This pilot study was undertaken to determine whether the NintendoCommunity-dwelling fallers over 70 years were recruited and attended for computer-based exercises (n = 15) or standard care (n = 6). Balance and fear of falling were assessed at weeks 0, 4 and 12. Participants were interviewed on completion of the study to determine whether the intervention was acceptable.Eighty percent of participants attended 75% or more of the exercise sessions. An improvement in Berg Score was seen at four weeks (p = 0.02) and in Wii Age at 12 weeks (p = 0.03) in the intervention group. There was no improvement in balance scores in the standard care group.WiiFit exercise is acceptable in self-referred older people with a history of falls. The WiiFit has the potential to improve balance but further work is required.ClinicalTrials.gov - NCT01082042 Community dwelling individuals, over the age of 65, fall at a rate of 35% per year , and thiUK/US falls guidelines support referral of fallers for multidisciplinary assessment ,4,5. Sig® WiiFit ("WiiFit") is a software/hardware game package for the Nintendo® Wii, designed to improve balance and fitness, whilst providing entertainment (further information in appendix 1). There is a lack of evidence showing the acceptability of interactive computer games in the older faller.The NintendoThis purpose of this pilot study was to determine the feasibility and acceptability of the WiiFit in community-dwelling older fallers and whether it has the potential to improve balance. Given the lack of published evidence it would be unethical to withhold standard care in a randomised controlled trial.This longitudinal intervention study was undertaken in community dwelling people over the age of 70 years who had fallen in the previous year. We compared 2 groups: an intervention group who attended for WiiFit exercise sessions and a standard care group who attended the local falls group.Age 70 years or older, living locally in the community (in sheltered accommodation or own home), fallen at least once in the preceding 12 months and have an abbreviated mental test (AMT) [Wheelchair bound individuals, people living in a care homes or long-term hospital care and current enrolment in an exercise or rehabilitation programme.A press release was issued to recruit members to the intervention group. This resulted in widespread press coverage. Fifty-three potential participants contacted the research team, of these 14 were excluded as they had not fallen in the preceding 12 months. The first 15 eligible people were included in the study.People referred to the local falls group were approached by the research team. Of 13 potential recruits, one was excluded as a result of low AMT, two refused to participate in standard care and four declined to participate in the study. Thus six participants were recruited. Due to poor uptake/high drop-out rates recruitment was abandoned after three months.The North of Scotland Research Ethics Committee granted ethical approval (Ref No. 08/S0801/186). The study was registered with ClinicalTrials.gov (NCT01082042).At an initial interview baseline information was obtained. Balance was assessed by a physiotherapist at weeks 0, 4 and 12. Functional balance was assessed by the Berg Balance Scale (BBS), a performance based measure using 14 activities of daily living (range 0-56) . A TinetThe Falls Efficacy Scale - International (FES-I), which is reliable and valid , was undAcceptability of the intervention was assessed by monitoring attendance, completion of an Attitude to Falls-Related Interventions Scale (AFRIS) and a qualitative interview. The AFRIS is a validated measure of the acceptability of falls-related interventions  . All memThe intervention group had their WiiFit Age calculated by the WiiFit software  undertaken at weeks 0 and 12 [The intervention group attended individual exercise visits supervised by a member of the research team twice weekly for 12 weeks. A walking frame was placed in front of the balance board, if the participant deemed this necessary. The exercise programme included balance and aerobic exercises on the WiiFit (appendix 1) and was modified at weeks 4 and 8. Participants were allowed to stop and move onto the next exercise at any point (including if they could not manage a particular exercise) and were allowed to terminate the session at any point. They were advised not to play the Wii out with the study. The actual time exercised was measured for each session.The standard care group was recruited from the local falls group, which is run by the local NHS Hospital. Patients attend a 12-week exercise/education programme supervised by NHS physiotherapists.SPSSv16.0  was used to analyse the data. Normally distributed data were compared using Independent samples T-Test between groups and equal variances were assumed. AMT scores were compared using Mann-Whitney U-Test. Fisher's exact test was used to compare living situation and use of walking aid, and Pearson-Chi Squared test was used to compare number of falls. Paired samples T-Test was used to assess change in the formal balance scores following the exercise programmes.Figure One member of the intervention group had previous experience of the Wii but had not played the WiiFit. Two of the intervention group had previously attended a falls group .On study completion there were 13 (86.7%) in the intervention group and 4 (66.7%) in standard care. Follow up is shown in figure The mean number of sessions completed by the intervention group was 19 . Attendance was good with 80% of participants attending 75% or more of the sessions. No significant difference in AFRIS score (p = 0.45) between the intervention group (Mean (SD) = 34.3 (7.2)) and the standard care group (Mean (SD) = 37.3 (3.3)) were found.At interview we found that 100% of the intervention group (inclusive of the two dropouts) found the intervention to be enjoyable and acceptable. Over half of participants felt that the exercise sessions were the right length and the right frequency (69%), with 23% expressing that the sessions were too short. The intervention group felt that "people like me" would participate in a similar exercise programme if it was more widely available (77%). Participants expressed a strong desire to exercise with the WiiFit in the future (92%), showing no preference towards exercising on their own/with company or towards supervised/unsupervised sessions. When asked for a preference to falls group or exercise with the WiiFit in the future, 61% chose the WiiFit, 8% chose falls group, 8% chose both and 23% did not know.A number of recurring themes were identified during the interviews. Positive themes were an improvement in confidence following the intervention, a sense of positivity about the researchers and the company the sessions provided and that the participants would like a Wii of their own. Negative themes identified were pain or discomfort, feeling tired and difficulties getting to the sessions.There was no difference between FES-I at the start and end of the study in the intervention group. However, there was a trend towards increasing FES-1 scores in the standard care group on completion of the study.For a future trial to be feasible the target population must be able to comply with the intervention. A low number of adverse events were recorded. One participant fell backwards (no injuries sustained) when stepping from the balance board, and another felt temporarily lightheaded on completion of one of the exercise sessions.We sought to determine whether the WiiFit has the potential to improve balance and whether this is comparable to standard care. The results are shown in figure This pilot study is one of the first to look at acceptability of the WiiFit as a falls intervention in older adults. There are low levels of willingness of older adults to participate in group strength and balance training . In our et al have suggested that a change of 7.7 BBS score points is required to reveal a genuine modification of function between 2 assessments [A randomised controlled trial would probably be feasible given our findings, and is essential to establish whether exercise with the WiiFit is, at least, as beneficial as standard care. As the standard care group represented a frailer population, we were unable to effectively compare standard care and the WiiFit intervention. However, the potential to improve balance in this specified population was seen, as there was a significant improvement in BBS at 4 weeks in the intervention group. Conradsson essments . In bothThere was no change in FES-I in the intervention group at 4 weeks despite improvement in BBS. Furthermore a significant deterioration in FES-I score was seen in the standard care group at 12 weeks. The lack of improvement in balance confidence despite improvement in balance ability has been previously shown . It is pFurther work must be undertaken to determine whether exercise with the WiiFit should be included in falls prevention programmes. If improvements in balance can be shown then the WiiFit could be used as part of supervised physiotherapy based exercise sessions or within the patients' own homes. With advances in telemedicine it is plausible that participants may be able to undertake exercise sessions within their own homes with the therapists monitoring progress from their base hospital. This may prove more cost-effective than standard care due to reductions in transport and staffing costs.The standard therapy group are likely to represent a frailer population in that there are statistically significant differences in weight, number of co-morbidities and AMT score, and a trend towards difference in baseline BBS. Difficulties in recruitment and a high drop out rate in the standard care group led to problems with comparison of the groups.et al have previously determined that a BBS of 45 or more indicates that an individual is likely to be safe in independent ambulation [A further limitation is that our intervention group had a mean BBS at baseline of 45 and Berg bulation . Thus anIn addition WiiFit sessions were undertaken in a supervised hospital environment and are unlikely to be reflective of people exercising with the WiiFit in their own homes. However participants indicated they were willing to exercise with the Wii in their own homes. This is important as research has found that older people are more open to exercise programmes undertaken in their own home .The WiiFit appears to be an acceptable falls intervention in the community dwelling older individuals who have fallen and has the potential to improve balance and self-perceived confidence. Future work must be undertaken to determine an acceptable exercise programme with the greatest potential to improve balance. A randomised controlled trial is required to determine whether the WiiFit is comparable to standard care.The authors declare that they have no competing interests.MAW contributed to the data collection, coordinated the study, contributed to the statistical analysis and led the writing of the manuscript. RLS participated in the design of the study, and contributed to the statistical analysis and manuscript writing. AMcEJ contributed to the design of the study and manuscript writing. AS conceived the study, participated in its design and contributed to the statistical analysis and manuscript writing. All authors read and approved the final manuscript.The Wii is a video game console which detects movement allowing the individual to play interactive games. WiiFit is an exercise game which combines a balance board on which the individual stands  with the Wii games system. The balance board incorporates pressure sensors to monitor centre of balance and shifts in weight/balance.http://www.nintendo.com/wiiSee the following for further information: http://wiifit.comhttp://en.wikipedia.org/wiki/Wiihttp://en.wikipedia.org/wiki/Wii_Fit1. Aerobic games - Jogging - 1 attempt to half way on the circuit2. Balance games - Tilt Table - 3 attempts3. Aerobic games - Step Basics - 2 attempts up to the point where the feet turn blue4. Balance games - Ski Slalom - 5 attempts5. Game of own choice from list above - 1 attempt6. Yoga game - Breathing Exercise - 1 attemptAll games played at beginner level.1. Aerobic games - Jogging - 1 attempt to half way on the circuit2. Balance games - Tilt Table - 2 attempts - option of advanced level3. Aerobic games - Step Basics - 2 attempts up to the point where the feet turn blue4. Balance games - Heading - 2 attempts - beginner level5. Balance games - Ski Slalom - 5 attempts - option of advanced level6. Balance games - Ski Jump - 2 attempts - beginner level7. Yoga game - Breathing Exercise - 1 attempt1. Aerobic games - Jogging - 1 attempt to half way on the circuit2. Balance games - Tilt Table - 2 attempts - option of advanced level3. Aerobic games - Step Basics - 2 attempts up to the point where the feet turn blue4. Balance games - Heading - 2 attempts - option of advanced level5. Aerobic games - Hula Hoop - 1 attempt - beginner level6. Balance games - Ski Slalom - 3 attempts - option of advanced level7. Balance games - Ski Jump - 2 attempts - option of advanced level8. Yoga game - Breathing Exercise - 1 attempt1. Do you feel your balance has improved following the balance training with the Wii?2. How did you feel about the exercise sessions?3. Was the Wii an acceptable form of exercise to you?4. What did you think about the length of the exercise sessions?5. What did you think about the frequency of the exercise sessions?6. Do you think people like you would participate in an exercise programme like this if it was more widely available7. Would you undertake exercise with the Wii again?a. Yes or No?b. On your own or with others?c. Supervised or unsupervised?8. Anything else you would like to say about the exercise sessions?9. If given the option of falls group or Wii in the future what would you chose?10. Any falls or problems during the study1. Why did you withdraw from the study?2. Do you feel your balance has improved following the balance training with the Wii?3. How did you feel about the exercise sessions?4. Was the Wii an acceptable form of exercise to you?5. What did you think about the length of the exercise sessions?6. What did you think about the frequency of the exercise sessions?7. Do you think people like you would participate in an exercise programme like this if it was more widely available?8. Would you undertake exercise with the Wii again?a. Yes or No?b. On your own or with others?c. Supervised or unsupervised?9. Anything else you would like to say about the exercise sessions?10. If given the option of falls group or Wii in the future what would you chose?


Less than half of U.S. hospitals meet guidelines for prompt treatment of ST-segment elevation myocardial infarction (STEMI). The Door-to-Balloon (D2B) Alliance is a collaborative effort of more than 900 hospitals committed to implementing a set of evidence-based strategies for reducing D2B time. This study presents data on (1) the prevalence of evidence-based strategies in U.S. hospitals that participated in the D2B Alliance and (2) identifies key hospital characteristics associated with their use.We conducted a cross-sectional study of U.S. hospitals that joined the D2B Alliance through a Web-based survey about their current practices for patients with STEMI who received primary percutaneous coronary intervention (PCI). We used multivariate logistic regression to identify hospital characteristics associated with use of each strategy.Of the 915 U.S. hospitals enrolled in the D2B Alliance as of June 2007, 797 (87%) completed the survey. Only 30.4% of responding hospitals reported employing at least 4 of the 5 key strategies ; 9.3% employed none of the strategies. There was no clear pattern of correlation between hospital characteristics and reported strategies.As of 2007, many hospitals had implemented few of the key strategies to reduce D2B time, suggesting substantial opportunity to improve care for patients with STEMI. Despite remarkable improvement in many quality indicators for acute myocardial infarction ,2, natioHaving contemporary data on the prevalence of these strategies is important for identifying the degree to which greater efforts in improvement are needed in this area. If most hospitals have adopted the strategies identified in 2005  as imporTo provide the most contemporary data on hospital strategies available, we used a baseline cross-sectional survey of hospitals completed at the time of their initial enrollment (November 2006–June 2007) with the D2B Alliance. In this report, we present data on the prevalence of these strategies and identify key hospital characteristics associated with their use.Between November 2006 and January 2007, we conducted a cross-sectional study of all U.S. hospitals that joined the D2B Alliance as of June 2007 (n = 915), all of which perform primary PCI for patients with STEMI. Hospitals completed a Web-based survey, which has been previously described , about tThe outcomes were the reported presence of key strategies recommended by the D2B Alliance to improve D2B time. The key strategies included emergency medicine activation of the catheterization laboratory, activation with a single call, expectation that the catheterization team is in the laboratory within 20–30 minutes of page, prompt data feedback on D2B times provided for emergency department and catheterization laboratory staff, and use of a pre-hospital electrocardiogram to activate the catheterization laboratory while the patient is still en route to the hospital. In addition, we created a summary measure of the total number of these key strategies that were implemented. The presence of strategies was measured with the Web-based survey, completed by the primary hospital contact person for the D2B Alliance campaign. The respondent was asked to consult with a variety of staff at the hospital in order to reflect the hospital perspective on strategies used for patients receiving primary PCI. The respondent who submitted the survey reflected the diversity of staff leading D2B improvement efforts, with roles as directors of quality improvement, nurse managers, catheterization laboratory directors, emergency department directors, emergency medicine physicians, cardiologists, and a range of clinical managers.Independent variables included hospital characteristics . All hospital characteristics were obtained from the American Hospital Association Annual Survey of Hospitals.We used standard frequency analyses to describe the hospital sample and to report the prevalence of each D2B Alliance recommended strategy, as well as the mean number of reported strategies. To examine if some hospital types were more likely to implement certain strategies, we used logistic regression models to estimate the adjusted associations between each strategy  and key hospital characteristics as independent variables . We used a multiple linear regression model to examine the adjusted association between the number of strategies implemented and all hospital characteristics. All models included all hospital characteristics as independent variables. All data analyses were performed using SAS software, version 9.1 (SAS Institute).Among the 797 respondent hospitals Table , the meaThe reported prevalence rates of key strategies recommended by the D2B Alliance are shown in Table There was no clear pattern of correlation between hospital characteristics and reported strategies Table . Larger We found that implementation of recommended strategies at the time of enrollment in the D2B Alliance (late 2006 through mid-2007) was limited for many enrolled hospitals, suggesting ample opportunity for improvement among these hospitals. For instance, emergency medicine physician activation of the catheterization laboratory and prompt data feedback about D2B times were being implemented by just about half of hospitals, and a single-call system of activation or use of pre-hospital electrocardiograms while the patient is en route to the hospitals were used by less than one third of hospitals. Given the strong evidence of the impact of these strategies on D2B times and the persistent underutilization of effective strategies, there remains tremendous opportunity to improve quality of care and reduce mortality for patients with STEMI.The study underscores the need for quality improvement initiatives and the potential impact of the D2B Alliance. Although a previous study demonstrated that many hospitals were not implementing these strategies in 2005, it was not known if there had been substantial adoption since that time. Our study indicates that even among hospitals that were committed to reducing D2B times by joining the D2B Alliance, the prevalence of some key strategies was modest, as recently as early 2007. Future evaluation of the D2B Alliance experience will be necessary to demonstrate if such a collaborative effort can facilitate adoption of these key strategies to reduce D2B times.The less commonly implemented strategies reflect particular challenges. Single-call systems are difficult without a unified on-call list for interventional cardiologists, an innovation that requires strong commitment and collaboration across potentially competitive clinical groups. Similarly, building the necessary systems of care to coordinate Emergency Medical Services and hospitals so that catheterization laboratories can be activated before the patient arrives at the hospital may require capital equipment, training of emergency medical personnel, and collaboration across service providers not under the control of the hospital. Nevertheless, recent studies have shown that such systems of care with Emergency Medical Services are feasible and effective ,18,19, sImportantly, traditional hospital characteristics, such as hospital size and teaching status, are not strongly correlated with reported approaches to reducing D2B time. Rather, hospitals that have been successful in reducing D2B time substantially demonstrate a clear organization-wide goal to reduce D2B time, strong administrative support for improvement efforts, uncompromising clinical leadership to promote improvement, and effective collaboration among departments and disciplines . These dAlthough this study provides the most current data on the implementation of recommended strategies for reducing D2B time, the data should be interpreted in light of their limitations. First, the data are self-reported, although we used an instrument that had been pre-tested and used in previous studies of this nature. Furthermore, we anticipate that self-reporting might bias the prevalence data upwards so that our reported findings may actually overestimate the true use of these strategies. Second, surveys were completed by a single respondent, although researchers were clear with respondents that they should consult with other hospital staff as necessary before reporting the existing processes in place at the hospital. The issue of documenting organizational practices is complex; however our approach was designed to reflect the combined views of key staff involved with treating patients with STEMI. Nevertheless, to the degree respondents knew what was recommended, this limitation may bias the prevalence upwards so that our findings may, again, overestimate the true use of these strategies. Third, although the sample is large and the response rate is high, especially for organizational studies of this type, the data reflect D2B Alliance hospitals rather than a random sample of hospitals that perform PCI. Experience in hospitals that did not choose to enroll in the D2B Alliance may be different. Finally, our study did not examine hospital strategies that might be linked to faster door-to-needle times, as it was beyond the scope of the study.In conclusion, this study establishes that key strategies associated with more rapid D2B times are underutilized across the country. Although many hospitals use some of these key strategies, few have adopted them all. Approaches to speeding adoption are varied; participation in inter-organizational learning efforts, such as the D2B Alliance, may be effective in promoting the efficient diffusion of such strategies. However, evaluation of its effectiveness awaits the completion of ongoing and future studies. Our findings highlight the opportunity to improve care and provide baseline data so that the effect of the D2B Alliance can be rigorously evaluated in the future.The authors declare that they have no competing interests.EB designed the study and analysis, and drafted the manuscript. BN designed the survey and analysis and revised the manuscript. AS and JB implemented the survey and revised the manuscript. EC collected and analyzed the survey data. YW analyzed the data and performed the data management. CY collected data. IN revised the manuscript. JB assisted in the study design. HK designed the study and analysis, and revised the manuscript. All authors have read and approved the final manuscript.


Blood spots collected onto filter paper are an established and convenient source of antibodies for serological diagnosis and epidemiological surveys. Although recommendations for the storage and analysis of small molecule analytes in blood spots exist, there are no published systematic studies of the stability of antibodies under different storage conditions.Plasmodium falciparum antigens, MSP-119 and MSP2, and calculated titres used to fit an exponential (first order kinetic) decay model. The first order rate constants (k) for each spot storage temperature were used to fit an Arrhenius equation, in order to estimate the thermal and temporal stability of antibodies in dried blood spots. The utility of blood spots for serological assays was confirmed by comparing antibodies eluted from blood spots with the equivalent plasma values in a series of samples from North Eastern Tanzania and by using blood spot-derived antibodies to estimate malaria transmission intensity in this site and for two localities in Uganda.Blood spots, on filter paper or glass fibre mats and containing malaria-endemic plasma, were desiccated and stored at various temperatures for different times. Eluates of these spots were assayed for antibodies against two Antibodies in spots on filter paper and glass fibre paper had similar stabilities but blood was more easily absorbed onto filter papers than glass fibre, spots were more regular and spot size was more closely correlated with blood volume for filter paper spots. Desiccated spots could be stored at or below 4°C for extended periods, but were stable for only very limited periods at ambient temperature. When desiccated, recoveries of antibodies that are predominantly of IgG1 or IgG3 subclasses were similar. Recoveries of antibodies from paired samples of serum and of blood spots from Tanzania which had been suitably stored showed similar recoveries of antibodies, but spots which had been stored for extended periods at ambient humidity and temperature showed severe loss of recoveries. Estimates of malaria transmission intensity obtained from serum and from blood spots were similar, and values obtained using blood spots agreed well with entomologically determined values.This study has demonstrated the suitability of filter paper blood spots paper for collection of serum antibodies, and provided clear guidelines for the treatment and storage of filter papers which emphasize the importance of desiccation and minimisation of time spent at ambient temperatures. A recommended protocol for collecting, storing and assaying blood spots is provided. When carrying out serological surveys, particularly in remote locations, it is of great advantage to have a method of collecting and storing blood samples which does not require that facilities for centrifugation are accessible, and which is relatively robust to irregular degrees of refrigeration, at least for short periods. One approach that seems to offer these advantages is to collect samples as dried blood spots, and to recover antibodies from the dried spots once transferred back to the laboratory. Blood spots have the advantage that they are quick, simple and inexpensive to prepare and to store, require very small blood volumes which can be obtained by finger- or heel-prick, and are likely to be more socially acceptable in cultural contexts in which larger volumes of blood are difficult to collect. Blood spots have been used routinely since the 1960s ,3. BloodAlthough the stability of low molecular weight analytes in blood spots has been extensively studied  and guidBlood spots for stability studies and protocol optimisation were artificially constituted from fresh erythrocytes and stored heparinized plasma from hyperimmune donors from Brefet, The Gambia . To deteFor spot size measurements, fresh heparinized blood was spotted onto sheets of Whatman no 1, Whatman 3 MM  or glass fibre  filter paper in volumes from 1 μl to 45 μl. For antibody stability and recovery studies, reconstituted blood was prepared by mixing individual African malaria-hyperimmune plasma samples or a hyperimmune plasma pool or a European (non-exposed) negative control pool  1:1 by volume with washed European blood group O erythrocytes and immediately spotting 10 μl of this mixture onto Whatman 3 MM or glass fibre filter paper. Spots were allowed to dry at ambient temperature and relative humidity (RH) overnight.Laboratory-generated blood spots and blood spots from Uganda were stored in individual self-sealing plastic bags (25 cm × 25 cm approx), which were then combined into sets and stored within three further successive plastic bags, the innermost of which contained approximately 3 g of self-indicating silica desiccant gel type III (Sigma). The bags were inspected regularly to confirm that the desiccant remained blue (RH < 20%), and the desiccant replaced if necessary.Fingerprick blood samples from lower Moshi were collected into EDTA-coated microtainers and as blood spots on Whatman 3 MM paper and transported daily to the laboratory at Kilimanjaro Christian Medical College. Filter papers were refrigerated (2–8°C) in individual plastic bags with silica gel. Plasma was separated from packed red blood cells after centrifugation and stored at -20°C. All samples were assayed within 8 weeks of collection., 1997–2005). Pixel counts were converted to mm2 and plotted against the volume of blood applied.Increasing volumes (1 μl – 45 μl) of fresh heparinized blood were pipetted (five replicates per volume) onto Whatman 3 MM, Whatman No 1 or Glass Fibre filter paper and the paper allowed to dry overnight at ambient temperature and RH. Next day the paper was optically scanned at 150 dots per inch (approx 5.9 dots per mm) in a desk-top scanner  as 256-level grey-scale images. The spot areas were measured by integration using the program ImageJ  overnight for 18 h with gentle mixing (rotary shaker at 2 revs per sec), giving a concentration of eluted serum proteins equivalent to a 1:100 dilution of the original blood. .Plastic bags containing blood spots were allowed to return to ambient temperature before opening. Discs approximately 2.5 mm in diameter were cut from the filter papers using a leather punch . We found that this was best done by placing the papers on a 0.6 mm thick cardboard backing. Individual filter paper discs were transferred to individual wells of a 96 well titreplate (Greiner PS) and antibodies eluted with 150 μl PBS/0.05% (v/v) Tween20/0.05% (w/v) NaN19.GST  were coated with recombinant MSP-1subclass ) or MSP-subclass ) and blosubclass ,37 excepA titration curve was fitted to the ODs obtained for the standard plasma dilutions by least squares minimisation using a three variable sigmoid model and the solver add-in in Excel (Microsoft), assuming an arbitrary value of 1000 Units/ml of antibody against each antigen in the standard pool. OD values for the spot extracts were converted to units/ml using this fitted curve.Recoveries for blood spots were estimated as follows  versus time) in the statistical package Stata . This set of rate constants was then used to fit an Arrhenius equation [k) on 1/T, the reciprocal of the absolute temperature. Values of ln(k) were weighted by the logs of the reciprocals of the variances of the first-order fits. Best estimated values and confidence intervals for different temperatures were calculated from this fitting exercize. For the glass fibre spots, the values were also fitted directly by least squares minimization without log transformation (which gave less weight to the most degraded samples) with essentially similar results.The stability of the immunoglobulin captured on Whatman 3 MM and GF filters was assessed by accelerated stability studies. Replicate sets of spots were prepared and triplicate spots stored at temperatures from 23°C to 65°C for between 1 and 16 weeks, at the end of which time they were transferred to -20°C and all samples were analysed together at the end of the study by ELISA. OD values for antibodies to MSP1equation  to derivAge-specific prevalence of anti-malarial antibodies is highly correlated with a number of measures of malaria transmission intensity . To dete2/μl respectively. These correspond, for a 2.5 mm diameter spot, to volumes of 0.77 and 1.4 μl respectively. The ratio of these figures agrees well with the relative density of the two papers .For fresh blood, the relationship between total spot area and volume was linear for Whatman No 1 and Whatman 3 MM Figure  with gra2, however, for volumes below 10 μl the relationship was not co-linear with that for volumes above 10 μl.For blood spots on glass fibre paper the relationship between area and volume was less regular Figure . For volBoth of the cellulose papers, 3 MM and No 1, wetted easily completely through the thickness of the paper, giving clean edges to the spot Figure . The gla19 and MSP-2 and, in one experiment, for MSP-119 specific IgG1 and MSP-2-specific IgG3. The ODs obtained for the eluates were compared with those obtained for the corresponding plasma samples tested at the equivalent dilution. For both antigens the correlation between the OD values obtained for plasma and blood spot eluates was >0.98, with a recovery of >95% for total IgG    of the original antibody activity would be lost or, conversely, the length of time for which spots could be stored at varying temperatures in order to recover 80, 90 or 95% of the original antibody reactivity (Table 19 (which are predominantly of the IgG1 subclass[Eluates were prepared from sets of blood spots from 3 MM and Glass Fibre paper that had been stored for differing lengths of time at temperatures between 23°C and 65°C and tested by ELISA for reactivity with MSP-1) Figure . When us subclass) and MSP subclass) decayedIn general, predicted stabilities for blood spots on glass fibre paper were slightly lower than for 3 MM paper, but for all temperatures the 95% confidence intervals for the two formats overlapped, so these differences cannot be considered statistically significant.Overall, the study showed that antibodies in blood spots should be stable for extended periods of time (years) if kept desiccated at temperatures below 4°C Table . When keThe state of degradation of the blood spots could be roughly assessed by visual inspection of the eluates after overnight incubation. Since elution of haemoglobin from the filter papers correlates with elution of plasma proteins including immunoglobulin, filter paper discs from freshly made spots, or from spots stored at -20°C, are very pale (looking white against the haemoglobin-containing eluate when the plates are viewed from below) Figure , whereas19 (r2 = 0.93) and MSP-2 (r2 = 0.92) . For MSP-2, however, the antibody recovery efficiency was noticeably lower  Figure . For MSPy Figure , althoug19 antibodies. The samples were carefully desiccated and kept cool, and the appearance of the eluates from the spots was excellent  or as a source of DNA for PCR-based detection and genotyping of parasites. However, whereas the ability to discern simply the presence or absence of parasites is frequently sufficient for DNA-based assays, antibody measurements tend to be of more value if information about the quantity of the antibodies present can be reliably obtained. This requires implementation of carefully validated protocols, for collection and storage of spots and elution of antibodies, designed to maximize the recovery of intact immunoglobulins.The utility of the procedures presented here rests on the assumptions that the blood spot is evenly impregnated with blood, so that a disc of a standard diameter represents a defined volume of blood, and the adsorbed proteins can subsequently be eluted with high efficiency. A number of alternative substrates have been used for collecting blood spots and we have not been able to evaluate all of these here. Some of these substrates have been optimized for the recovery of DNA, but it is not clear that treatments to reduce nuclease activity and improve nucleic acid recovery are as beneficial for protein recovery. Of those that have been evaluated here, glass fibre mats and Whatman No 3 MM had high adsorption capacities while 3 MM and Whatman no 1 were robust for handling in a field environment. Glass fibre paper, however, was fragile, especially when punching discs, had inferior wetting characteristics and the efficiency of antibody elution was non-linear. Although antibody recoveries from No 1 paper were not systematically compared with those from 3 MM, limited experience suggests that recoveries are similar when the different adsorption capacities are allowed for. On balance, Whatmann 3 MM paper appears to offer the ideal combination of strength, wettability and adsorption. Moreover, comparison of amounts of antibodies recovered from freshly-prepared blood spots on 3 MM showed good correspondence with levels determined in the equivalent plasma.The utility of this protocol also depends on establishing storage conditions under which immunoglobulins are stable in dried form, and can be quantitatively recovered from the paper at a later date. To estimate the stability of intrinsically stable preparations it is necessary to carry out accelerated stability testing at elevated temperatures, ideally under conditions where a substantial fraction of the activity has decayed during the period of measurement -32. Stab2 as indicator remains classified as safe for use in desiccation despite the recent classification of CoCl2 as being "possibly carcinogenic to humans" by the International Agency for Research on Cancer [The other major factor affecting the stability of antibodies in blood spots is the humidity at which they are stored. This was not examined in detail, partly because of the added experimental complexity of manipulating RH, but, more practically, because of the impossibility of monitoring and integrating changing levels of RH in field use. However, storage of spots for as little as two weeks at ambient humidity reduced the recovery of intact immunoglobulins by more than 50%, a far greater decrease than would have been expected if the spots had been scrupulously desiccated. Given the importance of keeping blood spots dry, the simple visual check provided by self-indicating silica gel appears to be the simplest if not the only practical method of monitoring storage humidity. Silica gel containing CoCln Cancer  and its 19 and MSP-2 in the accelerated degradation study were similar, recoveries from field samples suggested that antibodies to MSP-119 (predominantly IgG1) appeared to be more stable than antibodies to MSP-2 (predominantly IgG3). IgG3 antibodies have a greatly extended and unstructured hinge region [Although the rates of loss of antibodies to MSP-1e region ,45 and iOne unexpected advantage of this method of sample collection is that the quality of blood spots of questionable provenance (in terms of their storage conditions) can be judged visually by examining the colour of the filter papers discs after overnight incubation in elution buffer. Discs that have been properly stored, and from which there is a very high recovery rate of antibodies, turn from deep red to pale pink or white after incubation, indicating that the adsorbed proteins (including haemoglobin) have been eluted. By comparison, discs stored at high temperatures remain dark red, indicating that the indicator protein haemoglobin can no longer be readily eluted from the filter paper. When blood spots from different malaria field studies, which had been collected for diagnosis or DNA recovery and had been stored for extended periods at ambient temperature and humidity were examined, the eluates of spots which gave poor antibody recovery were uniformly pale in colour. Thus, whilst visual inspection may not be a fool-proof means of assessing blood spot quality it will allow seriously degraded samples to be identified and discarded prior to ELISA analysis.Finally, the utility of blood spot collection of serum antibodies has been demonstrated by showing that it is possible to derive serological estimates of malaria endemicity from blood spot samples and that these serological estimates agree well with entomological data. Close agreement was observed between the serological and entomological data for Lower Moshi and for Rukungiri; agreement was less good for Kabale which may be due to the longer gap between the two estimates (the only estimate of EIR available was from 1996/7) or the inherent inexactitude of entomological assessments at low transmission ,47.A protocol for collection of blood spots for serological use has been developed and validated. It is recommended that blood is spotted onto 3 MM paper and allowed to dry overnight at ambient temperature and humidity. The papers should then be interleaved with non-adsorbent paper (such as that used for interleaving photographic prints) or cardboard covers and immediately placed in a self-sealing plastic bag which is contained within a second bag containing desiccant. These bags should then be placed within two further self-sealing bags. Spots can be kept at ambient temperature for up to seven days without serious loss of activity provided they are desiccated. They can be kept, desiccated, for up to four weeks if refrigerated at 4°C or below. For storage of more than four weeks, the desiccated spots should be kept at -20°C. The desiccant should be regularly inspected and replaced if it changes colour. (A recommended protocol for collecting, storing and assaying blood spots is provided in Additional file The authors declare that they have no competing interests.PC Conceived study, developed protocol, carried out and analyzed experimental work, drafted paper. JC developed protocol, participated in experimental work, drafted paper. TB conceived and carried out surveys in Tanzania. HL carried out surveys and assays in Tanzania. AM carried out surveys and assays in Tanzania. CL conceived, organized and carried out surveys and analyses in Uganda. JC supervised and participated in planning and execution of work in Uganda. TA assisted in supervision, planning and execution in Uganda. JG devized fitting algorithms, provided statistical advice. AG devised fitting algorithms, provided statistical advice. CD supervized and participated in surveys and assays in Tanzania and analysis of data, drafting paper. ER supervized work and drafted paper. All authors read and approved the final manuscript.Estimation of recoveries from ODs. Outline of methods used to calculate relative recoveries from two sets of ODs of same samples.Click here for fileProtocol for obtaining, storing and reconstituting blood spots for serological purposes. A full recommended protocol for Preparing, sampling and storing blood spots for serological use, and instructions for reconstituting and assaying antibodies by ELISA.Click here for file


Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation 3.In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responsesin vivo tracking with non-invasive MR imaging techniques4.Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging. Cells are grown to 80% confluence at least 18 hours before labeling. During this time, labeling media is prepared by adding Endorem to the sample at a dose of 100 ug Fe/ml serum-free media. After cells are grown to confluence, culture media is aspirated and cells are washed 1x with PBS or serum-free media.2 for 4 hours. Next, the rinse solution is aspirated and the previously prepared labeling media is added. The cells are then incubated at 37°C, 5% CO After 4 hours incubation, labeling media is aspirated and cells are rinsed with PBS and trypsinized as usual. Once trypsinized, cells are washed 3x with PBS and centrifuged at 400rcf for 5 minutes. After this washing step, cells are resuspended, counted, assessed for viability, and used as needed for experimentation. After the cells have been labeled it is time to prepare the agarose solution. On a fine scale, 80mg of agarose powder are weighed and added to 2ml of PBS. This solution is then gently shaken and autoclaved. After approximately 40 min, the fluid agarose solution can be taken out of the autoclave. The volume should be measured and adjusted accordingly with preheated PBS. Then the solution is cooled down to 42°C When the agarose solution has reached the desired temperature, the cells are counted and centrifuged for the last time in a 1.5ml Eppendorf tube.6 cells/ml. After centrifugation, the supernatant is discarded and the cells are mixed with the DMEM to reach a concentration of 30 x 10 Now the cells are mixed with the 42°C warm agarose. A cold pipette tip may cause agarose to solidify inside the tip, therefore it is advised to preheat the tip by pipetting hot sterile PBS up and down Now, the same volume of agarose as media is taken up and mixed thoroughly with the cell-media-suspension so that a homogenous suspension is created. Keep the Eppendorf tube inside a preheated water bath while mixing so that the agarose does not cool down enough to solidify. When the suspension is homogenous, the cells are implanted in the defect.          Figure 1. Coronal T2 weighted SE images (TR 4000 ms/ TE 18.27 ms) of a patella specimen with implanted hMSCs in cartilage defects.          The described protocol provides a reproducible way to label MSCs with iron oxide nanoparticles and implant these labeled MSCs into cartilage defects. This technique permits non-invasive depiction of stem cell transplants in cartilage defects with MR imaging, which allows an early detection of MASI failure. A dislocation or efflux of the labeled cells can be diagnosed based on a disappearance of the label from the transplantation site on MR images. An early diagnosis of these events is desirable and would prevent the patient from unnecessary alternative invasive diagnostic procedures. The described non-invasive method for diagnosing MASI outcomes could aid in the successful development of cell based therapies for cartilage regeneration in osteoarthritis. Our protocol is currently applied in preclinical in-vivo studies, would be in principle clinically applicable and could serve as non-invasive outcome measure for the assessment of MASI therapies in clinical practice.


BMC Genomics. 9 (Suppl. 2), S4) revealed that HIV matrix protein p17 possesses especially high levels of predicted intrinsic disorder (PID). In this study, we analyzed the PID patterns in matrix proteins of viruses related and unrelated to HIV-1.A previous study  Protein intrinsic disorder toolbox for comparative analysis of viral proteins. mac and HIV-1 p17 proteins were predicted by PONDR VLXT to be highly disordered with subtle differences containing 50% and 60% disordered residues, respectively. SIVmac is very closely related to HIV-2. A specific region that is predicted to be disordered in HIV-1 is missing in SIVmac. The distributions of PID patterns seem to differ in SIVmac and HIV-1 p17 proteins. A high level of PID for the matrix does not seem to be mandatory for retroviruses, since Equine Infectious Anemia Virus (EIAV), an HIV cousin, has been predicted to have low PID level for the matrix; i.e. its matrix protein p15 contains only 21% PID residues. Surprisingly, the PID percentage and the pattern of predicted disorder distribution for p15 resemble those of the influenza matrix protein M1 (25%).Both SIVOur data might have important implications in the search for HIV vaccines since disorder in the matrix protein might provide a mechanism for immune evasion. The viral matrix protein underlies the envelope of a virion, representing essentially a link between the envelope and the nucleocapsid ,2. The fLentivirinae is among the genii of viruses that possess a matrix layer ,8. Viruscpz are more closely related to HIV-1, whereas most of the others, especially SIVsm, and SIVmac, are closer to HIV-2 [There are three known HIV viruses in the world today, HIV-0, HIV-1, and HIV-2 ,10,11. Tto HIV-2 . It doesto HIV-2 .Similar to HIV and SIV, EIAV is another retrovirus ,9, whichTherefore, these data clearly show that related viruses might affect their hosts differently, possessing variable virulence and different modes of interaction with their host's immune systems. A question then arises is whether some of the mentioned variability in the behavior can be reflected in some peculiar features of the corresponding viral proteins. This paper examines matrix proteins of several related viruses using computational tools such as intrinsic disorder predictors to search for the crucial differences in the levels and distributions of intrinsic disorder in the matrix proteins.® family of disorder predictors, VLXT and VL3 [® VL3 was chosen because of its high accuracy in the prediction of long disordered regions [® VLXT was shown to be extremely sensitive for finding function-related disordered regions [The concept of protein intrinsic disorder is used in this paper to investigate characteristics pertaining to the various viral matrix proteins. Intrinsically disordered proteins have been described by other names such as "intrinsically unstructured" ,20, "nat and VL3 -38, to e regions , whereas regions ,39,40. U® VLXT and VL3 respectively. Data for the four matrix proteins with known 3-D structures are further illustrated by Figure ® VLXT analysis as bar chart. High level of predicted intrinsic disorder in SIV and HIV-1 matrix proteins is clearly seen. In our earlier paper [® VLXT between 20–29% were considered moderately disordered; those in the range of 30–39% were considered as quite disordered by prediction; whereas, proteins that were disordered 40% and above were considered as very disordered by prediction. Therefore, the influenza M1 protein and EIAV p15 should be considered as moderately disordered by prediction. By the same rule, the SIVmac and HIV-1 p17 matrix proteins have to be considered as highly disordered by prediction.Table er paper , the fol® VLXT plots in Figure ® VLXT plots.While Figure ® VLXT scores suggesting that highly flexible regions of matrix proteins are responsible for protein-protein interactions. For example, contacts between the subunits of HIV-1 p17 are located near or within regions predicted to be disordered, whereas contact sites of the EIAV p15 are mostly located in regions with high B-factor. These observations are in a good agreement with earlier studies which established the usefulness of intrinsic disorder for protein-protein interaction [Analysis of Figure eraction ,40,43-46® VL3 , whereas the regions marked in red are those predicted to be disordered by PONDR® VLXT. Different colors such as yellow and green are used to denote different subunit regions. This presentation of structured proteins allows visualization of regions with the intrinsic propensity for being highly flexible.Figure mac is a subtype of SIV, which was first found in macaques and is known to be very closely related to HIV-2 [The HIV-2 and HIV-1 viruses, while related, differ in substantial ways in term of immune response, infection, and the onset of AIDS ,11,12. Sto HIV-2 . While dmac and HIV-1 are quite similar, even though the percentage of PID in SIVmac p17 (50% by PONDR® VLXT) is smaller than that in HIV-1 p17 (61%). The similarity in the level of PID is likely indicative of the ability of both viruses to evade the immune system. Further support for this hypothesis can be retrieved by analyzing the level of predicted disorder in the influenza M1 protein and in the EIAV p15 protein. Matrix proteins of both of the viruses have low percentage disorder rates, 25% in M1 and 21% in p15. Interestingly, effective vaccines were developed for both of these viruses, even though the mutation rates of the influenza virus is extremely high causing well-known difficulties in the development of new vaccines. Apparently, the PID rate is a good predictor of the ease of vaccine development of a virus. This should not be surprising as our earlier study [A brief glance at Table er study  suggesteer study . This himac and HIV-1 p17 proteins seem to be similarly high, the PONDR® VLXT plots revealed subtle differences in the disorder distribution within the protein sequences. Figures mac p17. Figures mac which seems to be crucial for inter-subunit contacts suggests that disorder-disorder protein-protein interactions are replaced by the order-disorder or order-order interactions.Even though the rates of predicted disorder in the SIV® VLXT and B-factor curves, as it is seen, e.g. in Figure Figure ® VLXT, see Table Matrix protein of EIAV was predicted to be less disordered than that of HIV  in their patterns of predicted disorder distribution, which also might contribute to the virus's ability to evade the host immune system.The question then arose: What are the potential implications of more rigid or more disordered matrix proteins? It is likely that more rigid p17 proteins may be less effective in evading immune response. This may be a reason why HIV-2 and SIVmac as in the search for HIV vaccination and drugs. SIVmac and SIVstm were the first subtypes found in laboratory macaques [Our findings might also have some implications to the search for HIV vaccines. One possibility is related to the use animal models and SIVmacaques . Asian pmacaques . For thimacaques . HoweverIt is established that the HIV envelope glycoprotein gp120 is one of the most glycosylated proteins in nature . Oligosamac p17 proteins. This feature may be attributed to the ways the viruses are evolved and are transmitted to their hosts. It should be reminded that EIAV is transmitted between horses via insect vectors. In other words, the virus experience dramatic change in the environment during the transmission. It is likely that this mode of transmission has evolutionary requirements similar to those of the influenza virus, which is transmitted via respiratory tract and mucus. HIV and SIV, on the other hand, spread by blood contact or sexual activities. Since it there lesser chance for the exposure to the outside environment in the transmission mode, there is hence lesser evolutionary pressure for the matrix proteins to be ordered. This highlights a role for the matrix protein in many viruses. In many instances, the matrix acts as an encasement for the virion, thereby protecting the virion from damage especially in adverse environments. We have also seen that disorder at the matrix is not an absolute characteristic of retroviruses.A peculiar finding of this paper is the pattern of predicted disorder of EIAV p15 matching more closely the disorder profile of the influenza M1 protein than those of the matrix proteins of its closer relatives, namely the HIV-1 and SIVA single nagging puzzle in the search for vaccines against HIV is the unknown mechanisms helping the virus to evade immune response. Our study suggests that this ability might arise from the abnormal levels of intrinsic disorder at the viral matrix. This hypothesis is supported by the fact that the matrix proteins of other viruses, where vaccines have been more easily found, were predicted to be more ordered. Therefore, there are several ways how disorder predictions can be utilized in the future strategies of the vaccine development. Particularly, one of the new directions in the anti-HIV drug development could be a search for the therapeutic agents able to stabilize the HIV matrix protein.Another puzzle of HIV viruses is the inability of virologists to account for the waves of the HIV strains seen, even after taking into account the fact that the mutation rate of HIV-1 is 25-times that of influenza. Yet another HIV puzzle is the greater pathogenicity of HIV-1 as compared to HIV-2. It has been generally understood that this is due to the fact that the HIV-1 affinity for CD4 is 28 times greater than that of HIV-2 . Our datWhile this paper has been largely focused on the study of immune evasion as applied to HIV and HIV-related viruses, it may provide a model for immune evasion by other entities, such as cancer cells. There are either very few or no studies done in this area. Perhaps, our results could invigorate interest in this area, given the models and approach used. Furthermore, the results of this paper likely have novel strategic implications for experimental studies on the use of viruses as oncolytic agents, which have often been observed to be rendered ineffective by the immune system. In fact, one of the greatest problems in using the oncolytic viruses is that they are detected by the immune system very quickly so they are only useful for localized treatment of tumors . Our datmac was used in lieu of HIV-2 since the two are genetically close and the X-ray diffraction data for EIAV matrix and capsid proteins were readily available.A full description of implementation techniques can be found in a previous paper . The seaGiven the appropriate accessions selected, JAVA programs were used to automatically place the necessary information into the MYSQL database. The data were often checked using the SQL (Sequel Query Language) .®  is a set of neural network predictors of disordered regions on the basis of local amino acid composition, flexibility, hydropathy, coordination number and other factors. These predictors classify each residue within a sequence as either ordered or disordered. PONDR® VL-XT integrates three feed forward neural networks: the Variously characterized Long, version 1 (VL1) predictor from Romero et al. 2001 [PONDRal. 2001 , which pal. 2001 , which p® VL3 combines the predictions of 30 neural networks for the entire protein sequence and was trained using disordered regions from more than 150 proteins characterized by the methods mentioned above plus circular dichroism, limited proteolysis and other physical approaches [PONDRproaches .In order to detect the locations of protein-protein contacts between the different chains of proteins , a JAVA program was written to check the interchain atom-atom distance. The program generated graphs with PONDR plots with locations of the protein-protein contacts.The JAVA programming language was used to generate codes readable by the molecular 3D software, Jmol . In resuB-Factor values were imported directly from the PDB files into the table access_seq  that wasThe authors declare that they have no competing interests.GKMG proposed the idea of the study, carried out the analyses and drafted the manuscript. AKD helped to design experiments and participated in the manuscript drafting. VNU coordinated the studies, participated in their design and helped to draft the manuscript. All authors read and approved the final manuscript.


Specific anti-influenza virus and normal transfer factors prepared in an experimental animal model, the pig, have been tested for their components, characteristics, and activity of known specificity. Two transfer factors are small molecular mixture which consist entirely or partly of polypeptides and polynucleosides. Moreover, the biological activity of transfer factors could be approved by Rosettes test and specific skin test. The study would lay a foundation for the research and development of other specific transfer factor. Transfer Factor (TF) was discovered in the 1940's and has been extensively studied for the past 50 years (1). In recent years, it has been known that Transfer Factor can transfer cell-mediated immunity (CMI) from an immune donor animal to a non-immune recipient. And now it is not only a scientifically recognized delivery system for transferring immune system advantages from one species to another but also most effective in regulating immunity to infections in which cell-mediated immunity  is important for controlling the infection . MoreoveTransfer factor, an immunomodulator of low molecular weight capable of transferring antigen-specific cell mediated immune information to T-lymphocytes, has been used successfully over the past quarter of a century for treating viral, parasitic, and fungal infections, as well as immunodeficiencies, neoplasias, allergies and autoimmune diseases. Moreover, several observations suggest that it can be utilized for prevention, transferring immunity prior to infection. Because it is derived from lymphocytes of immune donors, it has the potential to answer the challenge of unknown or ill-defined pathogens. Thus, iWe present arguments for the use of cell mediated immunity for the prevention of the infection as well as for the treatment of infected patients . SimilarMost of the original clinical trials with transfer factors ,5 used pIn this paper, we present the results of the methods of preparing and analyzing the specific transfer factor oral preparation in vitro experimental system. The data indicated that the methods were credible and the biological activity of the transfer factor resides entirely or partly in vitro.Pig spleens were obtained from slaughterhouse at Huanggang town in Zhaoqing city. Normal oral Transfer factors (H20013408) were bought from the drugstore in Zhaoqing city. Centrifuge , ultraspectrophotometer(UV-2550 made in Japan), central empty ultrafiltrition equipment (MOTIANMO company in Tientsin), tissue disintegrator, superlow temperature refrigerator and other essential apparatus exist in our Lab. Some chemical reagents were bought from the chemical reagent store in Zhaoqing city.10 Healthy pigs weighted 75 kilogram were chosen and vaccinated with influenza vaccine for human use, every pig was injected through muscle one unit, and 15 days after the first injection, the second injection would be performed in the same dosage as the first time. In 20 days after the second injection, the pigs would be slaughtered, and the spleens would be collected and stored in refrigerator.Specific and non-specific transfer factor were prepared from the vaccinated and normal pigs spleens through superultrafiltrator equipped with a membrane of 6000 dalton. The method was referred to literature [These frozen pig spleens was taken turns through mechanically crashed, frozen and thaw reduplicatively, centrifugation, filtration, superultrafitration, formulation and finally an oral normal and specific transfer factors would be prepared for the characteristics examined. The unit would be confirmed according to the criteria from the seventh international session on TF, that is, OD (ABS)240-260 nm was up to 10 as one unit. And the TF oral solution was formulated with some excipients.260/A280 was larger than 1.8 after detecting.Two kinds of samples would be detected by ultraviolet spectrum absorption. Normal saline would be as a control. It would be considered qualified if the ratio of AProtein would be detected with 20 percent of Sulfonic-Salicylic acid. It was considered as positive if the solution examined was cloudy whereas negative if lucidity.Three milliter of the sample of Specific and non-specific TF were added eight percent of Sulfonic-Salicylic acid for three milliter respectively and put at quiescence for 40 min at 4°. And then permitted them centrifuging at 18000 g for 40 min. The samples were diluted and analysed with type of 835-50 Amino acid auto-analysing instrument.Detection for content of polypeptides in TF including specific, non-specific and normal transfer factor sold on the medicine store would be performed by biuret reaction . And theContent of nucleosides (ug/ml) equal to value from the standard curve multiply multiples dilutedTo test for the heat sensitivity of the transfer factor solution, aliquots were diluted 20-fold with 50 mM Tris, pH 8, and then incubated for 10 min at various temperatures, ranging from -20°to 80°C. The lability of TFs would be detected according to ultraviolet spectrum absorption. The different TFs were put under the different temperature  for 10 days.Because transfer factor can make a mammalian immune system to elicit a secondary immune response, whereas infecting pathogen or antigenic agent to facilitate a secondary, or delayed type hypersensitivity, Thus the animals administered specific TF would be detected their delayed type hypersensitivity through skin test. 15 rabbits without any antigen infecting were chosen to divide 3 groups, 5 each group. Each rabbit in the first group would administered orally specific TF one unit once daily for 10 days, the second group would be orally given normal TF about dosage as above mentioned, the third group received the same amount of sterile saline diluent as control. And three days later the animal was shaved on back and injected intradermally with concentrations of 10 ul influenza vaccine and bcg vaccine respectively. Positive reaction would be considered according to the size of swollen nodus if it was larger than 1 mm. Contrarily, it was negative.Germ in the preparations were check up in terms of method  whether 15 healthy BALB/c mice were chosen for toxic test. And 3 groups were divided randomly, 5 mice in one group. The mice in group TF were infused with concentrated TF oral solution 40 units once daily for 7 days. Moreover, control group administering normal saline at the same dosage. Activation and appearance of the mice tested would be observed after administering high dosage TF with control group as comparation .6/ml)from human by Ficoll-Conray centrifugation by mixing them with red blood cells  from rat. And the action of peripheral lymphocytes of human was investigated. The concretive operations was followed as table E-rosettes test is an effective and simple method which identify the activity of T-cells from animals . The ideSmaller molecular weight molecules , including any transfer factor from the pig spleens, remained in solution. The physicochemical properties of TFs presented whatever specific or non-specific TF preparations were all transparency and light yellow fluid with a pH 6.5-7.0 and negative protein reaction. They contain sixteen amino acid residues without examining Ser Table . These vThe analysis of TF preparations in ultraviolet spectrum absorption indicated that the highest peak of specific and non-specific TF were similar without differences pertaining to the range of normal TF Table , 2. And Detection for content of polypeptides in TF indicated that content of specific was higher than that of non-specific and was also closed to normal transfer factor sold on the store Figure . And theTransfer factor is heat-sensitive. A solution of specific and non-specific transfer factor heated from 37 to 80° retained full biological Activity identified according to the varieties of TFs untravilet spectrum absorptions. When the TF solutions were at -20°C, 4°C and 20°C or heated from 37°C to 60°C retained partial biological activity  over that of the control rabbits  -10(-3) U) upon active rosette formation was studied to quantify T cells could significantly increase EAC percentage, but no significant difference between specific and non-specific TF including polypeptides of may amino acids components and a nucleoside portion.The specific pig transfer factor has the ability to generate an early secondary immune response in mammals as it could initiate an early delayed type hypersensitivity immune reaction in rabbit. However, it is clear that an appropriate in-vitro laboratory evaluation of each TF batch and of its destined recipient is essential in order to define the function and applications of the TF. Thus, what this suggests is that the transfer factor can not only use in treat influenza, but also prevent future breakouts as well for further study. This same action may apply to other viral infections like chronic fatigue and bronchitis.The authors declare that they have no competing interests.LH participated in the examination of STF, YW joined the preparation of STF, XL participated the examination of STF, SL and YZ also joined working on examinations of STF.All authors have read and approved the final manuscript.


AIDS is the main driver of young widowhood in southern Africa.The demographic characteristics of widows, their reported risk behaviours and the prevalence of HIV were examined by analysing a longitudinal population-based cohort of men and women aged 15–54 years in Manicaland, eastern Zimbabwe. The results from statistical analyses were used to construct a mathematical simulation model with the aim of estimating the contribution of widow behaviour to heterosexual HIV transmission.413 (11.4%) sexually experienced women and 31 (1.2%) sexually experienced men were reported to be widowed at the time of follow-up. The prevalence of HIV was exceptionally high among both widows (61%) and widowers  (54%). Widows were more likely to have high rates of partner change and engage in a pattern of transactional sex than married women. Widowers took partners who were a median of 10 years younger than themselves. Mathematical model simulations of different scenarios of sexual behaviour of widows suggested that the sexual activity of widow(er)s may underlie 8–17% of new HIV infections over a 20-year period.This combined statistical analysis and model simulation suggest that widowhood plays an important role in the transmission of HIV in this rural Zimbabwean population. High-risk partnerships may be formed when widowed men and women reconnect to the sexual network. The results from these statistical analyses were used to inform the construction of a mathematical model with the aim of estimating the importance of widow behaviour in the heterosexual transmission of HIV.The Manicaland HIV/STD Prevention Study includes a population-based open cohort study, full details of which can be found elsewhere.HIV serological testing was performed on dried blood spots using a highly sensitive and specific antibody dipstick assay.30For the purposes of prevalence calculations, the baseline and follow-up survey populations were treated as separate cross-sectional studies. Thus, all subjects interviewed in a given study round were included in the denominator of prevalence calculations. Men and women who had not yet started sex were included, but percentages are also presented based only on the sexually experienced population .Individuals who were part of a marital union at the time of the baseline survey were considered the population at risk of becoming widowed. The incidence of widowhood was calculated as the number of married people at baseline reporting to be widowed at the time of follow-up divided by the time at risk. The year of the widowing event was available from questionnaire data, but was sometimes missing or inconsistent . In either situation, we assumed that the person was widowed halfway (1.5 years) through follow-up. Widowing events where a husband and wife in a marital pair both died in the 3-year follow-up period may have been missed if the household dissolved following the deaths, potentially resulting in underestimation of widowing incidence rates. Rates were stratified by gender, age and HIV status at baseline. For the purposes of calculating the incidence of HIV, time-at-risk from all individuals negative at baseline who were found at follow-up were included in the denominator; individuals lost to follow-up were censored halfway through the study period.oisson regression models were fitted to the widowing incidence data and HIV incidence data and are presented with respect to age, gender and HIV status at baseline (widowing incidence only). Multiple logistic regression was used to model widowhood on HIV status at follow-up. Because age, marital status and HIV prevalence are correlated, we controlled for age in order to describe the independent association of marital status with HIV. Cox proportional hazards models were fitted to remarriage events and are presented with respect to gender, controlling for age. The combination of the questions “How many years is it since your last marital partner passed away?” and “How many years later did you remarry” were used to construct a Kaplan-Meier curve of remarriage. Data on individuals who had been widowed but not yet remarried were included as being censored at the end of the follow-up period.Based on the findings from the statistical analysis, a preliminary mathematical model was constructed. While individual risks of infection can be measured directly from cohort studies such as the Manicaland cohort, their contribution to HIV transmission at the population level cannot. The model presented here seeks to examine the importance of male and female widow behaviour in the transmission of HIV, rather than to quantify the impact of interventions targeted at this group. Four sets of alternative assumptions were used to define the baseline pattern of sexual behaviour of widows, where parameters could not be directly based on the survey data. Based on previously described models,Two parameters were estimated directly from the longitudinal data from the present study: the rates of being widowed, stratified by gender and HIV status, were based on the incidence of widowhood, and the rate of widow remarriage, stratified by gender, was estimated on the basis of the Kaplan-Meier survivor function. Other parameters were selected as described previously.31To illustrate the full contribution of widows to the spread of HIV, we compared a simulation where widows remain active with one where they do not, given different assumptions about their sexual behaviour.At baseline there were 4261 men and 5319 women whose marital status was known and at follow-up there were 3341 men and 5096 women resulting from loss to follow-up and new recruitments to the cohort. Among the subjects interviewed and tested for HIV at baseline whose marital status was known, 2242 men and 3265 women were followed up (follow-up rates: 54% for men and 66% for women). Thirteen men and 292 women who were widowed at baseline were identified at follow-up (follow-up rates: 39% for widowers and 80% for widows). An additional 180 men and 219 women with full data from the baseline study were known to have died.The proportion of sexually experienced men who reported to be widowed was 1.2% at baseline (n = 44) and follow-up n = 33, . The proAt follow-up the prevalence of HIV was high among ever-widowed men (54%) and women (63%). The prevalence of HIV was higher in widows than in those presently married and in those never widowed for both genders .For women, the incidence of HIV was not significantly higher in widows , 95% CI 2.2 to 10.9) than in married women . Of the 14 men who were widowed and who were HIV-negative at baseline, 2 had died and 7 were lost to follow-up. None of the remaining 5 seroconverted, thus precluding a full analysis of the incidence of HIV in widowed men.Mortality rates for both male and female widows (95 per 1000 pyrs and 39 per 1000 pyrs) were substantially higher than for other marital status categories . MortaliMortality rates were higher in HIV-positive widowers than in HIV-negative widowers (62 per 1000 pyrs vs 17 per 1000 pyrs); the same was true for widows (117 per 1000 pyrs vs 51 per 1000 pyrs). Even after controlling for HIV status at baseline, there was increased mortality in widows  and widowers  compared with individuals married at baseline. This suggests that mortality clustering, where one spouse’s death follows their partner’s death, is not completely attributable to HIV in this population.Seventy-three of 94 widowers (77%) reported sexual activity in the previous year compared with 104 of 297 widows (35%). Traditional practices in Manicaland discourage widows from taking another partner for 1 year after the death of their spouse. Thus, the high levels of reported sexual activity in the first year after widowhood  are surprising, but this figure may be influenced by relations with partners who died <1 year ago, as well as recall bias.The majority  of women widowed at baseline reported no (0) total sexual partnerships in the 3 years of follow-up compared with (1) for women who were separated, divorced, married or unmarried but sexually experienced. However, the mode masks the increased likelihood of extreme levels of sexual activity (⩾10 partners in 3 years) of widows compared with those married at baseline . Only 13 men who were widowed at baseline were found at follow-up, so no comparison could be made. However, separated and divorced men were much more likely to have high sexual activity (⩾10 partners in 3 years) than those married at baseline . A summary of the partnership history in the follow-up period is shown in 2 test).Twenty-four of 48 (50%) sexually active widowed women at follow-up reported that they had received money or gifts in exchange for sex with their last partner compared with 83 of 2794 (3%) married women  influences the age patterns of HIV in this population.2 test).Thirty-one of 45 widows (69%) reported that their last partner was married to someone else compared with 320/2281 married women , although HIV-positive widows reported condom use less frequently than HIV-negative widows (10/24 (42%) vs 8/11 (64%); p = 0.11, χ2 test).Condom use (either consistent or inconsistent) was more commonly reported by widowed women than by married women (21/40 (53%) vs 151/1921 (8%); p<0.001, χParameterisation of the rate of being widowed stratified by gender and HIV status was based on the incidence of widowhood shown in For the purposes of our baseline model, we assume that the rate of partner change among widows is equal to that of the non-widowed population, with widowed men from older age groups (⩾30 years) having a preference for women 5–10 years younger and widows preferentially selecting non-widows as their partners. Removing widower sexual activity from the model has a minor impact, whereas stopping female sexual activity results in a 2–3% decrease in adult prevalence within 10 years. This combined statistical analysis and dynamic simulation modelling suggest that widowhood may play an important role in the transmission of HIV in this rural Zimbabwean population. The prevalence of young widowhood, particularly among women, is high (11.4%) in Manicaland. In addition, the prevalence of HIV among widows (63%) and widowers (54%) is exceptionally high and is consistent with the finding that the incidence of becoming widowed is strongly associated with HIV status. However, we found no evidence that widowed men or women had a greater risk of becoming infected with HIV than married individuals, supporting the idea that the high prevalence of HIV among widows is a result of infections acquired while in marital unions rather than as widows. However, widows may be a source of infection as they resume sexual activity, since the sexual relations they form may be very risky for their partners. Men are likely to take on partners much younger than themselves, and women are likely to have relations with already married men and are disproportionately involved in high levels of sexual partner acquisition (⩾10 partners in 3 years) and transactional sex. These relationships may be important means by which HIV is transmitted in a sexual network to the partners of widows and their partners’ partners.The mathematical model simulations presented here serve to illustrate the relative importance of particular behaviours among widows in the transmission of HIV in this population. The model suggests that, at the population level, the impact of heterosexual transmission of HIV from widows is non-trivial. The cessation of male and widow sexual activity would result in the aversion of 8–17% of cases. Clearly, the cessation of such activity is neither possible nor necessarily desired, but this scenario illustrates the scale against which the impacts of other interventions targeted at widows  should be measured. Widow sexual activity is much more important than widower sexual activity in the models, but this prediction is simply explained by the higher observed prevalence of widows in the study population. As female mortality rises as a result of AIDS,Widows in Zimbabwe have an exceedingly high prevalence of HIV, mainly because of infection acquired in marriage.Many widows resume sexual activity following the death of their partner. Aspects of widow and widower sexual behaviour may put both themselves and their partners at risk.Widowhood plays an important role in the transmission of HIV in this rural Zimbabwean population and may be associated with 8–17% of all HIV cases.The strong sex difference, with widower prevalence at 1.2%, is notable and is likely to be a result of male mortality being generally higher combined with the impact of the early stages of the HIV epidemic which disproportionately affected men, and the pattern of husbands being older than wives.6Although participation rates were high, losses to follow-up were substantial. The most common reasons for loss to follow-up in our study were migration out of the study area and households not being found at follow-up.A limitation of this analysis is that we could only consider patterns of widowhood during a 3-year follow-up period. In order to see if remarriage patterns are changing, a longer study period will need to be analysed, which will be possible as data become available from further rounds. In this analysis we have focused on quantifying contributors to transmission probability and contact patterns with respect to widowhood. However, these individual characteristics and behaviours will depend on local social networks and the cultural milieu, which are predicated on demographic change, legal frameworks and other structural factors.36In the immediate term, counselling and testing services should reinforce efforts to provide widows with support and knowledge needed to make safe choices after the death of a spouse, and encourage participation in church or women’s organisation support groups where legal advice and support might be sought. In the longer term, if the levirate is declining, increasing financial independence through employment opportunities for widows will reduce their need for support from a new partner. Given the success of the “ABC” approach in helping to lower the prevalence of HIV in Uganda, church and community leaders as well as faith-based organisations should continue to promote safer sexual behaviours such as partner reduction and monogamy within the marital union. In future, as more longitudinal data become available from this population, these behavioural measures should be revisited to determine whether widow practices are changing. The impact of demographic changes on behavioural trends is hard to predict. One interpretation of higher levels of widowhood is that the prospect of remarriage is falling over time,


Schizophrenia is a complex disease with multiple factors contributing to its pathogenesis. In addition to environmental factors, genetic factors may also increase susceptibility. In other words, schizophrenia is a highly heritable disease. Some candidate genes have been deduced on the basis of their known function with others found on the basis of chromosomal location. Individuals with multiple candidate genes may have increased risk. However it is not clear what kind of gene combinations may produce the disease phenotype. Their collective effect remains to be studied.Most pathways except metabolic pathways are rich in protein-protein interactions (PPIs). Thus, the PPI network contains pathway information, even though the upstream-downstream relation of PPI is yet to be explored. Here we have constructed a PPI sub-network by extracting the nearest neighbour of the 36 reported candidate genes described in the literature. Although these candidate genes were discovered by different approaches, most of the proteins formed a cluster. Two major protein interaction modules were identified on the basis of the pairwise distance among the proteins in this sub-network. The large and small clusters might play roles in synaptic transmission and signal transduction, respectively, based on gene ontology annotation. The protein interactions in the synaptic transmission cluster were used to explain the interaction between the NRG1 and CACNG2 genes, which was found by both linkage and association studies. This working hypothesis is supported by the co-expression analysis based on public microarray gene expression.2+. Reducing the cation influx might be one of the disease mechanisms for schizophrenia. This PPI network analysis approach combined with the support from co-expression analysis may provide an efficient way to propose pathogenetic mechanisms for various highly heritable diseases.On the basis of the protein interaction network, it appears that the NRG1-triggered NMDAR protein internalization and the CACNG2 mediated AMPA receptor recruiting may act together in the glutamatergic signalling process. Since both the NMDA and AMPA receptors are calcium channels, this process may regulate the influx of Ca Schizophrenia is a severe mental disorder with grave personal and social costs . ApproxiIn this study, we provided a novel strategy by taking advantages of PPI to discover the regulatory mechanisms among disease candidate genes. We speculated that disease candidate genes may cluster together in a functional network at a protein level. Protein complexes interact with preferred partners to form a biological module serving a specific collective function . When usRecent molecular studies implicate neuregulin1 (NRG1) as the most promising risk factor for schizophrenia ,10. Liu CACNG2, also known as stargazin, was found to interact directly with AMPA receptor and allow interaction of the receptor with the scaffold proteins of the postsynaptic density, such as DLG4 ,20. In aThere are two major types of reactions which are complex formation and covalent modification in the signalling pathway. Both types of reactions have protein-protein interactions (PPI), which can be detected by high throughput methods. It has been shown that proteins which are involved in the same pathway, are likely to cluster together in the PPI network . If the In order to cluster proteins that are close to one another, the distances between every pair of these 831 proteins were computed by using a standard algorithm based on shortest-path of network topology . On the Discovering the protein interactions between NRG1 with CACNG2 according to biological interpretationIn order to explore the detailed relation between NRG1 and CACNG2, the cluster 2 sub-network was extracted from the original large network for further study. This cluster contains 7 gene products of candidate genes, which content DPYSL2, CRTC1, DISC1, CACNG2, KCNJ12, PTK2B and NRG1, and 204 interacting proteins; part of this cluster is shown in Figure Since functionally related genes are usually co-regulated, we went further to check whether these genes were co-regulated in the brain tissue. If disease is considered as a perturbation to the normal state, different brain tumors may perturb a given gene to different extent. The GSE4271 microarray data set deposited in Gene Expression Omnibus at National Center for Biotechnology Information contains 100 samples from 15 assigned subsets . These gAs shown in Figure As described previously, DLG4 appears to be a hub, which receives the NRG1-ERBB4 signal and then relays the signal to the NMDA receptor and the CACNG2.Hahn and colleagues suggest that schizophrenia is marked by increased NRG1-ERBB4 signalling and may lead to further suppression of NMDA receptor function by reducing NMDAR tyrosine phosphorylation . Yau and2+. IPIR has integrated protein-protein interaction information from BIND, DIP, HPRD, MINT, MIPS and IntAct databases. In this case, we chose brain, cerebellum, cerebrum and nervous as tissue filter. By using 36 candidate proteins as a data set to look for its primary protein neighbours and secondary protein neighbours, there were 831 proteins retrieved from databases. This network is displayed by Cytoscape which provides basic functionality for a visual representation of the graph and integrated data [Protein-protein interaction data were obtained from Integrated Protein Interaction Resource  [For each biological network investigated, relevant proteins (nodes) and the interaction among them (edges) were assembled as follow. Each edge in the network was assigned a length of one. A pairwise distance matrix contains the length of shortest path between every pair of proteins in the network. Each distance in the matrix was shown as an "association", defined as 1/ts (GAP) , which its (GAP) . GoMinerts (GAP) . GoMinerThe microarray data was obtained from Gene Expression Omnibus (GEO) , and theThe authors declare that they have no competing interests.Ueng-Cheng Yang has designed the approaches and made a preliminary analysis. Pei-Chun Hsu has extended the analysis and jointly proposed the working hypothesis with Ueng-Cheng Yang. Kuan-Hui Shih has performed the co-expression analysis for brain tissue by using public microarray data. Hai-Gwo Hwu and his research team have provided the disease candidate genes and the domain knowledge for schizophrenia.