and P = 0.035). Cholesterol levels decreased by 20% when mice were treated with Moab A7S8 compared to PBS. Furthermore, anti-oxLDL specific IgM and IgG antibody production increased significantly in the Moab A7S8 treated mice in comparison with PBS treated mice.Monoclonal mouse IgM antibodies were produced and the antibody with specificity for hypochlorite-oxLDL (HOCl-oxLDL) (Moab A7S8) was selected. VH sequence determination revealed that Moab A7S8 is a natural IgM antibody. Atherosclerosis in LDLrOur data show that passive immunization with a natural IgM antibody, directed to HOCl-oxLDL, can reduce atherosclerotic plaque development. We postulate that specific antibody therapy may be developed for use in human cardiovascular diseases. Atherosclerosis is the most important underlying cause of cardiovascular diseases and is a major contributor of morbidity and mortality in the western society. In large randomized clinical trials complications such as myocardial infarction and stroke, are reduced by less than 50% with current therapy. Therefore, development of novel therapeutic strategies is highly needed to complement or replace current treatments In vitro, IgM autoantibodies to oxLDL block the binding and degradation of oxLDL by macrophages Immune-modulation therapy via a passive immunization strategy aims to exploit the athero-protective aspects of the immune system to modulate the development of atherosclerosis OxLDL plays a pivotal role throughout development of atherosclerosis. Modification of LDL into its oxidized form is caused by several different mechanisms. One clinically relevant pathway is via myeloperoxidase (MPO) and its oxidant product hypochlorite (HOCl) −/− mouse model for atherosclerosis.We hypothesised that atherosclerotic plaque development can be reduced with passive immunization of IgM antibodies specific for HOCl-oxLDL. To test this hypothesis, we selected a monoclonal antibody that bound to hypochlorite-oxLDL and performed passive immunizations in our LDLrThe investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the directive of the European Parliament (Directive 2011/63/EU). Approval was granted by the ethics review board of Maastricht University (reference number: 2008–111) and carried out in compliance with the Dutch government.2O. Anti-oxLDL IgM producing clones were selected by enzyme-linked immunosorbent assay (ELISA) as described before Balb/C mice were immunized with hypochlorite-modified oxLDL Antibody-containing medium was generated by culturing the hydridoma cell line in IMDM containing 1% FCS. To IgM-containing medium, 0.75 M of amoniumsulfate was added and the medium was loaded on a thiophilic agarose resin. The column was eluded using a 50 mM Tris-HCl buffer at pH 8.9. Protein concentration was determined as absorption at A280. Purified IgM antibodies were dialysed against PBS and subsequently filtered by a 0.2 µM filter and stored at −80°C.Purified monoclonal IgM antibody recognizing HOCl-modified LDL was obtained as described above. The antibody preparation was dissolved in non-reducing sample buffer and analyzed by SDS- Poly-Acrylamide-Gel Electrophoresis (SDS-PAGE), using a modification of the method described by Laemmli The one-step mouse monoclonal antibody determination  procedure involves the capture of the mouse immunoglobulins by subclass specific rat anti-mouse monoclonal antibodies which are immobilized on the test strip.2 anti-mouse IgM (µchain)-specific conjugate , diluted 1∶2000 in incubation buffer for 1 hour at 37°C. After washing, 100 µl of freshly made substrate containing 1 mg/ml of nitrophenyl phosphate in di-ethanolamine buffer at pH 9.8 was added to each well. Plates were read at 405 nm.LDL was isolated according to Smook et al. To determine the VH sequence of the IgM monoclonal antibody, the heavy chain was cloned and sequenced after amplification with universal VH gene specific 5′ AGGTSMARCTGCAGSAGTCWGG 3′ −/− mice (n = 18 per group), obtained from the Jackson Laboratory, were operated after 2 weeks of high fat diet  to introduce a 2 mm long non-constrictive silastic tube around both carotid arteries, as described before Twelve-week-old male LDLrTo determine anti-oxLDL antibody titers, ELISAs were performed as described below. Blood samples were taken: before introduction HFD (T = −2), one day before collar placement (T = 0), one week after collar placement , 3 weeks after collar placement  and on day of sacrifice .Total IgM and IgG levels were measured in a chemiluminescent-based sandwich ELISA using polyclonal goat anti-mouse IgM for capture and alkaline phosphatase (AP)-labeled goat anti-mouse IgM (µ-chain specific) for detection. Sera were diluted 1∶10.000 for total IgM.Human LDL was isolated and MDA-LDL and copper-oxidized LDL were prepared as described ApoB-100 particles were captured on microtiter wells using LF3, a monoclonal antibody specific for murine apoB-100 which was coated on microtiter wells at 5 µg/ml in PBS. After washing and blocking steps, plasmas (1∶100 in BSA-PBS) were incubated in wells for 1 hour at room temperature and after further washing, bound IgM or IgG was detected using AP-conjugated goat anti-mouse IgM or anti-IgG by chemiluminescent ELISA. For detection of T15/EO6 antibodies bound to the captured apoB-containing particles, biotinylated AB1–2 was used, followed by incubation with AP-labeled NeutrAvidin and LumiPhos 530. In parallel wells, the relative amount of apoB captured in each sample was determined using biotinylated LF5, another monoclonal antibody specific for mouse apoB-100, followed by incubation with AP-labeled NeutrAvidin and LumiPhos 530. Because LF5 binds to only one epitope of apoB-100, the amount of each antibody used in this assay bound to the captured LDL was then normalized for the amount of captured apoB, and expressed as a ratio of antibody counts (RLU/100 ms) to apoB-100 counts (RLU/100 ms) 2 anti-mouse IgM (µchain)-specific conjugate , diluted 1∶2000 in incubation buffer for 1 h at 37°C. After washing, 100 µl of freshly made substrate containing 1 mg/ml of nitrophenyl phosphate in di-ethanolamine buffer at pH 9.8 was added to each well. Plates were read at 405 nm. Results are expressed as mean absorbance values (405 nm) from duplicate determinations and were calculated by subtracting binding to native LDL from binding to oxLDL.LDL was isolated from a plasma pool of healthy human subjects. Shortly, HOCl modification was performed by coating 100 µl of native LDL, diluted to 100 µg/ml in PBS, in microtitre plates  overnight at 4°C. After washing three times with PBS, half of each plate was incubated for 2 hours at 4°C in 36 µM sodium HOCl  in PBS; the other half of the plate was incubated in PBS. Incubation was followed by a washing with PBS to stop the reaction, and plates were stored in PBS at 4°C for a maximum of 2 h before starting the enzyme-linked immunosorbent assay (ELISA). Wells were then washed five times with a buffer containing 0.01 M Tris, 0.15 M NaCl and 0·05% Tween 20 (pH 8.0) followed by incubation in duplo with mouse plasma in a 1∶50 dilution, 100 µl/well, in a buffer containing 0.1 M Tris, 0.3 M NaCl and 0.05% Tween 20 (pH 8.0) and incubated overnight at 4°C. A positive control was used on each plate to test for intra-assay variation. The next day, plates were washed and incubated with alkaline phosphatase conjugated goat F(ab′)L regions was used as capturing antibody. For the assay, 2 µg/ml of AB1–2 in PBS was coated on microtitre plates overnight at 4°C. After washing and blocking steps, 50 µl of murine plasma diluted by 1∶100 in BSA-PBS was incubated overnight at 4°C. After further washing, captured T15-clonotypic antibodies were detected using an AP-conjugated goat-anti-mouse IgM  diluted in BSA-TBS, and a 50% aqueous solution of LumiPhos 530.For the detection of the natural IgM T15 clonotypic antibodies  in the plasma of mice, a chemiluminescent-based double antibody capture assay was used −/− mice. Collagen positive area was determined from the largest atherosclerotic plaque of each mouse and analysed by expressing the stained surface area as a percentage of the total area of the plaque.Picro-Sirius red staining was analyzed to determine collagen content within lesions in LDLrlasma samples for cholesterol determination were taken before start of the HFD, before collar placement and when mice were sacrificed. All plasma samples were taken after overnight fasting. Plasma cholesterol levels were measured with a commercially available kit (Sigma) following manufacturer’s instructions.All statistical analyses were performed using GraphPad Prism . Errors are shown as SEM and P<0.05 was considered as significant. Plaque volume measurements at the collar were done by a 2-way ANOVA. Differences between groups were analysed using a 1-way ANOVA when several groups were analysed; a two-tailed Student’s t-test or the Mann-Whitney U-test was used when comparing two groups with parametric or non-parametric data, respectively.To create an IgM monoclonal antibody to oxLDL, we immunized mice with human oxLDL. With ELISA techniques, we have selected and up scaled a monoclonal IgM antibody with specificity for HOCl-oxLDL, designated Moab A7S8. This Moab A7S8 was detected to be a pentameric IgM (κ) with no further degradation, which was shown by western blotting techniques by which a protein at approximately 800 kb (data not shown) was detected. The specificity of Moab A7S8 was shown using ELISA in which Moab A7S8 reacted strongly with HOCl-oxLDL and malonaldehyde (MDA)-oxLDL . No reacMoab A7S8 is composed of IgHV1–78*01, IGHJ3*01 and IGHD1–1*02 alleles, with 100% identity and no gaps. Except for N-addition, the alleles are in frame germline sequences, therefore B cell clone A7S8 produces a prototypical natural IgM antibody with an affinity for oxLDL .−/− mice were 8 times injected intraperitoneally with Moab A7S8, control-IgM or PBS. At sacrifice, plaques were located in the proximal area to the collar and in the aortic root. Atherosclerotic plaque formation in the right carotid artery was analyzed using haematoxilin-eosin staining. Treatment of anti-oxLDL natural IgM antibodies resulted in a significant reduced plaque volume formation at the collar , 295 µM2 (SEM 28) and 263 µM2 (SEM 17)  . The redAs shown in At T = −2 and T = 1 (in weeks), no differences could be observed in total IgM titers, HOCl-oxLDL and MDA-oxLDL IgM levels in mice treated with PBS or Moab A7S8. However, after 5 injections (T = 3) of Moab A7S8 antibodies a significant increase in total IgM, HOCl-oxLDL and MDA-oxLDL IgM antibodies could be observed compared to the PBS treated group. The total IgM levels also increased after this point in time in the PBS and C-IgM group, but to a lower extent compared to the Moab A7S8 treated group. Further, it could be observed that the difference between the A7S8 and the control groups were higher for the HOCl-oxLDL and MDA-oxLDL IgM antibodies compared to total IgM. Comparable but more pronounced differences could be observed after 3 more injections (T = 6) .Also, after 3 weeks and 6 weeks of Moab A7S8 treatment, Cu-oxLDL IgM, T15 IgM antibodies and IgM-apoB Immune complexes increased dramatically . These vAt T = −2, T = 0 and T = 1 (in weeks), no differences could be observed in HOCl-oxLDL IgG, MDA-oxLDL IgG and Cu-oxLDL IgG titers. However as also was demonstrated for the IgM anti-oxLDL antibodies, in addition, also IgG titers against HOCl-oxLDL and MDA-oxLDL significantly rose after 3 and 6 weeks of Moab A7S8 treatment .This study demonstrates that passive immunization with Moab A7S8 directed against HOCl-oxLDL results in decreased atherosclerosis development. By injecting Moab A7S8, we observed in our collar model a significant decrease of plaque development compared to PBS treatment. During lesion development, cholesterol levels decreased and anti-oxLDL IgM and IgG titers increased.2O2-catalyzed oxidation of chloride by the enzyme myeloperoxidase (MPO). HOCl is highly reactive and can result in the oxidation of the LDL particle in vivo oxidation pathway since MPO, released from plaque-infiltrating neutrophils and monocytes, is abundantly present in atherosclerotic plaques Several modifications of LDL into oxidized forms of LDL (oxLDL) can occur during atherosclerosis development. Hypochlorite (HOCl) is the product of the HBy VH sequencing, we could determine that our Moab A7S8 is a natural antibody. With their ability to recognize self, altered self and foreign antigens, natural antibodies comprise an important first-line defense against invading pathogens, but are also important for tissue homeostasis −/− mice −/− mice with PC linked to a carrier protein resulted in inhibition of atherosclerosis An athero-protective role of anti-oxLDL antibodies was demonstrated in studies in which treatment with different PC-specific related molecules reduced atherosclerotic plaque development. First, passive immunization with PC-specific T15 IgM antibodies reduced vein graft atherosclerosis in ApoEInterestingly, our control IgM reduced the plaque volume at the aortic root, although this difference did not reach significance. These findings are in accordance with Cesena et al. In our study, we observed a 20% decrease in cholesterol levels in Moab A7S8 treated mice. Decreased cholesterol levels contributed probably to the decreased atherosclerotic plaque development caused by passive immunization with Moab A7S8. We postulate that large amounts of IgM antibodies formed immune complexes with  oxLDL in plasma and resulted in enhanced clearance of oxLDL. During Moab A7S8 treatment IgM-ApoB immune complexes were strongly induced in our mice. In the pneumococcal vaccination study lower cholesterol levels were also found in the vaccinated mice in comparison with the PBS treated mice In conclusion, this study demonstrates an inhibition of atherosclerotic plaque formation by an intervention with Moab A7S8 specific for HOCl-oxLDL. Treatment resulted in decreased cholesterol levels and induction of specific IgM and IgG titers against oxLDL. This evidently shows the active biological consequences of the passive immunization strategy using Moab A7S8 in mice. Further research should be focussed on identification of the antigenic epitope(s) on HOCl-oxLDL which could induce these specific IgM and IgG antibody response against oxLDL. We postulate that therapeutic effects of IgM treatment should be further studied as a potential intervention for human cardiovascular diseases.


This study assessed the efficacy of using visual and auditory biofeedback while immersed in a tridimensional videogame to practice a stress management skill . All 41 participants were soldiers who had previously received basic stress management training and first aid training in combat. On the first day, they received a 15-minute refresher briefing and were randomly assigned to either: (a) no additional stress management training (SMT) for three days, or (b) 30-minute sessions (one per day for three days) of biofeedback-assisted SMT while immersed in a horror/first-person shooter game. The training was performed in a dark and enclosed environment using a 50-inch television with active stereoscopic display and loudspeakers. On the last day, all participants underwent a live simulated ambush with an improvised explosive device, where they had to provide first aid to a wounded soldier. Stress levels were measured with salivary cortisol collected when waking-up, before and after the live simulation. Stress was also measured with heart rate at baseline, during an apprehension phase, and during the live simulation. Repeated-measure ANOVAs and ANCOVAs confirmed that practicing SMT was effective in reducing stress.  American Psychologist by the U.S. Army Chief of Staff, General George W. Casey Mental resilience  Mental resilience draws on factors such as emotion regulation  Increasing resilience through the acquisition of self-regulation or, more narrowly, emotion regulation skills, are common objectives for psychological training programs aimed at military personnel. A few prevention programs have already been developed for soldiers and military personnel to build resilience and reduce psychological injuries: Battlemind Training In Canada, the Programme d'Entraînement à la Résilience Militaire (PERM) has been implemented in the past and has now evolved into the Road to Mental Resilience program In the U.S., one of the first largely implemented mental preparedness programs was Battlemind The main goal of the Comprehensive Soldier Fitness is to enhance psychological resilience in members of the Army community. This program not only focuses on the prevention of psychological disorders in soldiers but also aims at enhancing psychological strengths already present in military personnel The module on master resilience trainers is already being implemented The usual training method used to acquire the emotion regulation skills in the Road to Mental Readiness, the Programme d'Entraînement à la Resilience Militaire and the Comprehensive Soldier Fitness programs relies essentially on didactic seminars and short demonstrations in a classroom or a briefing room. Although the teaching method for the Comprehensive Soldier Fitness may differ slightly from the Canadian program As a set of techniques, SMT has been shown effective to control stress and reduce its negative impact  was developed where soldiers: (a) were exposed to stressful situations through immersions in a horror/first-person shooter game, and (b) could learn to master their skills with the use of biofeedback. Based on previous publications documenting the efficacy of SMT Soldiers were recruited from a list of volunteers provided by the Valcartier military base of the Canadian Forces. A total of 60 participants were initially recruited, but one was excluded (see selection criteria below) and some others dropped-out or their data were lost during the experiment due to technical problems  basic stress management program and (b) basic first-aid training. They also had to be physically and mentally fit for duty and, to reduce the risks of cybersickness, not suffering from vestibular problems, recurrent migraines, epilepsy, postural balance problems, cardiac or ocular problems, frequent and intense motion sickness in transports.Selected participants were randomly assigned to one of two conditions: (a) a “training as usual” control (TAU-Control) condition where no session of supervised practice was offered to participants, or (b) a practice condition (ImPACT) where three daily sessions were offered to practice stress management skills while immersed in a 3D game to induce stress and using biofeedback to inform the participant on their current level of arousal.The sample of 41 participants can be described as mostly single (68%) young man  with junior military ranks of Private (44%) and Corporal (48%). Sixty-two percent of them had been exposed to combat before. Comparisons between both conditions with Student t tests and chi-squared tests did not reveal any statistically significant difference.Upon arrival at the Canadian Forces Medical Simulation Centre for pre-screening (Day 1), participants were informed about the study and their right to refuse to participate. They completed an interview with an experimenter who administered the pre-screening survey, performed the Randot stereotest, and were told how to collect cortisol samples. All participants were gathered into groups to receive a 15-minute refresher briefing on SMT. The briefing focused on tactical breathing, a technique they had already learned in their basic usual training. Participants in the TAU-Control condition were told they would come back only on Day 5 for the final part of the study. Participants in the ImPACT condition remained on site and played the game on a 50-inch monoscopic television (TV) until they learned how to navigate in Left 4 Dead.For participants in the experimental condition, the ImPACT program was delivered on Day 2, 3 and 4 and cortisol samples were taken every day. The training program required participants to wear a system that monitored their heart rate and skin conductance while playing for 30 minutes a modified version of a 3D game. The immersive and biofeedback training was delivered inside two military ambulances located in the medical simulation center. Ambulances were chosen because they allowed the immersion to be conducted in a dark and enclosed environment. Two such ambulance-modified rooms were located about 10 meters from one another and were linked to a cluster of computers allowing participants to work in team (multi-player mode) and a research assistant to supervise all events occurring on the gaming computers and coach's computers. An experimenter, referred to as a coach, sat beside the participant, closed the ambulance door and launched the software. Coaches were experienced retired members of the Canadian Forces. While the game was loading, baseline physiology was recorded during two minutes. During each session, the participant was receiving continuous visual and audio feedback on his stress level. The coach applied the techniques described in the ImPACT trainer's manual. The program is broader than narrow training in tactical breathing. Participants were trained in tactical breathing and also in other associated SMT techniques, such as the recognition of signs of stress, increase in perceived self-efficacy to control stress and biofeedback.As part of the ImPACT program, tactical breathing was presented to the participant as a tool that can be used in at least three types of contexts: (a) before a stressor creates a peak in stress , (b) while stressed and performing his duties , or (c) while stepping back from the task . All three types of contexts were practiced during the training program. The coaches insisted on first recognizing stress as it increases , dressed on with their combat gear, took the pre simulation cortisol sample, entered the darkened simulation area and sat for two minutes to record baseline heart rate. They received their mission, which described a situation in Afghanistan, after sunset, with only a full moon providing limited lighting and an Islamic call for prayer being heard loudly in the background. A fellow soldier was caught in an ambush and they had to help him.After reading the mission, participants stood up in front of a curtain wall hiding the scene until they got the order to proceed. At the exact moment the curtain opened, a very loud explosion occurred from an improvised explosive device hidden in a garbage can and the fellow soldier was severely wounded at the head (blood), chest (open wound with blood), arm (blood) and leg (severely wounded and dismembered prosthetics). Eight medic actors were recruited to perform, one at a time, the wounded fellow soldier. They were randomized across conditions so for each pair of participants, one actor was randomly assigned to the participant in the ImPACT or the TAU-Control condition. The wounded soldier was lying on the floor, covered with blood, begging for help and suffering from severe pain. The very large room was filled with smoke and a second improvised explosive device exploded while the participants were providing first aid medical assistance. After 10 minutes, the simulation ended and participants took the post-simulation cortisol sample and completed the self-efficacy scale.The ImPACT program included a software system composed of: (a) a stressful game to be played by the participant, (b) a visual and audio feedback for the participant, (c) a biofeeback recording system to be used by the coach, and (d) a graphical user interface allowing the coach to fine-tune the biofeedback while the session was progressing. The stressful game was a modified version of “Left 4 Dead” . This horror/first-person shooter game was found in a previous study Feedback on arousal was provided in part with visual feedback. As participant's arousal was increasing, the display of the game on the TV was reduced with a red texture that partially obstructed the field of view, up to a point where only a small oval portion of the center remained visible. The field of view was not totally obstructed in order to avoid masking the action and stirring a sense of loss of control, and yet it interfered sufficiently with the game to require corrective actions from the user. It also illustrated the concept of tunnel vision that is associated with extreme stress The biofeedback recording system used by the coaches allowed visualizing in real time on a graph the evolution of the participant's heart rate and skin conductance. It used two channels/screens provided by the Biograph Infinity software from Thought Technology. This information was displayed on the coach's laptop computer and could be used to detect problems with the biofeedback device and, at the end of the training session, showed to participants' changes in their physiology.The graphical user interface provided on the coach's laptop, next the physiology level, allowed fine tuning the biofeedback delivered to the participant. By referring to a 2-minute baseline recorded while loading the game, both physiological parameters were integrated to provide feedback on arousal. The coach could adjust in real time: (a) the sensitivity of the feedback, (b) the relative weight of the heart rate versus the skin conductance, (c) the baseline level in order to maximise the chances participants could benefit from the biofeedback , and (d) the difficulty level of the game . At the end of the immersion, the weight and sensitivity settings were saved to be available for the following training sessions.The graphical user interface interfaced with Biograph Infinity and Left 4 Dead via a Direct X wrapper programmed in C# to create seamless, timely interaction between the programs. By setting a TCP connection from the host to the client running Left 4 Dead, the data was sent over the network to the appropriate computer running Left 4 Dead, and the modifications made to Left 4 Dead would take effect. Based on available literature on biofeedback By default, the heart rate and the skin conductance were given an equal relative weight “W” and the sum of both physiological parameters was multiplied by a constant “c” to allow increasing the sensitivity of the feedback.Based on Bouchard et al. In order to test the ability to perceive binocular disparity in distance from static stimuli (stereoscopic vision for depth perception), the participant performed a stereopsis test .The main measure of stress in this study was the concentration of salivary cortisol. Free cortisol response collected in the saliva is a reliable measure of stress and, although physical exercise can increase concentration of cortisol in the saliva Additional measures were also collected to document information relevant to the use of the program. To assess free cortisol levels, salivary samples were obtained with Salivette™ collection devices (Sarstedt AG & Co). Participants put the cotton sponge swab (without citric acid infusion) directly in their mouth, chewed and rolled the sponge around in their mouth for two minutes and spitted the sponge back into the tube without ever touching the tube. The tube were wrapped in Parafilm® M self-sealing laboratory film and identified by with participant's ID, date and time. Salivary sample were taken at awakening, before and after stressors . The samples were stored and frozen before assaying. The biochemical analysis of free cortisol in saliva was performed with a competitive immunosorbent assay  using a Sorvall Legend X1R™ centrifuge from Thermo Scientific (Thermo Fisher Scientific Inc) and BioTek ELx800™ microplate reader with GEN 5™ software (BioTek Instruments Inc).Heart rate was monitored and recorded using a ProComp Infinity™, a wireless-enabling Tele-Infiniti™ Compact Flash T9600 and a wireless EKG receiver from Thought Technology. The electrocardiograph sensor for heart electrical activity was a Polar T31™ wireless transmitter belt from Polar. Skin conductance was also monitored; not as an outcome measure but for biofeedback in the ImPACT program. Skin conductance electrodes were put on the participant's annular and auricular fingers of the hand manipulating the computer mouse. The ProComp Infinity™ sampled the data at 256 Hz. While data was acquired and recorded during the simulation on Day 5, a research assistant double checked that the wireless signal remained good and manually put markers in the series of physiological data at the instant events were starting and ending in order to define four separate blocks of heart rate data: (a) Baseline (2 minutes), (b) Apprehension 1 , (c) Apprehension 2 , and (d) Simulation per se (10 minutes).Heart rate data was analysed with the Biograph Infinity™ software version 5.1.0 and the Physiology Suite™. The heart rate data of 16 participants was highly erratic during the baseline or the simulation and, to compensate, the built-in algorithm of the Biograph Infinity™ software automatically generated a fixed heart rate . In five cases, the original data recorded by the ProComp™ could be salvaged with an, at the time, unreleased version of the Cardio Pro Infinity™ software and guidance from staff at Thought Technology. However, data from 11 participants see  could noPerceived self-efficacy The Trainees Evaluation Sheet was completed by professional military medical instructors while soldiers were delivering first aid during the live simulation. It consisted in a list of six essential tasks that had to be performed according to standard training protocol for delivering first aid in combat, otherwise the wounded patient would most likely not survive. It included: (a) assess the scene for safety, (b) assess breathing and correct it if needed, (c) assess blood circulation and correct it if needed, (d) identify appropriate treatment for the chest wound, (e) apply appropriate treatment for the chest wound, (f) assess patient's evolution and adapt to it if necessary. During the simulation, a medical instructor from the simulation center (blind to the participant's condition) remained less than a meter from the participant and continuously assessed the participant's performance in the application of the medical protocol with the wounded soldier. The simulations were video recorded with 16 cameras to allow reviewing the performance so medical instructors could revise their assessment if needed. Medical instructor's assessment were reviewed in a meeting after the experiment to cross-check the ratings. The Trainees Evaluation Sheet was expected to provide behavioural indices of stress. The percentage of participant who performed adequately each behaviour was calculated, as well as the number of participant who did a perfect performance .The Simulator Sickness Questionnaire The ImPACT coach checklist was completed by the coaches immediately after each participant's session. It included nine items about elements of the program that had to be addressed with the participant  and three items documenting the use of the controls in the graphical user interface .A 13-item survey investigated how much participants in the ImPACT program appreciated the experience and how useful they consider the program. Only descriptive results were analysed with this survey.Parametric inferential statistical analyses  were performed, except when variables were categorical . Normality of distribution, as measured with the Kolmogorov-Smirnov  and the Shapiro-Wilk  statistics, was a problem for the cortisol data only. A log transformation corrected the problem and was therefore used for the analyses. Data on the SSQ were skewed and not normally distributed. Since a Wilcoxon non-parametric test essentially confirmed the results of the repeated ANOVAs and this measure is reported only for descriptive purposes, the parametric analyses are reported.fCortisol was defined as the principal dependent variable. To control the error rate, a family-wise approach was taken To control the impact of experience in the military, all parametrical inferential comparisons for the efficacy of the program included the number of years in the military as covariable. It allowed generalizing the impact of the program independently from the number of years of duty and experience in combat, in applying first aid and in receiving SMT training seminars. To control for the impact of physical activity generated by providing first aid in an ambush  on cortisol, the maximum heart rate experienced during the simulation was used as a covariable in the analysis of cortisol response.The present study had been approved by the Defence Research and Development Canada Ethics Committee and the Ethics committee of the Université du Québec en Outaouais. All individual participants in this study gave written informed consent prior to their participation and were free to withdraw from the study without prejudice. = 7.31, p<.017, η2 = .17, Effect size = very large]. Both the Time  and Condition main effects  were not significant.When comparing efficacy of the ImPACT program on the main measure of stress, there was a significant difference in cortisol response see  document = 4.81, p<.017, η2 = .11, Effect size = almost large] during the apprehension phases. The Time main affect was significant  and the Condition main effect was not . The contrasts confirmed the superiority of dealing with stress following the ImPACT program compared to TAU-Control when comparing the baseline to the first apprehension phase  as well as the second apprehension phase .  = .65, p = .43, ns, η2 = .02, Effect size = small]. The Time main effect was significant , but not the Condition main effect .Heart rate was used to corroborate cortisol findings, although the physical efforts inherent to providing first aid in combat situation could blur the heart rate results. The analyses were performed independently for heart rate during the apprehension phases and the actual simulation phases. As shown in  = 15.8, p<.025, η2 = .31, Effect size = very large] and no significant Time and Condition main effects.Ratings of perceived self-efficacy to control stress were quite similar at the start of the study  but changed at the end . The repeated measures ANOVA revealed a significant interaction , session 2  and session 3 .The side effects induced by the immersions were small see  and did  = 11.01, p<.001] and skin conductance  when comparing participants' physiology 10 seconds before and after the beginning of the stressor.When systematically reviewing if each component of the program was implemented as intended in the manual, results confirmed the integrity of program delivery. In general, 100% of the content was delivered to each participant at each session, except on two occasions. Only once, during session 1, where a coach forgot to mention the contexts where tactical breathing could be applied and that stress management should be practiced with real stressors. After the last day of training, participants completed a survey describing how much they appreciated the program and its components. The descriptive statistics are reported in Stress management training encompasses a broad range of techniques that are used either very specifically to regulate emotions or as part of broader mental fitness and resilience programs. Acquiring the psychological skills to cope with stress requires practicing under stressful conditions and coaching. The innovation in the current study is not the demonstration that SMT is effective to reduce stress. This has been demonstrated in hundreds of studies  In order to corroborate the cortisol findings and trying to tease out if the program was effective both in times of apprehension and action, heart rate was examined during the simulation. Heart rate of participants who received only the default training increased significantly more while being briefed on their mission than the trained soldiers, as well as while waiting for the action knowing that a crisis was about to happen in a few moments and their performance would be formerly evaluated. Comparisons with results found in other studies suggest that, compared to physically trained men undergoing the TSST, our participant's heart rate was higher in both conditions during the apprehension phase and the simulation phase. Heart rate was also higher than in previous study The program had a statistically significant impact on medical actions performed by participants during the simulation. Significantly more participants treated adequately the patient's chest wound after having received the ImPACT program. The overall quality of the trainee's performance was much better, with perfect application of the medical protocol in 45% of the participants in the ImPACT condition compared to 10% in those in the TAU condition. This difference was not statistically significant when applying our stringent correction for the number of statistical analyses performed, although the probability was lower than .05. A slightly larger sample, or measuring a few less variables, would have made this difference significant. Interestingly, it appears that differences were more important in behaviours that required more cognitive attention. The program also had a clear positive impact on perceived self-efficacy to control stress. After the simulation, participants trained with the program showed an increase in self-efficacy while those in the control condition showed a decrease. Since self-efficacy is a very strong predictor of behavior change Data gathered during the application of the program per se suggests the immersions did not induce significant negative side effects. Interpretation of this result is still difficult given the skewed distribution of scores and the increase in some side effects likely due to anxiety instead of simulator sickness Not all results unanimously confirmed the efficacy of the program. The advantage of the program over training as usual was not significant when comparing heart rate during the simulation. The analyses revealed a very large and significant increase in heart rate, without meaningful differences between the conditions. It is suspected that heart rate, which was on average around 130 beats per minutes, may have been high in part because of the physical efforts required during the task. Yet, it remains possible that SMT was either: (a) not practiced in situations that were stressful enough for the participant's mastery of the skills to transfer adequately to very demanding stressors; (b) participants may not have applied SMT at all during the very demanding stressor; or (c) the program was not effective. These alternative hypotheses remain to be demonstrated. If the ImPACT program had been effective only for moderate stressors such as apprehension, and not the rest of the live simulation, the cortisol response would not have been so strongly supportive of the program's efficacy. Given the duration of the live simulation and the high heart rates, it would have most likely blunted the cortisol response and the effect of the program would not have been large and significant. As for being ineffective, this is highly unlikely given the significant difference in salivary cortisol and previous findings from the literature Further research is needed in at least three areas: (a) test the outcome of the ImPACT program on behaviors observed in other stressful situations, such as on a shooting range, when under enemy fire, dealing with hostile reactions from civilians, etc. The most recent and comprehensive stress management program developed in the US for the military population is deeply rooted in concepts such as resilience, positive psychology and self-regulation. The ImPACT program blends well with this approach since trainees are given the opportunity to practice and master a skill that fosters emotion regulation. It is not explicitly focusing on negative emotions, the prevention of mental disorders or building mental toughness. It could complement psychological resilience programs by offering the opportunity to practice under stress, which is hardly feasible otherwise. The costs are minimal and the buy-in factor is strong. The ImPACT program could also be used to complement training program used during military survival school To conclude, it is doubtful that the classical approach to SMT in the military, which is essentially based on providing information and teaching techniques in a classroom, would be sufficient to result in significant mastery of the techniques. Practice is essential; yet it may be insufficient unless it is performed under stress. Objective information about level of arousal and immediate feedback about the impact of the techniques can greatly improve learning. Soldiers should also be interested by the available learning strategy despite the strong masculine standards found in the military culture


The goal of this study is to examine scores on psychological measures and salivary cortisol level in these individuals both before and three months after the earthquake. The participants had been measured for these variables before the earthquake. After the earthquake, we carried out PTSD screening using CAPS for participants for another experiment, and then again conducted the aforementioned tests. We collected saliva samples from all survivors. Our results show that social relationship scores on the WHO-QOL26, negative mood scores of the WHO-SUBI, total GHQ score, POMS confusion scores, and CMI emotional status score after the earthquake showed scores indicating significantly decreased compared to before the earthquake. On the other hand, salivary cortisol levels after the earthquake was significantly increased compared to before the earthquake. Moreover, the result of a multiple regression analysis found that negative mood score on the WHO-SUBI and social relationship score on the WHO-QOL26 were significantly related to salivary cortisol levels. Our results thus demonstrate that several psychological stress induced by the earthquake was associated with an increase in salivary cortisol levels. These results show similar findings to previous study. We anticipate that this study will provide a better understanding of posttraumatic responses in the early stages of adaptation to the trauma and expand effective prevention strategies and countermeasures for PTSD.On March 11, 2011, the Pacific side of Japan’s northeast was devastated by an earthquake and tsunami. For years, many researchers have been working on ways of examining the psychological effects of earthquakes on survivors in disaster areas who have experienced aftershocks, catastrophic fires, and other damage caused by the earthquake On March 11, 2011, at 2:46 pm, the Higashi-Nihon Earthquake (the Great East Japan Earthquake), the largest disaster to occur in Japan since World War II, hit three prefectures  on the Pacific side of northeastern Japan, with lesser damage in several other prefectures. These regions were severely damaged by the magnitude 9 earthquake and ensuing tsunami. The coastal communities of Kesennuma, Ishinomaki, and Minamisanriku were particularly devastated by the earthquake. More than 20,000 people were killed or went missing as a result of the tsunami and fires after the earthquake, and over 240,000 homes were damaged or destroyed. Immediately after an earthquake, people affected are damaged both physically and emotionally. Even now, the region is fighting intermittent aftershocks.Over the past dozen years or so there have been several serious disaster events in Japan, including the Hokkaido Nansei-oki Earthquake of 1993, the Great Hanshin-Awaji earthquake of 1995, shocking cases such as the sarin gas attack on the Tokyo subway system in 1995, and so on. Many researchers have studied survivors’ mental health and physical changes after such events Previous studies have reported that based on individual difference and the type of disaster, the rate of psychiatric disorders among survivors either decreased after the second year or was prolonged and became chronic With regard to the relationship between stress and the body, Cannon (1929) and Selye (1956) provide the foundation for the current interest in this physiological phenomenon In this study, we examined that these changes by collecting samples before the earthquake and three months after the earthquake. We hypothesized that psychological measures regarding the mental health of survivors, such as depression, anxiety would worsen and the saliva cortisol level of survivors would increase, three months after the earthquake, compared to before the earthquake. Our hypothesis was based on the previous studies mentioned above.Written informed consent was obtained from each subject, in accordance with the Declaration of Helsinki (1991). This study was approved by the Ethics Committee of Tohoku University.The study was performed three months after the Great East Japan Earthquake occurred. A total of 14 participants  were recruited from among undergraduate and postgraduate students at Tohoku University. All participants experienced the Great East Japan Earthquake. They live in Sendai and the surrounding areas, including the areas closest to the epicenter of the earthquake. All participants had participated in past psychological experiments and saliva cortisol experiments conducted in our laboratory, and had undergone psychological tests and saliva cortisol tests within the past six months and before the earthquake . All participants were screened for absence of neuropsychiatric disorders using the Mini-International Neuropsychiatric Interview (MINI) To assess any change in quality of life, happiness, and mental health of participants from before to after the earthquake, we administered the following questionnaires to participants.The World Health Organization Quality of Life 26 (WHO-QOL26) is a 26-item self-report measure designed to assess quality of life (QOL). Twenty-four items measure the four domains of QOL–physical, psychological, social, and environmental–and the other two items measure overall QOL and general health. The score for each question ranges from 1 to 5; higher scores reflect higher QOL. This study used the Japanese version of the WHO-QOL26, which was created by Tasaki and Nakane (1997) The World Health Organization Subjective Well-being Inventory (WHO-SUBI) is a 41-item self-report measure designed to assess subjective well-being The General Health Questionnaire (GHQ30) is self-report measure designed to assess underlying psychological distress. This questionnaire comprises 30 questions covering a range of neurotic symptoms, with an emphasis on those typical of anxiety and depression and with a deliberate avoidance of those that might also reflect physical illness The Profile of Mood States (POMS) is a 65-item self-report measure designed to assess seven aspects of mood . Factor analyses by the developers failed to confirm the friendship domain, and although the guidelines for administration that were followed in this trial continue to include the seven friendship items, they are no longer reported as a POMS subscale or included in the TMD score The Cornell Medical Index (CMI) consists of 18 sections and 195 items. The A–L sections (144 items) represent physical state and the M–R sections (51 items) represent mental state. Participants answered “yes” or “no” to indicate the presence or absence of a symptom or disorder. If the answer was “yes,” it indicated that the patient had symptoms and received a score of 2. On the other hand, a “no” answer indicated that the patient had no symptoms and was scored at one point We collected saliva samples from participants to measure salivary cortisol level. In consideration of the circadian cortisol rhythms of the participants, we collected saliva samples across the board at 5 pm on weekdays, both pre-examination (before the earthquake) and post-examinations (after the earthquake). The reason we selected 5 pm is because people at this time of day are less affected by circadian cortisol rhythms Saliva samples were collected using the salivette  and centrifuged at 3,000 rpm for 5 minutes. We stored the supernatant solution in an airtight container at minus 80 degrees and measured cortisol using the solution. We measured the cortisol with a semi-microcolumn High Performance Liquid Chromatography (HPLC) system . For reagents, we used cortisol and cortisone .The acetonitrile and methanol used were those available on the market. As for the standard solution of CS and CZ, the CS and CZ were dissolved in methanol so that 0.1 mg/ml each  was used as the original liquid; this was then diluted with 100% methanol or 10% methanol  to be the standard solution used. As for the semi-microcolumns, we used the Capcell Pak MF Ph-1 (4.6 micrometer i.d. times 50 mm) from Shiseido Co., Ltd. as the semi-microcolumn for the preprocessing column, Capcell Pak C18 UG120 (1.5 micrometer i.d. times 250 mm) of Shiseido Co., Ltd. as the semi-microcolumn for the analytical column, and Capcell Pak C18 UG120 (2.0 micrometer i.d. times 35 mm) of Shiseido Co., Ltd. as the semi-microcolumn for the concentrating column.For HPLC analysis, we set the following conditions: the mobile phase for preprocessing used 5 millimoles per liter phosphoric acid buffer solution (pH = 6.9) and acetonitrile at a ratio of 98/2, and the solution was sent at the flow rate of 1 ml/min. The mobile phase for measurements used 10 millimoles per liter phosphoric acid buffer solution (pH = 6.9) and acetonitrile at a ratio of 78/22, and the solution was sent at the flow rate of 0.1 ml/min. The column temperature was kept constant at 35 degrees Celsius with the detection wavelength set at 242 nm. The duration of salivary cortisol levels was 50 min per sample.In this study, participants were measured within subject, pretest (before the earthquake) and posttest (after the earthquake). Both of these experiments followed the same procedure. Participants were tested in the lab of university at weekday afternoon. Participants received experimental suggestions from experimenter and were answered all psychological measures prepared by experimenters in the time given. Participants were allowed 60 min as response time to psychological measures. Then, participants took a saliva sampling at 5 pm. Saliva sampling time was for about five minute. Total experimental time was 65 minutes.p<0.05.The statistical analyses of psychological and salivary cortisol data between participants the high PTSD score group and the low PTSD score group were analyzed using the statistical software PASW Statistics 18 for Windows . Pre- and post-levels of measured variables were analyzed by a paired t-test, and exacerbating factors in salivary cortisol were analyzed by multiple regression analysis. We set the significance level at First, we examined sex differences in salivary cortisol level, because several previous studies have indicated that it shows a sex difference in stress response t = 2.166, degree of freedom [df] = 13, p = 0.05; negative score of the WHO-SUBI: t = 3.183, df = 13, p = 0.01; social relationship score of the WHO-QOL26: t = 2.222, df = 13, p = 0.05). Emotional status per the CMI and confusion score per the POMS also significantly increased .Next, the comparison results for the data obtained from these tests are shown in t = −2.745, df = 13, p<0.05).Next, we measured cortisol levels from after the earthquake. The results of the comparison of salivary cortisol level before and after the earthquake are given in To pinpoint the exacerbating factors in salivary cortisol level, a multiple regression analysis was conducted using the difference between salivary cortisol level before and after the earthquake as the objective variable. The CAPS score, positive score on the WHO-SUBI, negative score on the WHO-SUBI, and the social relationship score of the WHO-QOL26 were used as explanatory variables. The results of the multiple regression analysis are shown in To investigate psychological changes among the survivors in the disaster area before and three months after the earthquake, we used psychological measures and salivary cortisol level. The results show that salivary cortisol levels increased after the earthquake. The exacerbating factors in this increase were negative scores on the WHO-SUBI and (higher) social relationship score of the WHO-QOL26. Our findings were consistent with the results of previous studies, such as those on the Hanshin-Awaji earthquake of 1995 and the Wenchuan earthquake of 2008 which were associated with higher cortisol level Some exacerbating factors in salivary cortisol level may have included anxiety as a result of the disruption of access to daily information, damage to homes or businesses, and lifeline damage such as interruption of water supply, gas supply, or electrical power supply. Another possible factor was logistical disruption and resultant shortages of food, drinking water, and gasoline. Many people who were in the disaster still spend their days feeling stress, fear, fatigue, helplessness, disappointment In the way of limitations of this study, four points should be noted. 1) The sample size of this study was very small. This is because the psychological and saliva cortisol data from before the earthquake were conducted for the purpose of collecting baseline data for other preliminary experiments, and the number of participants in the preliminary experiment was 14. This study compared the data of these 14 participants before the earthquake and three months after the earthquake. 2) We could not consider gender-differentiated salivary cortisol levels or psychological measures, because the numbers of men and women in participants was very small and because the salivary cortisol levels of participants showed no significant difference by gender. 3) Because this study compared data before the earthquake three months after the earthquake, we did not use new depression scales such as the Center for Epidemiologic Studies Depression scale (CES-D) or the Self-rating Depression Scale (SDS). 4) We did not check or consider coping behavior or resilience of survivors, for the same reason as in point 3.In the future, we plan to examine the following three points: 1) First, we will examine change in activity of natural killer cells before and after the earthquake, because these cells are associated with cortisol levels and immune activity in stress conditions In conclusion, more than half a year after the earthquake, rebuilding efforts in the disaster area are very slow. Many survivors still have to confront painful situations such as life stress after the earthquake, disparity between survivors in the disaster area, employment problems, and the effect of radiation. Especially in the worst-affected coastal area, survivors have become extremely unstable. Our overarching mission is to assess about change over time in physical and mental health of many people damaged by earthquake using scientific methods.