Government-led pollution prevention programs tend to focus on large businesses due to their potential to pollute larger quantities, therefore leaving a gap in programs targeting small and home-based businesses. In light of this gap, we set out to determine if a voluntary, peer education approach led by female, Hispanic community health workers (promotoras) can influence small and home-based businesses to implement pollution prevention strategies on-site. This paper describes a partnership between promotoras from a non-profit organization and researchers from a university working together to reach these businesses in a predominately Hispanic area of Tucson, Arizona. From 2008 to 2011, the promotora-led pollution prevention program reached a total of 640 small and home-based businesses. Program activities include technical trainings for promotoras and businesses, generation of culturally and language appropriate educational materials, and face-to-face peer education via multiple on-site visits. To determine the overall effectiveness of the program, surveys were used to measure best practices implemented on-site, perceptions towards pollution prevention, and overall satisfaction with the industry-specific trainings. This paper demonstrates that promotoras can promote the implementation of pollution prevention best practices by Hispanic small and home-based businesses considered “hard-to-reach” by government-led programs. The United States Environmental Protection Agency (US EPA) defines pollution prevention P2) as, “reducing or eliminating waste at the source, promoting the use of non-toxic or less-toxic substances, implementing conservation techniques, and re-using materials rather than putting them into the waste stream” . Formal  as, “redThe unique features of small and home-based businesses need to be considered when crafting P2 interventions to better target these businesses. Small businesses account for 99% of the total employer firms in the USA . Small bLow-wage workers typical of small and home-based businesses are disproportionately foreign-born, Hispanic, and women . Furtheretc.) [Given the challenges stated, P2 strategies need to be developed and customized for these types of businesses. We set out to determine whether a voluntary, peer education approach led by female, Hispanic community health workers (promotoras) can influence small and home-based businesses to change behaviors and apply P2 strategies on-site. In the literature, P2 outreach programs that target businesses are typically government agencies  or university groups that communicate P2 information unilaterally . Some P2etc.) ,15,16. Getc.)  suggest etc.)  determinetc.) , comprehBarriers in the implementation of best practices for long-term sustainability of P2 programs are important to study. These barriers highlight the potential pitfalls that can be considered when implementing a new P2 program. Identified barriers include declining public support, competing business priorities, and difficulty in documenting P2 progress . The lasi.e., politics, ethnicity, language, culture, and economics) partly determines how health disparities develop and how solutions can be applied [What is missing from traditional P2 approaches is a clear understanding of the social constructs in which small and home-based businesses reside. The eco-social context needs to be considered to implement effective P2 measures with long-term effects. A community’s ecology  grant, promotoras working for the Sonora Environmental Research Institute, Inc.  received complaints from community members about unusual chemical odors in their neighborhoods during promotora routine home visits. As part of this CARE grant, SERI developed a community-mapping project to identify high-risk areas based on environmental hazards by having promotoras walk neighborhoods and georeference tag areas of concern using global positioning system units. The maps developed showed the large number of small and home-based businesses in and around the neighborhoods and overlapped this information with the potential hazards based on the chemicals used at the businesses. Subsequent business interviews conducted by promotoras indicated a shortage of language and culturally appropriate information, lack of trust in governmental enforcement agencies, and perceived high cost of “becoming green” as barriers to implement P2 activities . Based oThis initial work pinpointed a community need, but for the program to become more formalized, promotoras needed technical trainings. To address this educational need, new collaborations were formed with academic and government organizations to move the program forward. The resulting multidisciplinary collaboration of academic, industry, and government environmental programs became an essential component of the promotora-led P2 program. Each collaborator’s contribution is reported in detail in the Experimental Section . This prIn Tucson there are an estimated 37,000 registered businesses of which 85%–90% are considered to be small businesses and many of which are Hispanic owned . The proThe promotora-led P2 program consisted of diverse activities that specifically included promotora-focused P2 technical trainings, P2 industry-specific workshops, P2 educational materials tailored to business and language, and face-to-face, peer education visits to small and home-based business. In addition, P2 outcomes and perceptions that were derived from the activities were captured via survey instruments . The University of Arizona (UA) Human Subjects Protection Program determined that this program is exempt due to the use of the data for evaluation and improvement purposes.The P2 technical trainings were developed to be accessible by promotoras and provided real-world applications (connecting basic science with action-oriented examples that could be easily translated to the targeted businesses). They consisted of a formal classroom-style lecture (one to two hours), hands-on activities, question and answer period (15 min), and business tour. Content experts carried out the trainings and all trainings were provided in Spanish (simultaneous translation into Spanish was provided if needed). SERI collaborated with UA’s Department of Atmospheric Sciences, Superfund Research Program (SRP), Dean Carter Binational Center for Environmental Health Sciences , and Arizona Water Institute to strengthen technical P2 trainings for promotoras. Government-based environmental and conservation collaborators housed in Pima County  and the City of Tucson (Tucson Water and Tucson Fire Department) provided additional technical trainings to promotoras as well as literature on existing small business assistance programs. This multidisciplinary component of the program worked to leverage P2 resources, expand the expert pool, and increase the knowledge base of promotoras. No evaluation tools were implemented to assess promotora knowledge gained or readiness.SERI promotoras worked closely with UA SRP and Binational Center to coordinate industry specific workshops for small and home-based businesses. Meetings were coordinated with industry-specific P2 experts to discuss the informational needs and accepted best practices of specific industries. Partnerships were developed with the Automotive Services Association, Printing Industry Association of Arizona, Arizona Lithographers, The Source Beauty Salon and Spa, The Green Edge Group, and Pure Aesthetics Natural Skincare School.At each industry-specific workshop, P2 technical advice, environmentally preferable product samples, and P2 informational packets were distributed. In general, an industry representative involved in either implementing P2 at his/her business or promoting P2 through a professional association delivered the majority of the technical advice. Bilingual (English and Spanish) P2 packets provided suggestions for reducing business waste, factsheets on industry-specific hazardous chemicals, and information on local P2 assistance services. Much of the outreach information was readily available from the US EPA Design for the Environment program, Arizona Department of Environmental Quality’s P2 for Automotive Maintenance and Repair Shops manuals, Occupational Safety and Health Administration safety information, and California Department of Toxic Substances Control P2 materials. This information was not always available in Spanish and lacked cultural nuances. The program translator not only worked with promotoras to develop culturally appropriate linguistic translations, but also had technical expertise that allowed for appropriate science translation. In addition, simultaneous translation into Spanish was provided at each industry-specific workshop.UA SRP and Binational Center partners developed a brief survey to apply at the industry-specific workshops. It was comprised of eight questions consisting of four-point Likert scale, three-point Likert scale, and open-ended. The purpose of this survey was to understand the workshop benefits, potential connections, practical application, and content satisfaction. Surveys that were not completed were not included in the analysis.SERI promotoras partnered with UA SRP and Binational Center to implement small and home-based businesses visits. Technically trained promotoras were the leads during these P2 peer education visits. Promotoras began each business visit by introducing themselves, providing the project goal, and discussing general concepts of P2. They used a survey to collect business information and observations . The survey also allowed promotoras to document their observations of the businesses as well as highlights from their conversations.The follow-up visit survey was structured to gauge the business’s progress towards implementing P2 practices, plans, and policies, obtaining measureable emission reductions, and utilizing the industry-specific packets and/or samples distributed during the initial visit. This follow-up survey was designed similar to the initial business visit survey, containing questions in the form of a script and checklist (yes or no). When an in-person follow-up visit was not feasible, the promotoras called the business and conducted the survey over the telephone.During follow-up business visits, behavior changes were self-reported and used to determine emission reductions. The emission reduction calculations for nail salons were based on average use of acetone per station. The calculations for solvent degreasers at auto repair shops were based on estimates for uncontrolled organic emissions from cold cleaner units.i.e., an established procedure for a given operation, can reduce emissions by 15%–45% [Control devices (methods and procedures used to reduce or prevent emission of pollutant) can reduce emissions 13%–38%, while operational procedures,  15%–45% . Reducti 15%–45% . A conseWe set out to determine whether the promotora-led P2 program, a voluntary peer education approach, would influence Hispanic small and home-based businesses to change behaviors and apply P2 strategies on-site. Survey instruments applied to both the small and home-based business visits and industry specific workshops allowed for analysis of outcomes. The results have been divided into the P2 activities mentioned A total of 17 SERI promotoras participated in basic P2 trainings for the auto repair, printing, and nail salon industries. Fifteen additional technical trainings were designed for these promotoras that included environmental exposure, contaminant transport of industrial solvents, air quality, water conservation, and business-specific P2 best practices .The promotora-led P2 program completed a total of 11 industry-specific workshops for auto repair, hair and nail salons, and printing and lithography. A total of 313 business representatives participated in these workshops of which 74 requested on-site visits. Eight industry-specific packets were developed that targeted auto repair, hair salons, woodworking, business offices, nail salons, dry cleaning, print shops, and auto body and paint shops.Industry-specific P2 workshop surveys were provided at both the printing and lithography (28 participants) and hair and nail salon (17 participants) workshops. Survey responses highlighted that participants were satisfied with the workshops and that the P2 best practice information provided was relevant to their business. Of the 17 attendees of the hair and nail salon workshop, 11 stated that they were going to switch to ammonia-based hair dye and all printing and lithography workshop attendees reported that they would implement P2 suggestions. Information on whether these businesses actually implemented P2 strategy was not collected.Of the 17 technically trained promotoras, 10 of them conducted business visits and became actively involved in the P2 program. These participating promotoras engaged a total of 640 different small and home-based businesses in face-to-face peer education through initial visits. Of the 640 businesses, 105 were initially contacted under the CARE grant. After promotoras received technical training under this P2 program, they revisited these 105 businesses to share their newly acquired P2 technical knowledge.In addition to the initial business visits, 72 businesses received follow-up visits and 493 received follow-up telephone calls. As already mentioned, eight industry-specific packets were developed for the targeted businesses. A total of 662 of these packets were distributed at either initial or follow-up visits. All businesses received an industry-specific packet during the initial visit, and 22 businesses requested an additional packet during the follow-up visit. Of the total businesses, fewer than 20 were determined to be home-based businesses. Although the number is low, interacting with home-based businesses is noteworthy and was not originally anticipated as a business type. The businesses that participated in the survey identified themselves as managers (52%), owners (32%), or employee/receptionist/worker (14%). Two percent did not identify themselves at all. All businesses that received an initial visit completed the survey. The initial business visit survey revealed that 90% of the businesses were willing to take part in the voluntary promotora-led P2 program. Also, the initial business visit survey identified preferred incentives that would entice them to implement P2 best practices .Qualitative observations were also made during initial business visits. Promotoras detected that businesses did not want to participate in workshops because of the perceived lack of time and value. To encourage participation, promotoras personally visited businesses and invited them to events. Events were scheduled at different times throughout the day, and when possible, events coincided with new air quality requirements. Most small businesses believed that it took money to implement P2 measures and requested grants to purchase equipment. We emphasized low-cost methods that any business could implement and distributed information that demonstrated the cost savings over time; however, we found it difficult to convince many small businesses to purchase new equipment based solely on P2. Promotoras observed that many business owners and managers did not see the value in taking time to develop new plans or polices, as they were busy completing day-to-day operations and in general did not have business policies. As part of the program, examples of business plans were provided that could be easily modify for their facilities.During follow-up business visits, promotoras documented using the survey  any reported or observed P2 changes. When it came to actually actively implementing any P2 practices suggested, 288 (45%) of the total businesses accomplished this goal. These practices ranged from recycling to substitution of products and were variable from business to business. The follow-up business visit survey was used to determine reported emission reductions using recommended best practices provided to the business via the program . A totalFor automotive repair and paint and body shops, 203 businesses reported they began covering solvent degreaser containers when not in use. Covering a cold solvent degreaser and associated drainage facility reduces emissions between 13% and 38% . Using tThe promotora-led P2 program reached 640 small and home-based businesses in Tucson and 288 of these businesses reported implementing some kind of P2 practice that they had learned about through the program. Promotoras who are technically trained can influence small and home-based businesses’ environmental operation decisions. In this program, using the right culture and language, small and home-based businesses were encouraged to participate in the program and as a result became aware of the opportunities that P2 could offer. Through the promotora-led P2 program, businesses trusted both the messenger and the information, making them more amenable to suggested P2 practices. Since the information was “translated” with the community’s ecology in mind, the businesses could both better understand the information and engage in P2 practices more readily. P2 programs in other settings can benefit by applying similar community ecology principles. If promotora groups have not been established, community leaders also known as “sparkplugs” or “champions” can provide the structure needed to develop such a program. The benefits of incorporating a community’s ecology, demonstrated in the Tucson promotora-led P2 program and in the Boston Safe Shops model, are apparent—trusted community members could work successfully with small businesses to promote P2.Program findings reinforce and echo previous P2 findings, specifically the importance of cost avoidance and profitability ,39. It wi.e., follow-up in-depth interviews and content analysis) to obtain information not previously anticipated or code interviews to derive additional conclusions. In addition, more detailed information could have been gathered pertaining to how the promotoras reduced specific barriers and what were the motives for adopting practices after taking part in the P2 program (long-term perspective).Surveys were the primary method of gathering program information and evaluating outcomes. As mentioned previously, these surveys helped gather the perception of participants in the initial and follow-up business visits and the industry-specific workshops. The usefulness of these surveys was limited since only prescribed information was obtained. The program evaluation could be improved by applying a mixed methods approach  can result in improved P2 outreach. Specifically, technically trained female, community health workers can be effective at convincing small and home-based businesses in applying P2 practices on-site and demonstrate that emission reductions can be achieved by low-cost interventions. This type of voluntary approach is complimentary to government-based programs, since it extends the reach of existing resources and infrastructure.We recommend that similar programs should be implemented to fill the gap left by large-scale government programs. This process described here to achieve P2 can assist in decreasing community-wide pollution emissions that are greatly amplified in minority neighborhoods. It is important to keep in mind that this type of intervention is only one piece of the diverse partnerships needed to implement a sustainable P2 culture.


The Graduate Program in Biotechnology and Biodiversity (PPGBB) is a consortium of public and private education institutions of the Center-West Region of Brazil committed to offer academic studies at the doctorate level. The Program was created in 2012 as an initiative of the "Pró-Centro Oeste Network". The coordination of the Program belongs to Universidade de Brasília. The following institutions participate in the Program:- Universidade de Brasília (UnB) - DF- Universidade Católica de Brasília (UCB) - DF- Universidade Federal de Goiás (UFG) - GO- Pontifícia Universidade Católica de Goiás (PUC-Goiás) - GO- Instituto Federal Tecnológico Goiano (IFGoiano) - GO- Universidade Federal de Mato Grosso do Sul (UFMS) - MS- Universidade Católica Dom Bosco (UCDB) - MS- Universidade Federal da Grande Dourados (UFGD) - MS- Universidade do Estado de Mato Grosso (UNEMAT) - MT- Universidade Federal de Mato Grosso (UFMT) - MTThe course has a duration of 48 months and currently there are 50 students enrolled. The faculty is composed of 70 investigators. The Program encourages students from multiple disciplines to experience research in close relation to several fields of biotechnology.Goals of the program:1. To provide graduate education at the doctorate level;2. To promote innovation in the fields of basic science and technology in order to contribute to the sustainable development and to improve the quality of life of the Center-West Region of Brazil;3. To contribute to the local bioindustry through the development of products, processes and services based on the rational use of the regional biodiversity.Lines of research:1. Science, tecnology and innovation for the sustainability of the Center-West Region.2. Bioeconomy and conservation of natural resources.3. Development of biotechnological products, processes and services.http://redeprocentrooeste.org.br/More information on the Program can be found at


Despite over a decade of research and programming, little evidence is available on effective strategies to reduce HIV risks among Central American men who have sex with men (MSM). The Pan-American Social Marketing Organization (PASMO) and partners are implementing a HIV Combination Prevention Program to provide key populations with an essential package of prevention interventions and services: 1) behavioral, including interpersonal communications, and online outreach; 2) biomedical services including HIV testing and counseling and screening for STIs; and 3) complementary support, including legal support and treatment for substance abuse. Two years into implementation, we evaluated this program’s effectiveness for MSM by testing whether exposure to any or a combination of program components could reduce HIV risks.PASMO surveyed MSM in 10 cities across Guatemala, El Salvador, Nicaragua, Costa Rica, and Panama in 2012 using respondent-driven sampling. We used coarsened exact matching to create statistically equivalent groups of men exposed and non-exposed to the program, matching on education, measures of social interaction, and exposure to other HIV prevention programs. We estimated average treatment effects of each component and all combined to assess HIV testing and condom use outcomes, using multivariable logistic regression. We also linked survey data to routine service data to assess program coverage.Exposure to any program component was 32% in the study area (n = 3531). Only 2.8% of men received all components. Men exposed to both behavioral and biomedical components were more likely to use condoms and lubricant at last sex , and those exposed to behavioral interventions were more likely to have tested for HIV in the past year .PASMO’s strategies to reach MSM with HIV prevention programming are still achieving low levels of population coverage, and few men are receiving the complete essential package. However, those reached are able to practice HIV prevention. Combination prevention is a promising approach in Central America, requiring expansion in coverage and intensity. Despite several decades of work , 2, therThat MSM in Central America experience elevated HIV risks is well documented. High levels of unprotected anal intercourse have been reported in Nicaragua and El Salvador. Condom use is frequent, but the picture of consistent condom use is not clear, and in 2013, 60.2% of MSM reported sometimes or never using condoms with their partners , 9. Use HIV risk behaviors among MSM occur in a context of stigma and violence. Central America experiences high levels of generalized violence along with homophobia targeted against MSM and transgender women –15. StigGiven structural factors, such as homophobia and violence, it is difficult to reach targeted populations at impactful levels of coverage and challenging to maintain these contacts. In Central America and elsewhere, the HIV community is increasingly mobilized around combination prevention strategies, recognizing that it is not possible to get traction on the epidemic through isolated efforts that do not address contexts of risk , 19. A cWhile combination prevention is increasingly the consensus framework for HIV prevention, there is as yet little evidence on the effectiveness of different combination prevention strategies . The neeWith support from USAID , the Pan-American Social Marketing Organization (PASMO) and Population Services International (PSI)/Mexico began implementing a Combination Prevention Program for HIV with community-based partners in 2011 across Belize, Guatemala, El Salvador, Nicaragua, Panama, Costa Rica, and Mexico. The program is designed to increase access to HIV prevention interventions in order to reduce risk behaviors among key populations and reduce social hostility that contributes to stigma and discrimination.PASMO has defined an essential package of interventions for MSM following a combination prevention approach . To receThe behavioral component includes integrated behavior change communications activities conducted by outreach workers. Based on the transtheoretical model and Population Services International’s PERForM framework, these activities were designed to generate demand for the program’s products and services, while also encouraging individuals reached to assess their health risks and build skills in HIV prevention –30. ActiThe biomedical component is core to the Combination Prevention program’s design. Services provided include HTC, STI screening and treatment, and for people living with HIV, referrals for care and treatment, including antiretroviral therapy. A variety of service providers participate in the program, including private laboratories, private and NGO clinics, and some public sector facilities. HTC is provided through fixed and mobile services, according to where the program is operating .The addition of complementary services was designed to support program clients to locate resources that help them negotiate potential stigma and discrimination . These sIntegrated HIV prevention teams conduct behavioral interventions and referrals to biomedical and complementary services in identified high-risk zones in program cities. For MSM, these zones are areas with large concentrations of nightclubs, bars, parks and areas where community organizations have identified that MSM are likely to meet each other. Teams include outreach workers, PASMO sales staff responsible for ensuring availability and access to condoms, community leaders, and service providers. When clients are contacted by an outreach worker, they receive an in-person behavioral intervention plus a packet of vouchers for other behavioral interventions, condoms, biomedical services, and complementary services. The program tracks client contacts and use of referral vouchers via unique identifier codes assigned to each client contacted . While cWe aimed in this paper to provide early results on PASMO’s combination prevention program, as targeted to MSM in Central America. Two years into implementing the program, our objectives were to 1) assess population-level coverage of specific and combined intervention components and 2) determine whether program exposure to any or a combination of components was associated with HIV risk reduction behaviors, specifically condom and water-based lubricant use, HIV testing and counseling, and appropriate treatment of STIs.We used behavioral survey data and data extracted from PASMO’s program monitoring system for this analysis.From September-November 2012, PASMO recruited MSM from 10 cities in six Central American countries into a survey using respondent-driven sampling n = 4231). The survey was conducted in areas where the Combination Prevention program operates: Belize City in Belize, San Jose in Costa Rica, San Salvador and Santa Ana in El Salvador, Guatemala City and Quetzaltenango in Guatemala, Managua and Chinandega in Nicaragua, and Panama City and Colón in Panama. These cities were selected because they had on-going program operations and national level evidence indicated high projected HIV prevalence in each city 1. The su. Men werData was collected through in-person interviews administered by a male enumerator in a location that ensured the respondent’s privacy. The survey instrument included questions on socio-demographic characteristics, sexual behaviors, STI symptoms, use of STI treatment and HTC services, mass media and program exposure, knowledge and attitudes regarding HIV prevention, and experience with violence. The PSI Research Ethics Board provided ethical approval of this survey along with Ministries of Health in Belize, El Salvador, Guatemala and Nicaragua. The Panama National HIV/AIDS Program and the University of Medical Sciences in Costa Rica approved the study as well. Individuals were asked to provide informed consent before participating in the interview. No identifying information was collected from respondents.For this analysis, we did not use data from Belize because program operations were not completely comparable. We also excluded transgender women (n = 400 self-identified cases) from this analysis because transgender women are increasingly considered to be a separate population programmatically and epidemiologically . Our finWe also used data extracted from PASMO’s routine program monitoring system. PASMO’s system for applied monitoring (SAM) tracks BCC activities and service utilization across all program implementers in six countries. A key feature of the SAM is the use of unique identifier codes (UICs) to track individual clients across program contacts. Unique identifier codes are based on personal information that does not vary over time and that clients can easily recall. Monitoring data are collected on BCC activities by type of activity and target population reached, messages and behaviors, referrals initiated and completed, plus client use of HTC, and STI treatment, and any referrals to other services, such as CD4 count and viral load testing for people living with HIV. Data on program contacts and service utilization are aggregated by country and reported to partners on a monthly basis. For this analysis, we extracted data from October 1, 2011-September 30, 2012. No identifying information on program clients was collected, and analysis of this data was determined to not constitute research with human subjects. From extraction of routine data, we identified 15031 program contacts across the five countries.Five outcome measures were used for this analysis, all taken from behavioral survey data: 1) condom use at last sex, 2) condom use with water-based lubricant at last sex, 3) consistent condom use in the past 30 days, 4) receipt of an HIV test in the past 12 months, and 5) seeking medical care for a suspected STI symptom in the past 12 months. Consistent condom use was defined as using a condom from start to finish for all sex acts with any partner type for the past 30 days. We measured consistent condom use by partner type for commercial, casual and regular partners. Commercial partners were partners from whom the respondent received money or to whom the respondent gave money for sex. Casual partners were defined as partners with whom the respondent had sex with no emotional attachment and no financial transaction. Regular partners were defined as partners with whom the respondent had engaged in sex for more than 3 months and felt connected to, either by love or friendship.We created several measures of program exposure from behavioral survey data. These measures were designed to capture specific components of the combination prevention approach in order to capture program operations and assess potentially additive effects of the different components. Exposure to behavioral interventions was defined as having been contacted by an outreach worker representing the project. Exposure to biomedical interventions was defined as having received a voucher for HIV testing and counseling, to capture the referral process. Exposure to complementary interventions was defined as receiving a voucher for other services. We defined any exposure to the program as receipt of any one of these components. We also created a measure of having received all three components, defined as the combination package.After extracting individual records from the program monitoring system, we created measures of receipt of behavioral interventions , receipt of biomedical services (y/n and number of services), and receipt of complementary interventions (y/n and number of services) for this analysis.We considered a range of other factors from the survey data for this analysis. We looked at socio-demographic factors of age, education , and an index of socio-economic status. The index of socio-economic status was constructed following methods of the Mexican Market Research Association and grouped individuals into categories of high, medium and low socio-economic status . We alsoWe considered a series of factors assessing men’s social interactions with gay, transgender and other MSM communities. These included: friends’ knowing the respondent’s sexual orientation, having participated in a gay/transgender event in the past 12 months, and going to a gay bar/disco in the past 30 days. We also considered exposure to other HIV/AIDS programming targeting MSM, measured as whether the respondent received a condom for free in the past 12 months. All factors were treated as dichotomous variables.For descriptive sample characteristics, we present un-weighted data. We conducted weighted analyses to assess program effects using multiple logistic regression. Because of our sampling and analysis strategy, we calculated weights using pooled data. Several types of weights were calculated and adjusted for in regression models. First, we calculated RDS weights using RDSAT software, which accounts for network size and recruitment patterns for each city . Second,We next used a statistical matching technique, coarsened exact matching (CEM), to improve the quality of our estimates of program effects –48. Coar-16, indicating minimal imbalance between respondents exposed and not exposed [We matched survey respondents on factors likely to influence exposure to PASMO’s combination prevention program, given the strong likelihood of selection bias in a setting where MSM are highly stigmatized . We matc exposed .We calculated un-weighted descriptive statistics for key measures using country data and pooled data. Descriptive statistics in the pooled dataset were calculated for the full and the matched samples. We next compared distributions of program exposure in the survey data to program exposure from monitoring data by country. We then used weighted multivariable logistic regression to estimate odds ratios of average treatment effects for each measure of program participation, applying all three types of weights . We first estimated unadjusted odds ratios for each outcome of interest. We next adjusted for age and socio-economic status – factors not included in the matching procedure. We also investigated the effects of receiving two combination prevention channels , with and without controls. The same modeling strategy was used in the full sample and with the matched sub-sample to estimate how estimates of treatment effects changed following matching.Finally, we created a new sub-sample of cases where we could link unique identifiers between routine data and survey data. Because of the limited sample size for these linked cases, we did not apply any sampling weights, and we did not match because all cases were exposed. We conducted chi-square tests of association between type of program exposure and the outcomes of interest by country.More than 80% of respondents across all countries were under 30 Table . Half ofOf the outcomes of interest, 85% of men reported having used a condom at last sex, and 48% used a condom with water-based lubricant at last sex. Regarding consistent condom use, 82% reported consistent condom use in the past 30 days with all partners. Consistent condom use was substantially higher when respondents reported having casual partners (90%) and commercial partners (92%). Just over half of respondents reported having had an HIV test in the last 12 months (52%), and of those with STI symptoms, 76% sought medical treatment.Almost a third 32.2%) of respondents reported exposure to any component of the Combination Prevention program , while El Salvador and Nicaragua had the greatest reported contacts with complementary services (both at 3.4%).Table We were able to link a total of 520 unique individuals between monitoring and survey data Table . These cIn light of the need for a larger evidence base on the effectiveness of combination prevention, particularly from Latin America, we have aimed in this study to systematically evaluate a program strategy that seeks to link multiple program components for a priority population in Central America. We aimed to assess population-level coverage of a combination prevention program targeted to men who have sex with men and to determine whether program exposure was associated with HIV risk reduction in this population. We present preliminary evidence here that suggests this strategy is associated with condom use with water-based lubricant at last sex, consistent condom use with regular partners, and HIV testing.Two years into implementation, PASMO’s Combination Prevention program has achieved fairly modest levels of coverage in the target population of MSM, a population that is notably difficult to reach , 55. ConProgram coverage varied across countries, indicating that the program was at different stages of penetration into the target population. Exposure to the behavioral component of the program was most common at a population level, followed closely by exposure to referral for biomedical services Panama and Costa Rica both had relatively lower levels of exposure to the behavioral component, reflecting that these countries were relatively newer sites for program implementation than Guatemala or El Salvador. Variation in biomedical program exposure was likely due in part to differing periods of program operation.The picture of program exposure based on records of contacts from monitoring data is somewhat different. Several countries showed high service utilization, but trends differed from survey data. This suggests issues with under-reporting in the monitoring system, likely due to problems with voucher redemption, and is compounded by likely under-reporting of program exposure through recall bias in survey data. Despite the potential for discrepancies, exposure to the complementary component was consistently low across data sources and countries. The notable exception was Guatemala, where complementary exposure was 45% of exposure amongst survey respondents with any exposure. This suggests that the program was more successful in implementing the voucher mechanism and service linkages in that country.Modalities of program implementation also account for some of the differences in exposure trends. Specifically in Costa Rica, the program operates solely through referrals to the public sector Social Security Institute, following government policy on maintaining HTC services as a public sector service. Rapid HIV tests are not yet available in Costa Rican clinics, which may have influenced outcomes there. Based on coverage levels in other countries, including in Panama where the program also only recently began implementation, loosening policy guidelines on service providers and types of HTC services that can be provided in Costa Rica may be merited to increase the use of HTC.Despite modest levels of coverage and inconsistencies across data sources, there is evidence that participation in the Combination Prevention program was associated with HIV risk reduction. The behavioral component was associated with consistent condom use with regular partners and with using condoms and water-based lubricants at last sex. Both of these behaviors are focuses of messaging during behavioral interactions. Print materials are distributed that demonstrate the importance of using condoms with water-based lubricant, and counselors are trained to talk about lubricant use in pre- and post-counseling. Effect estimates more than doubled for men who had received both the behavioral component and referral to biomedical services, suggesting that a greater dose of exposure was particularly effective at promoting water-based lubricant use with condoms.Based on previously collected data showing comparatively lower levels of condom use with regular partners, compared to with casual or commercial partners, PASMO specifically focused messaging within BCC activities on condom use with regular partners during the period evaluated by this study –61. The There was also evidence that program exposure was associated with HIV testing, particularly the exposure to the behavioral component. The effectiveness of referral to complementary services was attenuated in the matched sample, and having received both program components was associated with an even higher likelihood of HIV testing, but these results were not statistically significant, likely due to the small numbers of men who reported both components. These results suggest that BCC strategies are effectively able to promote use of HIV testing and counseling services. The program’s emphasis on improving quality of care within testing service providers, adapting service hours to be convenient for the population being served, and helping service providers to treat client respectfully likely contributed to these results, along with making referrals to these service providers. Subsequent evaluations of this program should continue to investigate whether service linkages and making services MSM-friendly are able to achieve increases in demand for and use of HTC services. This study was not designed to assess links between HIV testing and care and treatment services, but other data from the Combination Prevention program suggest that the program has contributed to improved access to care and treatment for people living with HIV .After linking cases from monitoring with survey data, we found no evidence that program exposure was associated with any of the outcomes. Again, this was likely due to the rarity of exposure, a result of challenges in locating men exposed to the program during survey data collection and perhaps inconsistencies in reporting of unique identifiers. Future survey research to evaluate this program may be able to address these challenges of recall during data collection.The Combination Prevention program results are aligned with recent evidence from the HIV prevention literature and contribute unique results on condom use with water-based lubricants, especially in regards to the doubling of effect size for men who were exposed to both the behavioral and biomedical components in the program. Several evaluations from large-scale HIV prevention efforts focused on MSM show an increase in consistent condom use with regular male partners, some with all male partners –65. CommThis analysis has several limitations. In the survey data, recall of outcomes may have been biased. However, levels of condom use and of HIV testing were comparable to other reports from the region . ProgramAnother potential study limitation was in the coarsening of variables before matching. The matching procedure we used faces a trade-off in increasing the number of matched pairs at the expense of less exact matching , 51. HowThe measurement approach taken here primarily assesses program exposure and outcomes based on individual-level measurement. It has been argued that combination prevention programs require a multi-level exposure measurement approach or evaluation platform approach to assess the effects of structural factors , 26. We We have early evidence that a combination prevention strategy is associated with HIV risk reduction among MSM in Central America. Additional strategies are needed to expand program coverage into a population that is difficult to reach due to stigma and discrimination. Service linkages appear to strengthen program impacts, but measurement may be hampered by under-reporting. Future evaluations should take a multi-level approach to account for complex program operations and the multiple levels  at which a combination prevention strategy operates.


The purpose of this study was to develop a new Internet-based computerized cognitive behavior therapy (iCBT) program in Manga format, the Japanese cartoon, for workers and to examine the effects of the iCBT program on improving subthreshold depression using a randomized controlled trial (RCT) design among workers employed in private companies in Japan.All workers in a company (n = 290) and all workers in three departments  at the headquarters of another large company were recruited by an invitation e-mail. Participants who fulfilled the inclusion criteria were randomly allocated to intervention or control groups (N = 381 for each group). A six-week, six-lesson iCBT program using Manga (Japanese comic) story was developed. The program included several CBT skills: self-monitoring, cognitive restructuring, assertiveness, problem solving, and relaxation. The intervention group studied the iCBT program at a frequency of one lesson per week. Depression  was assessed as a primary outcome at baseline, and three- and six-month follow-ups for both intervention and control groups were performed.The iCBT program showed a significant intervention effect on BDI-II  with small effect sizes .The present study first demonstrated that a computerized cognitive behavior therapy delivered via the Internet was effective in improving depression in the general working population. It seems critical to improve program involvement of participants in order to enhance the effect size of an iCBT program.UMIN000006210UMIN Clinical Trials Registry  Occupational stress has been considered a major risk factor for a wide range of health outcomes An innovative way to deliver CBT-based treatment widely is with a computerized CBT (CCBT). CCBT programs can teach information and techniques based on the same CBT principles as face-to-face CBT programs do, with a highly structured format comprising educational lessons, homework assignments, and supplementary resources Previous iCBT programs have been shown to have a significant positive treatment effect on depression and anxiety mostly in the clinical setting. A meta-analysis of RCT reported that iCBT can improve depressive symptoms of patients who have major depressive disorder (Hedges' g = 0.56–0.99) and anxiety disorders  more effectively compared with TAU or waiting list Despite a number of benefits of using CCBT and iCBT, the application of these programs in the workplace setting has been limited. Grime (2004) conducted a randomized controlled trial (RCT) to evaluate the effect of an eight-week CCBT program to improve emotional distress among employees who had recently experienced stress-related absenteeism. A month before the end of the intervention, the depression and anxiety among the participants in the intervention group improved significantly compared to that of the TAU group The purpose of this study was to develop a new six-week iCBT program for workers in Japan and to examine the effects of the iCBT program on improving the symptoms of depression at three- and six-month follow-ups using a randomized controlled trial design among workers employed in private companies in Japan. In the iCBT program, we applied a “Manga” (the Japanese cartoon or comic story) format, that is now frequently used to communicate medical information in JapanThe study was a randomized controlled trial. The allocation ratio of the intervention group to the control group was 1 to 1. The study protocol was registered at the UMIN Clinical Trials Registry (UMIN-CTR) (ID = UMIN000006210). The protocol for this trial and supporting CONSORT checklist are available as supporting information; see All workers in company A (N = 290) and all workers who belonged to three selected departments  at the headquarter of company B  were recruited by an invitation e-mail. Both companies developed information technology systems and related services as their products. Those who were interested in participating in the study were asked to go to a research website to obtain full explanation of the study's aim and procedure and to input their baseline information if they agreed to participate. The inclusion criteria stated that participants could 1) not be diagnosed with a major depressive disorder in the past month  Internet CBT program; Useful mental health solutions series for business. The program was a six-week web-based training course to provide stress management skills. This program was structured into six lessons, with one lesson given per week. Learning one lesson required about 30 minutes, including homework. This program can be used anywhere the Internet is available.Participants who were allocated to the intervention group studied the new iCBT program called the One of the unique features of the program was that the training was provided along with a Manga (Japanese comic) story of a psychologist and a client worker to facilitate the understanding of the participants . SeveralIn terms of the CBT components of the program, included were self-monitoring skills, cognitive restructuring skills, assertiveness, problem-solving skills, and relaxation skills. At the end of each lesson, participants were asked to submit homework to facilitate their understanding, although it was voluntary. Participants who submitted their homework received feedback from trained staff .The iCBT program included self-monitoring skills (in lesson 2), cognitive restructuring skills (in lessons 3 and 4), assertiveness (in lesson 5), problem-solving skills (in lesson 6), and relaxation skills (in lesson 4). In this study, a cognitive restructuring method was adopted as the primary outcome and main cognitive approach, and this has been shown to be effective in reducing depression Participants completed six weekly lessons and homework within the iCBT program. They were allowed to complete the six lessons within 10 weeks after the baseline survey. Participants were reminded by e-mail to complete each lesson and/or to submit homework if they had not already done so. Reminders were sent from the research office to the participants every Monday.Participants in the control group received an e-mail message once a month titled “Useful information for stress management.” Each e-mail message consisted of about 500 words in Japanese and included stress management tips. The contents were as follows: 1) How to have a good sleep, 2) The effects of your diet and ingesting alcohol for stress, 3) How to have a good holiday, 4) Relaxing by listening to music and taking a bath, and 5) The effects of exercise on stress. Participants were also able to use an internal employee assistance program service. In addition, an e-learning program on stress and stress management was provided to all employees at company B. This e-learning program contained only a few descriptions of CB-based approaches.The BDI-II is a 21-item self-report inventory that measures depressive symptoms such as sadness, pessimism, suicidal thoughts or wishes, tiredness or fatigue, loss of energy, and loss of pleasure, among others Psychological distress was measured with the Japanese version of the K6 scale The 24-item Dysfunctional Attitude Scale (DAS-24) is a short version of the Dysfunctional Attitude Scale, which is a self-report inventory measuring depressogenic schemata. Each item is scored on a scale ranging from one  to seven , with a higher score indicating a more dysfunctional attitude Respondents were asked to rate their improvement of knowledge and self-efficacy regarding five components of the iCBT program . Knowledge improvement was assessed by asking participants, “How much knowledge do you have about…” and self-efficacy improvement was assessed by asking respondents “How confident are you that you can do….” Both items were scored on 5-point scale ranging from 0 (none) to 4 (enough).Demographic data, such as age, gender, marital status, occupation, education, chronic disease, and overtime hours during the past month were also collected.A systematic review of psychological treatments, mostly CBT for subthreshold depression, yielded a Cohen's d of 0.42 (95%CI: 0.23 to 0.60) at post-test Participants who fulfilled the inclusion criteria were randomly allocated to intervention or control groups (N = 381 for each group). Stratified permuted-block randomization was conducted. Participants were stratified into four strata according to two factors: K6 score (5 or greater or less than 5) at baseline survey and company (A or B) to which they belonged. A stratified permuted-block random table was generated by an independent biostatistician. Enrollment was conducted by a clinical research coordinator, and assignment was conducted by an independent research assistant. The stratified permuted-block random table was password-protected and blinded to the researcher. Only the research assistant had access to it during the work of random allocation.A mixed-model for repeated measures conditional growth model analyses were conducted using a group (intervention and control) * time  interaction as an indicator of intervention effect. Intention-to-treat analysis (ITT) was conducted. The Linear Mixed Model in the PASW Statistics 18.0 was used. The Number Needed to Treat (NNT) to reduce depressive symptoms or psychological distress to achieve one improvement from subthreshold depression was calculated. Referencing the cutoff scores of BDI-II and K6 of previous studies The Research Ethics Review Board of Graduate School of Medicine, the University of Tokyo (No. 3083) approved the study procedures. We prepared a website which contained full explanation of the study. Before the baseline survey, participants were invited to read the explanation on the website, and asked to click an “agree” button to show their consent to participate in the study; then they proceeded to the baseline questionnaire page. Written consent was not required by the National Ethical Guidelines for Epidemiologic Research, Japan; the Research Ethics Review Board of Graduate School of Medicine, the University of Tokyo approved this procedure to obtain participant's consent.One major change made to the protocol is the inclusion criteria. Originally it was planned that the participants who scored 5 or more for the K6 on the baseline survey would be allocated. Before the commencement of this study, this criterion was canceled due to the anticipation of a low participation rate. With this, sample size was recalculated. Another change made to the protocol is the method of statistical analysis. Originally the per-protocol analysis was planned, but the ITT analysis was conducted in the present study due to the expectation to be conservative in superiority trials. In addition, NNT was calculated due to the description of the difference between a treatment and a control in achieving a particular outcome.Participant flowchart is shown in At 3-month follow-up, 270 (70.9%) participants in the intervention group and 336 (88.2%) in the control group completed the follow-up survey. At the 6-month follow-up, 272 (71.4%) participants in the intervention group and 320 (84.0%) in the control group completed the follow-up survey. At each follow-up, the response rate of the control group was higher compared to that of the intervention group. Reasons for dropping out were not assessed in this study.Recruitment and the baseline survey were conducted during the period from September to October 2011. The intervention and control groups were assessed at approximately three months (from December 2011 to January 2012) and approximately six months (from March to April 2012) after the baseline survey.Demographic characteristics are presented in The iCBT program showed a significant effect on DAS  and all knowledge and efficacy variables (p<0.05) except for the efficacy of problem solving. The program showed a marginally statistically significant effect on K6  and efficacy of problem-solving . Effect sizes for K6, DAS, and improvement of self-efficacy were small, while effect sizes for knowledge variables were medium.Among respondents at the 3-month follow-up, 32.1% and 20.2% had a remission from subthreshold depression defined by BDI-II and K6, respectively. At the 6-month follow-up, 30.1% and 19.7% of respondents had a remission from subthreshold depression on BDI-II and K6, respectively. No significant difference was observed in the remission based on BDI-II between the intervention and control groups: relative risk (RR) of having subthreshold depression in the intervention group were 0.79 (p = 0.22) at the 3-month follow-up and 0.77 (p = 0.20) at the 6-month follow-up; the NNTs to achieve one remission from subthreshold depression were 14 at the 3-month follow-up and 13 at the 6-month follow-up. The probability of the remission based on K6 was not significantly different between the groups at the 3-month follow-up  and was marginally significantly higher in the intervention group than in the control group at the 6-month follow-up ; the NNTs were -279 at the 3-month follow-up and 12 at the 6-month follow-up.The subgroup analysis was conducted for participants who scored 5 or more on K6 at the baseline. The iCBT program showed a significant effect on DAS , all knowledge variables, efficacy of stress management, assertive communication, and relaxation training (p<0.05). While the effect sizes of DAS and self-efficacy were small, those for variables on knowledge were medium.The present RCT examined the effects of a newly developed iCBT program for improving the symptoms of depression and other outcomes at three- and six-month follow-ups among workers employed in private companies in Japan. In the total sample, the iCBT program showed a significant intervention effect on depression, as measured by BDI-II, with the effect sizes of −0.16 at the six-month follow-up. The iCBT program also showed a marginally statistically significant intervention effect on psychological distress measured by K6 and significant intervention effects on improving dysfunctional attitude, knowledge, and self-efficacy of most components of CBT. This iCBT program may effectively improve depressive symptoms among workers with the universal approach , even though the effect size may be modest.To our knowledge, the present study first demonstrated that an Internet-based computerized CBT was effective in improving depression in a non-clinical working population. In the intervention group, BDI-II scores improved at the three-month follow-up and returned to the baseline level at the six-month follow-up. In the control group, BDI-II scores stayed at almost the same level at the three-month follow-up and increased slightly by the six-month follow-up. This difference yielded effect sizes of −0.14 and −0.16 on BDI-II scores at the three- and six-month follow-ups, respectively. After the baseline survey, one of the two companies that participated in the study merged with another company. The observed increase in depression in the control group was partly attributable to the increased workload associated with the organizational change. The current iCBT program seems to improve depression to some extent under severe working conditions. With consideration for the fact that an iCBT can be delivered to many workers at a low cost, this iCBT may be a promisingly cost-effective approach for improving depression among workers. The effect size of the current iCBT program for improving depression was small (around 0.15). Grime (2004) reported that the effect sizes (Cohen's d) of CCBT on depression among workers who recently had stress-related absenteeism were −0.25 (p = 0.04) at one month after the end of intervention and −0.1 (p = 0.56) at the three-month follow-up In the present study, the NNTs to achieve one remission from subthreshold depression were 14 at the 3-month follow-up and 13 at the 6-month follow-up for BDI-II. These were −279 at the 3-month follow-up and 12 at the 6-month follow-up for K6. These figures were larger than those of the previously reported NNTs of iCBT for improving depression among patients with major depressive disorder (2 to 4), when compared with TAU or waiting list The iCBT program showed a significant intervention effect on dysfunctional attitude measured by the DAS in terms of knowledge and efficacy of most CBT components except for self-efficacy of problem solving. Dysfunctional attitude is one of major targets of cognitive restructuring, a component of the current program. The present findings are consistent with previous ones that a CCBT program was effective in improving dysfunctional attitudes among medication-free participants with major depressive disorder The process evaluation of the iCBT program indicated that proportions of those who completed lessons and submitted homework decreased for the later lessons. About two-thirds of the intervention group completed all six lessons, while only a quarter of them submitted all six homework assignments. The completion rates of self-help programs without clinician contact were generally low This study has several limitations. First, participants were recruited from two IT companies in Japan. Most participants were males, working as professionals, and university graduates. They had their own PCs in their offices or homes. The participants were also supposed to have experience using a PC and studying through online programs. Higher education level may also help participants learn from the iCBT program. The generalization of the present findings to the general working population is limited. Second, the rate of completing lessons and homework was low. Only 93 participants submitted all homework assignments, and the average number of homework assignments submitted was 2.7 per respondent. This may weaken the findings. Third, the dropout rates in the present study were 29.1% and 28.6% at the three- and six-month follow-ups, respectively. In their systematic review, Kaltenthaler et al. (2008) reported that average CCBT dropout rates were 18–35% Checklist S1CONSORT 2010 checklist of information to include when reporting a randomized trial.(DOCX)Click here for additional data file.Protocol S1The original protocol in Japanese.(DOCX)Click here for additional data file.Protocol S2English translation of the original Japanese protocol.(DOCX)Click here for additional data file.


Some IgG-NAA are produced by B2 cells that have escaped tolerance mechanisms and we show how such pathogenic IgG-NAA are regulated to prevent autoimmune disease. The Fc region of IgG-NAA can influence inflammation and B cell function in vivo by binding to activating and inhibitory FcγR. IgM-NAA has therapeutic potential. Polyclonal IgM infusions can be used to abrogate on-going inflammation. Additionally, inflammation arising after ischemic kidney injury, e.g., during high-risk elective cardiac surgery or after allograft transplantation, can be prevented by pre-emptively infusing polyclonal IgM or DC pretreated ex vivo with IgM or by increasing in vivo IgM with a vaccine approach. Cell therapy is appealing as less IgM will be required.We review how polyreactive natural IgM autoantibodies (IgM-NAA) protect the host from invading micro-organisms and host neo-antigens that are constantly being produced by oxidation mechanisms and cell apoptosis. Second, we discuss how IgM-NAA and IgM anti-leukocyte antibodies (IgM-ALA) inhibits autoimmune inflammation by anti-idiotypic mechanisms, enhancing removal of apoptotic cells, masking neo-antigens, and regulating the function of dendritic cells (DC) and effector cells. Third, we review how natural IgM prevents autoimmune disorders arising from pathogenic IgG autoantibodies, triggered by genetic mechanisms  or micro-organisms, as well as by autoreactive B and T cells that have escaped tolerance mechanisms. Studies in IgM knockout mice have clearly demonstrated that regulatory B and T cells require IgM to effectively regulate inflammation mediated by innate, adaptive, and autoimmune mechanisms. It is, therefore, not surprising why the host positively selects such autoreactive B1 cells that generate IgM-NAA, which are also evolutionarily conserved. Fourth, we show that IgM-ALA levels and their repertoire can vary in normal humans and disease states and this variation may partly explain the observed differences in the inflammatory response after infection, ischemic injury, or after a transplant. We also show how protective IgM-NAA can be rendered pathogenic under non-physiological conditions. We also review IgG-NAA that are more abundant than IgM-NAA in plasma. However, we need to understand if the (Fab) Poly-reactivity with low binding affinity, but with high avidity, has enabled these IgM-NAA-producing B cell clones to be rapidly activated by a foreign or an auto neo-antigen for deploying protective mechanisms to the host. Such a response provides time for the adaptive immune system to mount a highly specific immune response to foreign antigens and, in addition, lessens the burden on the host to maintain diverse B cell clones producing highly specific IgG auto-antibodies, which has the potential of causing autoimmune disease owing to their high-affinity binding. Second, since both foreign and self neo-antigens can induce an inflammatory response, these IgM-NAA have taken over another task of subduing an excessive inflammatory response that can injure the host. This regulation of inflammatory cells without damage is made possible by the low binding affinity of IgM-ALA to live leukocytes, combined with its inability to effectively activate the lytic component of complement at body temperature 37°C). Hence, it is not surprising why these IgM-NAA antibodies, which first arose in cartilaginous fish, have been conserved during evolution [reviewed in Ref. ] and why7°C. Henc in mice –6.in vivo is rapid and can be driven by TLR agonists independently of antigen binding to their BCR ] , 39, Fc arasites –57 Hencearasites , 56. Addarasites , 50.In mice, B1 cells are rare in the bone-marrow, lymph nodes, and splenic B cell follicles (white pulp) while significant numbers of B1 cells are located in the splenic marginal zone as well as in the peritoneal and pleural cavities. However, under normal conditions, these peritoneal B1 cells do not contribute significantly to circulating IgM-NAA, but during sepsis, peritoneal B1 cells rapidly migrate to the splenic marginal zone , 58 wherOne can demonstrate the existence of IgG-NAA in normal adult murine or human serum under conditions where serum IgM is either removed or diluted out , 68–70, Importantly, IgG-NAA-producing B cells, unlike IgM-NAA-producing B cells, are in an inactive state at birth and in mice these B cells start producing IgG-NAA after exposure to bowel bacteria or foreign antigens –73. In hlpr murine model of SLE, IgG anti-dsDNA has been shown to be produced by B2 cells under the influence of excess T helper cells as a result of Fas deficiency 2 region and the Fc region of IgG-NAA. In these in vitro studies, the (Fab)2 region has been shown to (i) block function of activating FcγR receptors expressed by monocytes/macrophages. Such activating FcγR are involved in phagocytosis of IgG autoantibody complexed to platelets, (ii) block idiotypic determinants on IgG autoantibodies, thus neutralizing these antibodies, e.g., their binding to Factor VIII or platelets, (iii) inhibit function of certain pro-inflammatory cytokines by binding to these cytokines or their receptors, and (iv) provide protection against bacterial infection by binding of IgG-NAA to lectins on bacterial surfaces thus enhancing opsonization , macrophages, and neutrophils by binding to the inhibitory FcγRIIB receptor expressed by these cells. Activation of FcγRIIB by Fc region of IgG-NAA inhibits production of autoantibodies by B cells and plasma cells. Additionally IVIG ameliorates cell-mediated inflammation, e.g., in Kawasaki’s disease, by activating FcγRIIB and inhibiting the function of dendritic cells, macrophages, and neutrophils.Under normal conditions, it is unclear whether the (Fab)activity , 50. Hen in Ref. ]. One canization . Similarin vivo anti-idiotypic IgG has been associated with amelioration of disease activity mediated by pathogenic IgG autoantibodies, thus indicating that under pathological conditions, in vivo IgG with anti-idiotypic activity is functionally active  patients, there are data to show that production of IgG polyreactive autoantibodies results from breakdown of B cell tolerance mechanisms where during B cell development, B cells producing high-affinity IgG autoantibodies have not been removed or silenced either centrally in the bone-marrow or in the periphery before they mature into naïve immuno-competent lymphocytes , 96. Stu(i)lpr mice Genetic predisposition that permits some antigen-driven B1 cell clones to acquire the ability for isotype switching and somatic mutation as, for example, in the NZB/W murine lupus model and in SPA-1 deficient mice , 106, 62(iii)Repeated immunization that induces isotype switching in B1 cells that normally generate IgM autoantibodies with germline genes. For example, repeated immunization with hapten or a streptococcal antigen can induce B1 cells to generate IgG antibodies of different isotypes that are polyreactive and bind to the immunizing antigen as well as to self-antigens, such as DNA or myosin –101.(iv)+ T cells in thymoma-associated myasthenia gravis where such autoreactive T cells have escaped tolerance mechanisms Deficiency of IgM-NAA, especially IgM-NAA with anti-idiotypic activity to a specific IgG autoantibody, thus incompletely neutralizing the increase in pathogenic IgG autoantibodies, such as antibodies to neutrophil cytoplasmic enzymes , which are commonly referred to as ANCA, dsDNA, and glomerular basement membrane (GBM) that are present in normal individuals , 87, 112Based on observations in the preceding paragraph, it appears that different mechanisms could operate in generating pathogenic IgG autoantibodies depending on the murine disease model. Additionally, the mechanisms may differ in humans even though the disease has similar disease manifestations. A summary of the existing evidence would indicate that IgG autoantibodies can be induced by the following mechanisms:Figure SLE is the most studied autoimmune disorder where high levels of IgM and IgG autoantibodies, directed against nuclear antigens, especially dsDNA, are produced in the majority of patients. Before we discuss the role of IgM-NAA in protecting against pathogenic IgG anti-dsDNA, it would be important to indicate that a subset of lupus nephritis (approximately 25%) can occur in the absence of detectable circulating IgG anti-DNA and this is commonly referred to as C1q nephropathy , 114. Th/lpr lupus-prone mice is ameliorated when rendered IgG deficient by crossing these mice with either activation-induced deaminase (AID) or CD40L-deficient mice Countering the pathogenic effects of the normally present IgG autoantibodies that escape B cell tolerance mechanisms. Potential strategies employed by IgM-NAA for this purpose include (a) neutralization of these IgG autoantibodies by anti-idiotypic mechanisms (lpr mice (by crossing MRL-lpr mice with transgenic mice expressing IgM anti-DNA) ameliorated disease activity  maschanisms , and (c)chanisms , 126. Weactivity , 126.(ii)Providing the first line of defense against pathogens while the adaptive immune system, i.e., B2 and T cells, are being deployed to mediate a more specific and effective immune response that is long lasting and has memory. The role of NAA in mediating protection against pathogens was first brought to light by the observation that NAA recognize bacterial toxins and several viral antigens Inhibiting IgG autoantibody production and inflammatory responses by clearing apoptotic cells and binding to oxidized neo-determinants. Apoptotic cells that are normally produced in large numbers, express oxidized neo-determinants. Several homeostatic mechanisms, including binding of C1q and mannose binding lectin (MBL) to apoptotic cells, are involved in inducing macrophages and DC to phagocytose apoptotic cells and nuclear antigens . IgM di Figures D (138).in vivo studies (to be presented in the next section), we show that IgM pretreatment of LPS-activated BMDC switches these activated BMDC to a regulatory phenotype possibly by a mechanism involving downregulation of CD40 and NF-κB.Since there are <1.5% of DC in murine splenic leukocytes, we used 7–8 day cultured murine bone-marrow DC (BMDC) to investigate the functional effects of IgM on DC . We showin vitro studies to test the effect of polyclonal IgM on Type1 NKT cells, which together with DC, have an important role in inducing innate inflammation, e.g., after renal reperfusion injury (IRI). In these studies, we used α-gal-ceramide, a glycolipid that is taken up by DC and presented via the CD1d receptor to Type 1 NKT cells, which get activated and secrete IFN-γ. This assay is specific for determining Type 1 NKT function as only Type 1 NKT, but not T effector cells, will secrete IFN-γ after exposure to α-gal-ceramide, which is recognized, in context of CD1d presentation, by the invariant TcR on Type 1 NKT cells. We show that physiological doses of IgM inhibits α-gal-ceramide induced IFN-γ production of splenic leukocytes even when IgM is introduced 1 h after α-gal-ceramide . . in vitrin vitro data with both human and murine cells would indicate that IgM-ALA regulates leukocyte function by binding and downregulating certain leukocyte receptors  and inducing regulatory function in DC. Importantly, IgM does not decrease Foxp3+ Tregs. IgM regulates leukocyte activation, proliferation, and chemotaxis to attenuate excess inflammation [Figure In summary  that lack circulating IgM but have normal levels of other immunoglobulins. These mice have normal or increased levels of Tregs, B regs, and IL-10 and their normal functioning B cells express membrane IgM or BcR but are unable to secrete IgM. Unlike their WT counterpart, we demonstrated that these mice are very sensitive to renal ischemia, developing AKI with mild ischemia (26 min clamp time) that is insufficient to cause AKI in their WT counterparts Figures A,B. ReplIn the second approach, a single dose (150 μg) of normal purified polyclonal IgM was administered intravenously to wild-type C57BL6 (WT-B6) mice to increase baseline circulating IgM by about 30–50% and to determine if increasing IgM would protect these WT-B6 from severe renal ischemia (32 min clamp time). These studies clearly indicated that increasing circulating IgM levels protected mice from severe renal IRI Figure C. We nexin vitro data, in vivo IgM-ALA could mediate protection through several mechanisms, including regulation of NKT and DC and maintaining or enhancing Tregs, which also mediates protection in this model of innate inflammation  were used (6 BMDC (6 cells) to UV irradiation. Such apoptotic LPS + IgM pretreated BMDC failed to protect mice from ischemia induced AKI, thus excluding the role of apoptotic cell/IgM complexes in inducing protection  inhibited alloantigen-activated T cell proliferation and differentiation into Th-1 and Th-17 independently of DC   T cells . Two app T cells . Histolo T cells . The T cs Figure D.In the second approach, we wanted to determine whether increasing circulating levels of IgM in WT-B6 mice inhibited the severe and rapid rejection that occurs in the setting of fully MHC-incompatible donor hearts . In this model, rejection in WT-B6 recipients is detectable by day 5 and graft loss occurs by 7–9 days . In thesIn summary, we show that physiological doses (175 μg) of polyclonal IgM can subdue inflammatory responses mediated by an adaptive immune mechanism. Potential mechanisms include the following (a) a direct inhibitory effect of IgM-ALA on T effector cells, but not Tregs, possibly by binding and down-modulating CD3/TcR and certain specific co-stimulatory receptors, such as CD4 and CD2 but not CD8. As a result, T effector cells are inhibited from proliferating or producing certain specific cytokines  or from differentiating into TH-1 and TH-17 pro-inflammatory cells. Importantly IgM-ALA does not affect levels of Foxp3+ Tregs, but prevents Foxp3 + cells from differentiating into TH-17 cells under pro-inflammatory conditions, (b) by binding to CD40 and switching activated DC to a regulatory phenotype with downregulation of CD40 and p65NF-κB, and (c) by inhibiting chemotaxis. It is highly unlikely that IgM anti-PC could have a significant role in inhibiting allograft rejection in our studies as we used small doses of polyclonal IgM (175 μg) while 1.5–2.0 mg of a monoclonal IgM anti-PC (T15 idiotype) was used to inhibit an arthritis model of inflammation mediated by adaptive immune mechanisms .in vitro studies demonstrated that polyclonal IgM inhibited T cell proliferation and differentiation into Th-1 and Th-17 cells  became diabetic, while 0% of mice (n = 30) treated with IgM beginning at 5 weeks developed DM. Importantly, only 20% of pre-diabetic mice (n = 20) treated with IgM beginning at 11 weeks of age developed DM at 25 weeks of age. This latter observation is particularly notable as prior studies using co-stimulatory blockade failed to prevent DM in this murine model. At 18–25 weeks of age the pancreas revealed no or minimal insulitis in NOD mice treated with IgM beginning at 5 weeks of age. Other investigators using monoclonal polyreactive natural IgM in the neonatal period have also obtained similar results (We wanted to determine the effect of increasing IgM levels on development of DM . NOD mic results , 184.In summary, these studies indicate that polyclonal IgM inhibits insulitis via several potential mechanisms, including inhibition of autoimmune T effectors and possibly countering IgG autoantibodies via anti-idiotypic mechanisms and by inhibiting the B cells that produce them. Additionally, IgM by switching activated DC to a regulatory phenotype and maintaining Tregs could enhance this protective effect.Figures The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


When membrane and membraneless EBCs are compared, MPD of the membraneless EBC that is run at the similar operating condition to EBC including membrane is speculated as about 134 μW · cm−2.Mediatorless and membraneless enzymatic biofuel cells (EBCs) employing new catalytic structure are fabricated. Regarding anodic catalyst, structure consisting of glucose oxidase (GOx), poly(ethylenimine) (PEI) and carbon nanotube (CNT) is considered, while three cathodic catalysts consist of glutaraldehyde (GA), laccase (Lac), PEI and CNT that are stacked together in different ways. Catalytic activities of the catalysts for glucose oxidation and oxygen reduction reactions (GOR and ORR) are evaluated. As a result, it is confirmed that the catalysts work well for promotion of GOR and ORR. In EBC tests, performances of EBCs including 150 μm-thick membrane are measured as references, while those of membraneless EBCs are measured depending on parameters like glucose flow rate, glucose concentration, distance between two electrodes and electrolyte pH. With the measurements, how the parameters affect EBC performance and their optimal conditions are determined. Based on that, best maximum power density (MPD) of membraneless EBC is 102 ± 5.1 μW · cm Enzymatic Biofuel cells (EBC) are device that converts chemical energy into electrical energy using (i) enzyme as biocatalysts and (ii) glucose/alcohol as biofuels since it was reported in 196411345678Regarding the enzyme immobilization strategy, although physical adsorptions like physical entrapment and chemical bonding like covalent coupling and enzyme cross-linking were attempted, using Layer-by-Layer (LbL) has been recently emerged. According to the LbL structure, (i) multiple repetitive layers are stack together by electrostatic interaction between oppositely charged species and (ii) the stack layers are formed on the nano-sized supporters like carbon nanotube (CNT)21011122 to H2O without production of intermediate H2O2 can be considered the meritAs the possible enzyme catalysts, glucose oxidase (GOx) catalyzing glucose oxidation and laccase (Lac) catalyzing oxygen reduction have been considered14To fabricate the viable enzyme-included LbL catalysts, using proper conducting polymer (CP) and/or cross-linker can be affordable option. In this study, poly(ethylenimine) (PEI), glutaraldehyde (GA) and CNT were used as the CP1719Regarding cost and size of EBC system, optimizing role of membrane included in the EBC may be critical. For doing that, we evaluate two things; first, inspecting optimal membrane thickness and second, removing membrane although the latter is likely to be more effective because the removal of membrane can reduce the size and cost of EBC in a more straightforward way. The membrane usually plays two different roles as separator (i) to alleviate occurrence of mixed potential and (ii) to increase ohmic resistance so that it should be designed (i) what is appropriate membrane thickness if the membrane is utilized and (ii) how to modulate associated parameters if membrane is not used.2/CNT] biocatalyst that is an enzyme structure based on our previous research for glucose oxidation reaction (GOR) and (ii) cathode consisting of three different biocatalysts including GA, Lac, PEI and CNT for oxygen reduction reaction (ORR)In this study, we fabricate EBC system using (i) anode consisting of GA/[[GOx/PEI]2/CNT] used as a catalyst for anode and (ii) Lac-based three different catalysts  used as catalysts for cathode. Regarding the activity of anodic catalyst, GOR occurring at flavin adenine dinucleotide (FAD) that was deep inside GOx molecules was investigated by CV measurements in N2-state and air-state (with O2)  GA/[[GOx/PEI]with O2) . For the2-state. It is due to occurrences of reduction reaction of O2  and redox reaction of FAD (GOx (FAD) + 2H+ + 2e− ↔ GOx (FADH2), existence of redox reaction peaks appeared at −0.469 V vs. Ag/AgCl)2 decreased with decrease in cathodic current. In this case, O2 was consumed for redox reaction of FAD (GOx (FADH2) + O2 → GOx (FAD) + H2O2), not for reduction reaction of O2, producing cathodic current. Due to the change in role of O2, CV curve downshifted before supply of glucose was upshifted with increase in glucose concentration2224There are two observations about the CV measurements. First, according to the 2-state (without O2) was also measured . In the N2-state, redox reaction peaks of cupric ions within Lac molecules of the Lac-based three different catalysts were observed at 0.272, 0.274, and 0.282 V vs. Ag/AgCl. The small reaction peaks were included in inset of the 2 was supplied, current densities of the three catalysts were down-shifted from 0.1 V vs. Ag/AgCl, while redox peak of cupric ions within Lac molecules was shifted to positive direction and appeared around 0.6 V vs. Ag/AgCl. These observations indicate that ORR of the catalysts takes place appropriatelyIn contrast, regarding the activity of cathodic catalyst, ORR occurring at cupric ions within Lac molecules of the three different Lac-based catalysts was also measured. For comparison, the reaction occurring at Nss) and apparent Michaelis-Menten constants (Kms) were measured using Laviron’s formula and Michaelis-Menten plot, respectively 2227s of GA/[[GOx/PEI]2/CNT] catalyst was 12.7 s−1, while that of GA/[Lac/PEI/Lac/CNT] catalyst was 8.6 s−1, (Kss of Lac/CNT and Lac/PEI/Lac/CNT were 6.5 and 8.4 s−1).To investigate and compare reaction rate of the catalysts, their electron transfer rate constants  . There are two noticeable things for 22s of the catalysts was higher than that of other similar catalysts, meaning that the catalysts played their role well and second, we swept the entire potential ranges using very slow potential scan rate (5 mV·s−1)22Even in comparison of K2/CNT] and the three different Lac-based structures were measured. Also, to inspect effects of enzyme catalysts on EBC performance, those of EBCs adopting catalysts fabricated without enzyme molecules  were measured (−1 (anode) and 100 cc · min−1 O2 gas was provided (cathode). There were two things to mention about the results. First, regarding EBC performance, maximum power density (MPD) of EBC adopting GA/[[GOx/PEI]2/CNT] and GA/[Lac/PEI/Lac/CNT] catalysts was highest . It indicates that GA plays a critical role in immobilizing more Lac molecules and activating ORR, followed by improving EBC performance. Second, based on −2 respectively), it was proved that both enzymes (GOx and Lac molecules) were well acted as catalysts for EBC.To evaluate performances of EBCs including membrane between anode and cathode electrodes, polarization curves of the EBCs adopting GA/[[GOx/PEI]measured . For thects) of the EBCs used in 2 gas. According to the Nyquist plots, when enzyme catalysts were included, Rct was highest when GA/[Lac/PEI/Lac/CNT] was employed in EBC, while the value was lowest when Lac/CNT was employed . On the other hand, when enzyme catalysts were not included, Rct of the EBC was very low .To further investigate effect of enzyme catalysts on charge transfer of EBC, charge transfer resistances  because the Lac molecule is assemble of proteins that have non-conductive propertyct trend of ss) of EBCs are not affected by catalyst types .These results make sense. As reported previously, R2/CNT] catalyst for anode) and 2/CNT] catalyst was measured, while cupric ion redox reaction peaks of Lac-based catalysts were measured for the It is also important to investigate whether our enzyme catalysts can work well for a long time. To evaluate the long term stability of enzyme catalysts, catalytic activities of the enzyme catalysts were measured every week for four weeks and the results are represented in 2/CNT] catalyst was maintained to 87% of initial value, while those of Lac-based catalysts were maintained to 89, 75 and 64% of initial value. It can be explained that (i) long term stabilities of all the catalysts are better than that of other similar catalysts, implying that EBCs adopting these catalysts may also keep their stabilities22According to the Figs S4a,b, the peak current density of GA/[[GOx/PEI]The results mentioned above discernibly mean that enzyme structures suggested for both anode and cathode electrodes play their role as catalyst. It is now time to determine how performances of membraneless EBCs adopting the enzyme catalysts are affected by four main parameters like glucose flow rate, glucose concentration, interval between two electrodes and electrolyte pH. For doing that, polarization curves of the corresponding EBCs were mainly measured.2 fuels are properly acted for GOR and ORR, inducing superior OCP and MPD. However, as glucose flow rate increases, the fuels become easy to access to the opposite electrode, lowering ORR potential and producing self-discharge current by occurrence of mixed potential. Such decreased ORR potential induces decrease in OCP, followed by decrease in MPD of membraneless EBC. More specifically, when glucose flow rate was 0.5 cc · min−1, MPD of EBC was 34 ± 2.6 μW · cm−2, while the MPD was plummeted to 10 ± 0.9 μW · cm−2 with the glucose flow rate of 90 cc · min−1. This result was well matched with one reported by Jayashree et al., who studied effect of fuel flowrate on performance of microfluidic membraneless fuel cellet al., who investigated effect of fuel flowrate on performance of proton exchange membrane fuel cell (PEMFC) previously reported and their result was also compatible with oursRegarding the glucose flow rate effect, according to polarization curves , as gluc−1. Their polarization curves are presented in As another parameter affecting performance of membraneless EBC, glucose concentration was regarded and its effect on membraneless EBC performance was evaluated. For the EBC tests, glucose concentrations of 10, 25, 40, 100 and 200 mM (pH 7.4) were provided with glucose flow rate of 0.5 cc · minet al., who investigated effect of ethanol concentration on performance of direct ethanol fuel cell (DEFC)2 concentration .In detailed evaluations, it is clear that under different glucose concentrations, performances of EBCs measured by polarization curves mainly depend on the difference in current density region occurred by electrolyte resistance and mass transfer with little change in OCP of the EBCs . According to the Effect of distance between anode and cathode on performances of membraneless EBCs was also critical and related polarization curves were measured . For the−2). The linear relationship among ohmic resistance, distance between two electrodes and MPD of the EBC is explained that the ohmic resistance loss is probably one of dominant factors to determine MPD of membraneless EBC because MPDs of membraneless EBCs follow up trend of ohmic resistance loss in all three different distances of 1, 2 and 3 mm.Second, as a distance between two electrodes was reduced, MPDs of the EBCs increased in a linear progression  using different MEA thickness, as distance between electrodes in DMFC decreased (decrease the membrane thickness), its performance increased due to reduction of ohmic resistance between electrodesSuch results are reasonable and compatible with previously reported ones−1 and glucose concentration of 40 mM. Their polarization curves are represented in −2). Generally, GOx  was mostly activated near to pH 7~8, while activity of Lac  was best near to pH 3~5. Thus, it means that the electrolyte pH effect is mostly dominated by catalytic activity of Lac molecules, confirming that performance of this membraneless EBC depends on cathodic reaction.Effect of pH of electrolyte (glucose solution) on performance of membraneless EBCs was also investigated because the pH of glucose solution filling empty acrylic separator placed between two electrodes affected the EBC performance. For the EBC tests, glucose solutions showing five different pHs like 2, 3, 5, 7.4 and 9 were adopted with distance of 1 mm, glucose flow rates of 0.5 cc · min−) controlled by electrolyte pH. When the pH is higher than 5, OH− ions also increase with activated reduction reaction. Such increased OH− ions then make activity of Lac molecules weak because the OH− ions are stuck to the active sites of Lac molecules, preventing O2 molecules from reacting at the active siteset al. and Tan et al.40This pH dependence on performance of membraneless EBC may be ascribed to change of hydroxide anion (OH−2) is also pretty excellent value compared to that of other membraneless EBCs. For instance, MPD of membraneless EBC consisting of GOx/PPy/CNT (anode) and Lac/PPy/CNT (cathode) was 30 μW · cm−2 −2 −2 −2 It is noticeable that the best MPD of the EBC (102 ± 5.1 μW · cm−2. Although the MPD is still less than that of EBC including membrane (171 ± 5.5 μW · cm−2), it is obviously viable result.As we already explained, EBCs including membrane and membraneless EBCs showed their own performances. However, since there is a difference in the distance between two electrodes , it is not possible to compare performances of the two EBCs in the status quo. To compare them in a proper manner, we estimated the regression curve between MPDs of the membraneless EBCs gained under the condition of 0.04 M glucose solution (pH 3) and distance between two electrodes . Based o−2). Although further efforts are required to improve the stability, it is reasonable to say that our membraneless EBC system is relatively stable.Stability of membraneless EBC was also measured for four weeks by tracking variance in its MPD. For the tests, optimal conditions of the aforementioned parameters were used and three samples were experimented for determining MPD. According to the MPD results represented in −2), meaning that (i) GA promoted immobilization of more Lac molecules, followed by activation of the ORR and (ii) cathodic reaction (ORR) was a rate determining step to affect performance of EBC.In this study, performances of mediatorless/membraneless EBCs adopting enzyme catalysts and catalytic activities of the enzyme catalysts were investigated. When the catalytic activities of catalysts for GOR and ORR were evaluated, they showed reasonable reaction trend with excellent activity values. In EBC tests, performances of EBCs adopting Nafion 117 membrane were evaluated. According to the EBC tests, performance of EBC adopting GA/[Lac/PEI/Lac/CNT] as cathodic catalyst was best , 40 mM (glucose concentration), 1 mm  and pH 3 were determined.When performances of membraneless EBCs were measured, four different parameters like glucose flow rate, glucose concentration, interval between two electrodes and electrolyte pH were considered. In the polarization curve measurements, how the parameters affected the performances of EBCs was clarified and the best condition of the parameters was obtained. As a result of that, MPD of membraneless EBC was reached to 102 ± 5.1 μW · cm−2 was calculated by the extrapolation equation. Although this value is still lower than that of EBC including membrane, it is clearly promising result.To compare performances of EBCs including membrane and membraneless EBC in equivalent state, MPD of the membraneless EBC was expressed as distance between electrodes. With that, a linear progression of MPD = −0.037 mm + 0.14 was obtained and the MPD value of 134 μW · cm−1 solid), Laccase , glutaraldehyde (GA) solution, and polyethylenimine  were purchased from Sigma Aldrich . Sodium-acetate buffer pH 5 was made from mixture between sodium acetate and acetic acid and that was purchased from Sigma Aldrich .Multiwall carbon nanotubes (MWCNT) (its purity is higher than 90%) were obtained from NanoLab . Glucose oxidase  were mixed. The mixture was further dissolved into 0.4 mg·mL−1 Lac solution under 0.1 M sodium-acetate buffer (pH 5). The mixture was then incubated for 2.5 h and centrifuged at 13,000 for 10 min. Lac/PEI/Lac/CNT catalyst was prepared by additional process of the Lac/CNT catalyst. The suspended Lac/CNT catalyst was mixed with 2.5 mg·mL−1 PEI solution (in 0.5 M NaCl (pH 5)). Supernatant of the mixture was then removed and washed with DI water to remove excess PEI. 0.4 mg·mL−1 Lac solution (in 0.1 M sodium-acetate buffer pH 5) was added to the mixture and the final mixture underwent the same incubation process to Lac/CNT catalyst. GA/[Lac/PEI/Lac/CNT] catalyst was prepared by additional process of the Lac/PEI/Lac/CNT catalyst. The suspended Lac/PEI/Lac/CNT catalyst was mixed with 0.5 w/v % GA solution. The mixture was then centrifuged at 13,000 rpm for 10 min. The excess GA was removed by using DI water 2 times to avoid further cross-linking process. All the catalysts were dipped in sodium-acetate buffer (pH 5) at 4 °C when not in use.Three different types of catalysts including Lac molecules were prepared for cathode. The catalysts are denoted as Lac/CNT, Lac/PEI/Lac/CNT, and GA/[Lac/PEI/Lac/CNT], respectively. Same to anodic catalyst, by carrying out preliminary optimization process, it was confirmed that 0.4 mg·mL2O and then, 10 μm of catalytic ink was dropped on the GCE. The catalytic ink-loaded working electrode was dried for 45 min. After drying, 5 wt % Nafion solution was coated on the working electrode to complete configuration of working electrode. As for electrolyte, 0.01 M PBS (pH 7.4) was considered.A computer connected potentiostat  was used for electrochemical measurements. For the half-cell tests like CV, Pt wire and Ag/AgCl (soaked in 3.0 M KCl) were served as counter and reference electrodes respectively, while catalysts loaded on glass carbon electrodes (GCE) were acted as working electrode. For loading of catalysts, catalytic powder was mixed with 1 mL of Hs/Rct), new EBC kit was designed with active surface area of 1.0 cm2. When membrane was included, the kit was consisted of catholyte reservoir, cathode electrode, membrane, anode electrode and anolyte reservoir. On the other hand, when used as membraneless EBC kit, membrane was removed and instead, acrylic separator was inserted. As the membrane, Nafion 117 (150 μm thick) was chosen.For measuring polarization curve and membrane/charge transfer resistances  and placed in cathode part of the EBC kit. After the cathode was ready, it was stuck to the membrane, while GA/[[GOx/PEI]s/Rct) in the corresponding EBC single cells, its Nyquist plot was measured. Nyquist plot produced is available for anode and cathode, indicating that semicircle of the Nyquist plot reflects Rct of the full cell, while intercept of x axis stands for Rs.To measure polarization curves from the EBC single cell, Bio-Logic  potentiostat was used. It was also connected with a frequency response analyzer (FRA). By coupling FRA with the potentiostat, the power output was determined. The EIS measurements were performed at the open cell potential condition of the EBC. The impedance spectra were decided in the frequency range between 2 MHz and 10 Hz with 10 steps per decade and modulating potential was fixed as 10 mV. To attain the membrane/charge transfer resistances .