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1079836

General practitioners' satisfaction with and attitudes to out-of-hours services

Background In recent years, Dutch general practitioner (GP) out-of-hours service has been reorganised into large-scale GP cooperatives. Until now little is known about GPs' experiences with working at these cooperatives for out-of-hours care. The purpose of this study is to gain insight into GPs' satisfaction with working at GP cooperatives for out-of-hours care in separated and integrated cooperatives. Methods A GP cooperative separate from the hospital Accident and Emergency (A&E) department, and a GP cooperative integrated within the A&E department of another hospital. Both cooperatives are situated in adjacent geographic regions in the South of the Netherlands. One hundred GPs were interviewed by telephone; fifty GPs working at the separated GP cooperative and fifty GPs from the integrated GP cooperative. Opinions on different aspects of GP cooperatives for out-of-hours care were measured, and regression analysis was performed to investigate if these could be related to GP satisfaction with out-of-hours care organisation. Results GPs from the separated model were more satisfied with the organisation of out-of-hours care than GPs from the integrated model (70 vs. 60 on a scale score from 0 to 100; P = 0.020). Satisfaction about out-of-hours care organisation was related to opinions on workload, guarantee of gatekeeper function, and attitude towards out-of-hours care as being an essential part of general practice. Cooperation with medical specialists was much more appreciated at the integrated model (77 vs. 48; P < 0.001) versus the separated model. Conclusion GPs in this study appear to be generally satisfied with the organisation of GP cooperatives for out-of-hours care. Furthermore, GPs working at the separated cooperative seem to be more satisfied compared to GPs working at the integrated cooperative.

Background During the last decade, out-of-hours care by general practitioners (GP) in the Netherlands has changed substantially. Formerly, GPs performed out-of-hours care in small locum groups in which they joined a rota system. In recent years, large GP cooperatives have been set up following British and Danish examples [ 1 , 2 ]. Currently, about 124 GP cooperatives are operational in the Netherlands, taking care of more than 90% of the Dutch population during out-of-hours. In the current Dutch out-of-hours primary care, roughly two types of organisation models can be distinguished; a separated model and an integrated model. In the separated model the GP cooperative is located separate from the hospital's accident and emergency (A&E) department, indicating that there is no functional integration of out-of-hours services. In this model patients with a medical problem can choose between attending the GP cooperative or the A&E department, during out-of-hours. In the second organisation model the GP cooperative is integrated with the hospital A&E department. In this model, all patients utilising out-of-hours primary and emergency care without referral are first seen by a GP or practice nurse. It is known that a substantial number of self-referred patients at the A&E department exhibit minor injuries or non-urgent ailments that can be treated by a GP [ 3 , 4 ]. As a consequence, GPs of the integrated model will generally have to handle more patients than GPs at the separated model [ 5 ]. Patients with a referral or brought in by ambulance always bypass this system and will be directed to the emergency department without interference of the GP on call. The initiative of the out-of-hours primary care reorganisation has come mainly from the medical profession itself, motivated by increased dissatisfaction with the former out-of-hours services. However, as indicated by a recently published systematic review, there is little evidence available on current GPs' satisfaction with out-of-hours services [ 6 ]. The authors of this review identified only one study with respect to GPs' satisfaction with out-of-hours care. That study showed high levels of satisfaction with cooperative based primary care services [ 7 ]. However, only a part of the GPs interviewed actually participated within this service. Other studies have reported of beneficial effects to GPs with the introduction of GP cooperatives for out-of-hours care, like improved GPs' health [ 8 ] or decreased levels of stress [ 9 ]. We have also identified one Dutch study that showed increased satisfaction after reorganising out-of-hours care from practice based to cooperative based [ 10 ]. No further information is available on GPs' satisfaction with cooperative based out-of-hours care. There were two important reasons to conduct this study. First, because GP satisfaction has been shown to be an important contributor to quality of care [ 11 ]. GP satisfaction, besides patient satisfaction and costs, should be taken into account when evaluating out-of-hours care services. Second, during the time of the study, the integrated GP cooperative was still in its trial period and insight had to be gained in experiences and opinions of GPs working at this cooperative to support the decision whether this way of organising out-of-hours care should be continued. The purpose of this study is to gain insight in the satisfaction of GPs with out-of-hours primary care organised in cooperatives. In addition, this study investigates potential differences in the relationship of satisfaction and other out-of-hours care related opinions between GPs working in an integrated model and GPs working in a separated model. Methods This study investigates two specific elements: GPs' satisfaction with the organisation of two types out-of-hours care and the GPs' opinions related to working at either of two GP cooperatives. Two differently organised out-of-hours cooperatives are involved; a separated model and an integrated out-of-hours care model in two adjacent geographic regions in the Netherlands. Setting The separated cooperative is located in the centre of the city of Heerlen, the Netherlands, about 5 km and 9 km away from the only two A&E departments in this region. This cooperative was first set up in 1999, and covered at that time a population of approximately 100,000. In 2001, more GPs joined the cooperative and the population was increased to 278,000. The number of participating GPs increased to 120. In this system patients are stimulated to make a phone call before attending the GP cooperative. This allows the GP cooperative to triage patients at urgency levels of their medical complaints in order to prioritise treatment. During out-of-hours, patients with a medical problem can choose which out-of-hours service to attend, i.e. the GP cooperative or the hospital A&E department. The integrated GP cooperative is located in the city of Maastricht, the Netherlands, at the region's only A&E department of the University Hospital Maastricht. This cooperative was set up in January 2000. During the first one and half year, this cooperative covered only the population of the city of Maastricht (approximately 120,000). In August 2001, the surrounding area of Maastricht also joined the cooperative, increasing the coverage area to 190,000 inhabitants. In total, 83 GPs participate in the integrated GP cooperative. At this GP cooperative patients are allowed to attend the cooperative without an appointment, although it is preferred that they make a phone call first. All patients attending the integrated out-of-hours care facility without referral are first seen by a GP, who refers, if necessary, the patient to the A&E department. At both GP cooperatives, telephone triage is performed by doctor's assistants who are supported by guidelines and protocols, and are supervised by a GP. GPs of these cooperatives perform telephone consultations, consultations at the cooperative, and home visits. Regarding home visits a chauffeured care is at their disposal. Both regions comprise rural as well as urban areas. Development of the questionnaire Topics relevant to out-of-hours primary care were identified in interviews with three GPs participating in the two GP cooperatives under study. We have developed a set of items to enable us to measure and test multi-item scales. The items are related to relevant themes with respect to working at a GP cooperative. In total the questionnaire consisted of 86 items. (Some items are excluded from the analysis because they are only of local interest.) We investigated opinions on: overall satisfaction with the GP cooperative for out-of-hours, reorganisation of out-of-hours care, perceived workload, out-of-hours care as being an essential part of primary care, anonymity of care, gatekeeper function, availability of patient dossiers, cooperation with medical specialists during out-of-hours, and safety. We used a Likert five point scale (strongly agree, agree, neutral, disagree, strongly disagree) to record responses. Sample In both GP cooperatives (separated and integrated) a random sample of 50 GPs was taken. In case one of these GPs was not able or refused to participate, we had a substitution list of 25 GPs for each cooperative. This list was a random sample of the remaining GPs who were not selected by the first sampling. The questionnaire was administered by telephone to ensure high response rates. Two research assistants administered the questionnaire and received instructions, prior to the study, by FN. The study was conducted from November 2001 to February 2002. Statistics Beforehand, the 86 items of the questionnaire were divided into four blocks. These blocks represented 'satisfaction with out-of-hours care organisation', 'perceptions and subjective evaluations on working conditions in the present organisation', 'opinions and beliefs on professional philosophy', and 'evaluation of the cooperation with medical specialists at the local hospital'. The most important block concerns the one in which satisfaction with out-of-hours care organisation was measured: this was operationalised with 12 items. Principal component analysis with oblimin rotation was performed on the items of this block and after removal of items with weak factor loadings (lower than +0.60 or -0.60) and/or ambiguously loading items (on more than one factor) two factors remained in analysis. Oblimin rotation can be clarified as an oblique axis-rotation technique in finding the proper factor solution within data. By using this method we wished to come to a more 'natural' solution. With oblimin rotation no orthogonal assumptions are made to the correlation(s) between factors. In this study we strove for unidimensionality as a prerequisite for independently measured scales. Four items measured satisfaction with the current cooperative (scale 1: response variable) and three items measured satisfaction with the state of affairs before the cooperative was set up (out-of-hours care in a rota system). Next, per intended scale the test stability of each factor was measured by Cronbach's alpha, and again items could be removed from this scale, if this did increase the value of the alpha coefficient. In constructing the scale "overall satisfaction with the GP cooperative" one of the four items was removed following inspection of the Cronbach's alpha, while all four had high loadings on the same factor in the principal component analysis. The item was left out of table 2 and concerned the notion that the current organisation should be restructured. Alpha went up from 0.852 on four items to 0.893 on three items. In constructing the other scales, items which were loading unambiguously high in the principal component analysis were all included after being tested on reliability by Cronbach's alpha. Table 2 GP questionnaire. Description of scales and items. (Original items are in Dutch*) BLOCK 1. Satisfaction with out-of-hours care organisation Scale 1 . Overall satisfaction with GP cooperative (Cronbach's α = 0.90; mean (SD) = 65.0 (21.6)) I am very satisfied about the functioning and the structure of the GP cooperative (+) I am pleased with the current GP cooperative (+) I am very satisfied with the current organisation of out-of-hours primary care (+) Scale 2 . Current out-of-hours care is better organised than formerly (Cronbach's α = 0.92; mean (SD) = 89.8 (19.5)) Out-of-hours care was better organised before the establishment of GP cooperatives (+) I prefer the former organisation of out-of-hours primary care (-) The current organisation of out-of-hours care is not an improvement compared to the former organisation (-) BLOCK 2. Perceptions and subjective evaluations on working conditions in the present organisation Scale 3 . Experienced a high workload (Cronbach's α = 0.87; mean (SD) = 65.5 (18.4)) The workload at the GP cooperative is too high (+) Out-of-hours care during daytime on Saturday and Sunday is very aggravating (+) Usually, out-of-hours service is much too aggravating (+) I do not experience such a high workload at the GP cooperative (-) Performing out-of-hours care is absolutely not aggravating (-) Out-of-hours care during daytime in the weekends is not too high (-) Scale 4 . One feels safe at the cooperative (Cronbach's α = 0.87; mean (SD) = 76.9 (18.7)) Sometimes, I feel unsafe at the GP cooperative during out-of-hours (-) During my shifts at the GP cooperative, I never feel unsafe (+) Regularly, I feel unsafe at the GP cooperative during out-of-hours (-) Scale 5 . One feels safe during home visits (Cronbach's α = 0.84; mean (SD) = 76.0 (18.4)) Regularly, I feel unsafe when performing home visits during out-of-hours (-) Sometimes, I feel unsafe when performing home visits during out-of-hours (-) Usually, I feel safe when performing home visits during out-of-hours (+) BLOCK 3. Opinions and beliefs on professional philosophy Scale 6 . Out-of-hours care is an essential part of primary care (Cronbach's α = 0.97; mean (SD) = 60.3 (31.7)) These days, out-of-hours care should no longer be an essential part of primary care (-) Out-of-hours care is definitely an essential part of primary care (+) Out-of-hours care should always be a part of general practice (+) There is no place anymore for out-of-hours care in general practice (-) Scale 7 . Anonymity of care is a problem (Cronbach's α = 0.90; mean (SD) = 32.9 (21.8)) Because the GP of the cooperative and the patient are not familiar with each other, there is a risk for inadequate treatment (+) Because of anonymity of care there is a risk that diagnostics and treatment are not adequately adjusted for the patient's needs (+) One of the big disadvantages of the GP cooperative is the anonymity of care, because the GP is not familiar with the patient (+) Because the GP of the cooperative and the patient are not familiar with each other, there is a risk for inadequacy of care (+) Scale 8 . Gatekeeper function is well guaranteed (Cronbach's α = 0.74; mean (SD) = 64.5 (16.8)) I think the GP gatekeeper function at the GP cooperative is well guaranteed (+) I am afraid that the GP gatekeeper function during out-of-hours will disappear (-) I believe that in the near future the GP gatekeeper function will be put under too much pressure (-) I think the GP gatekeeper function at the GP cooperative is well protected (+) Scale 9 . Availability of patient dossiers is important (Cronbach's α = 0.93; mean (SD) = 57.4 (24.1)) As far as I am concerned, I do not need the availability of my colleagues' patient dossiers (-) I think it is a serious problem that my colleagues' patient dossiers are not at my disposal (+) The availability of patient dossiers of the other participating GPs during out-of-hours is absolutely unnecessary (-) During out-of-hours I am hindered in my practice, because of lack of information about my colleagues' patients (+) BLOCK 4. Evaluation of the cooperation with medical specialists at the local hospital Scale 10 . Cooperation with medical specialists is good (Cronbach's α = 0.94; mean (SD) = 62.7 (23.0)) Sometimes, the cooperation between GPs and medical specialists is not so good (-) I think that during out-of-hours the understanding between GPs and medical specialists is sometimes pretty bad (-) I think that during out-of-hours the cooperation between GPs and medical specialists is always fine (+) Generally, the cooperation between myself and the medical specialists of the hospital is good (+) * The provisional translation into English is meant to inform the reader of the content of the scales and cannot be seen as a definite one. Scale constructions were performed under specific rules for missing item data: in summating to a total for each case, scores had to be valid on at least half of the items, if the number of items was even, and on at least half of the items plus one half, if the number of items was uneven. Otherwise, scale scores were set to 'missing'. Finally, a transformation of the total scale score to a 0–100 score was made [ 12 ]. After that, the remaining three blocks were analysed in a similar way. In total this procedure produced ten scales. The relationship between individual scales and overall satisfaction (scale 1) was analysed using multiple regression analysis. Upon finding the final 'direct effects' model for both regions of interest we have tested all possible interactions of pairs of significant predictors for statistical significance by the forward selection technique. Next to this, interactions were computed by multiplying predictors significant within the 'direct effects' model by the dummy 0–1 (Heerlen or Maastricht GP cooperative) coding of region. In case of missing data, listwise deletion of missing cases was applied. To test differences between GPs from either two GP cooperatives we performed independent Student's t-tests per scale. In case of non-normality, which was assessed visually by histogram analysis and by the Kolmogorov-Smirnov test, a Mann-Whitney test was used. A P -level of less than 0.05, was considered to be statistically significant. All data were analysed using SPSS-pc, version 10.0.5. Results In total 100 GPs participated; 50 GPs per each cooperative. One respondent of the Maastricht GP cooperative (integrated model) refused to participate and was substituted by a GP from the reserve list. The mean duration of the interviews was 22 (± 6.6) minutes. The characteristics of the respondents of both models do not differ statistically (Table 1 ). Table 1 Characteristics of respondents. GP cooperative Heerlen (n = 50) GP cooperative Maastricht (n = 50) Age 48.0 ± 7.5 47.3 ± 6.6 Gender Male 42 43 Female 8 7 Employed Part-time 11 16 Fulltime 39 34 Size of practice (GPs) Mean (range) 2.5 (1 – 6) 2.0 (1 – 7) Participation in GP cooperative Fully 37 37 Partly 13 13 We tested ten scales related to aspects of current out-of-hours primary care (Table 3 .). Internal reliability of these scales was considered appropriate; Cronbach's alpha's ranging from 0.74 to 0.97. An overview of all scales and related items is presented in Table 2 .). Table 3 Scales scores for GPs opinions on different aspects of out-of-hours primary care. Separated Model Integrated Model (Heerlen) (Maastricht) Scale score Scale score Significance Scale Mean (95% CI) Mean (95% CI) Overall satisfaction 70.0 (64.0 – 76.0) 60.0 (54.0 – 66.0) 0.020 Current out-of-hours care is better organised than formerly b,† 90.1 (84.7 – 95.5) 89.5 (83.7 – 95.3) 0.569** Experience a high workload b 63.4 (58.1 – 68.8) 67.6 (62.5 – 72.7) 0.256 One feels safe at the cooperative b 76.5 (71.1 – 81.9) 77.3 (72.0 – 82.7) 0.825 One feels safe during home visits b,‡ 74.8 (68.7 – 81.0) 77.2 (73.1 – 81.4) 0.532 Out-of-hours care is an essential part of primary care b 52.9 (43.7 – 62.1) 67.8 (59.4 – 76.1) 0.018 Anonymity of care is a problem b 31.8 (25.1 – 38.4) 34.0 (28.3 – 39.7) 0.609 Gatekeeper function is well guaranteed b,†,‡ 66.8 (62.2 – 71.2) 62.2 (57.1 – 67.3) 0.180 Availability of patient dossiers is important b 55.4 (48.4 – 62.4) 59.3 (52.6 – 66.0) 0.422 Cooperation with specialists is good b,† 48.5 (42.1 – 54.8) 76.6 (72.9 – 80.4) <0.001 * Scale score ranges from 0 to 100 points b 100 points represents strong agreement † One case missing at the separated model, ‡ one case missing at the integrated model ** Because of non-normal distribution, the Mann-Whitney test was used GPs' overall satisfaction score with the current organisation of out-of-hours care was 65 points (95%CI: 60.7 – 69.3) on a scale from 0 (absolutely not satisfied) to 100 (highly satisfied). However, GPs from the separated model were more satisfied compared to their colleagues of the integrated model (scale score 70.0 vs. 60.0; P = 0.020). GPs from both cooperatives reported that the new organisation of out-of-hours primary care is better compared to the former practice-based out-of-hours care (mean scale score 89.8). Most GPs experience a high workload (mean scale score 65.5). A minority of all interviewed GPs think that the anonymity of patient care – many patients are not known to the GP because care is organised on large-scale – endangers adequacy of out-of-hours primary care (mean scale score 32.9). Furthermore, a small majority feels that the patient's medical file should be available at the cooperative (mean scale score 57.4). In both cooperative models (integrated and separated) of out-of-hours care, GPs think that their gatekeeper's role to secondary care is sufficiently guaranteed (mean scale score 64.0). Most GPs feel relatively safe at the cooperative or during out-of-hours home visits (mean scale score 76.9 and 76.0 respectively). GPs from the separated model were neutral about out-of-hours care as being an essential part of their job as a GP, in contrast with the integrated model GPs who were more convinced that out-of-hours care is an important part of their job (scale score 52.9 vs. 67.8; P = 0.018). GPs at the integrated model experience a better cooperation with medical specialists during out-of-hours care (scale score 76.6 vs. 48.5; P < 0.001). The regression analysis identified three scales that are significantly related to overall satisfaction (Table 4 .). Effects on satisfaction for two of these scales, experienced workload and whether the GP thinks that his gatekeeper function is well guaranteed during out-of-hours, are different for both cooperatives. Experienced workload is mainly related to overall satisfaction of GPs from the integrated model. Whereas, the GP's opinion about the gatekeeper function is mainly related to overall satisfaction of GPs from the separated model. Increased experienced workload will lead to a decreased overall satisfaction, and the better the GPs valued the guarantee of their gatekeeper function during out-of-hours the higher their overall satisfaction will be. Table 4 Regression analysis with overall satisfaction with the organisation of out-of-hours care as dependent variable (0 = not satisfied, 100 = very satisfied) (n = 98; R 2 = 0.36). Unstandardised coefficients Standardised coefficient Scales B SD Beta t Significance Constant 106.352 22.154 4.801 Age -0.034 0.262 -0.012 -0.130 0.897 Gender b -2.188 5.228 -0.037 -0.418 0.677 Cooperative a -75.980 26.073 -1.840 -2.914 (0.005) Gatekeeper function 0.101 0.149 0.081 0.677 (0.500) Out-of-hours care is an essential part of primary care -0.140 0.063 -0.212 -2.216 0.029 c Experienced workload -0.597 0.152 -0.518 -3.915 (< 0.001) Gatekeeper * cooperative 0.737 0.247 1.253 2.980 0.004 c Workload * cooperative 0.487 0.219 0.799 2.225 0.029 c a Cooperative: Maastricht = 0; Heerlen = 1; b gender: male = 0, female = 1 c Only effects that are interpretable Gatekeeper function (0 = not guaranteed, 100 = highly guaranteed) Out-of-hours essential part (0 = not essential, 100 = highly essential) Experienced workload (0 = very low, 100 = very high) The third scale that is significantly related to overall satisfaction is the GP's opinion about out-of-hours care as being an essential part of his task as a GP. GPs who indicated that they believed that out-of-hours care was an essential part of their job as a primary care physician had a lower overall satisfaction with respect to current out-of-hours care. Neither gender nor age was significantly related to overall satisfaction. The regression model explained 36% of the variation in overall satisfaction. Subgroup regression analysis for GPs of the integrated and separated model separately (see Table 5 ), showed that for the GPs of the integrated model workload was the main factor that influenced overall satisfaction (variance explained: 34%). With respect to the GPs of the separated model, the guarantee of the gatekeeper function was of great importance to the overall satisfaction (variance explained: 35%). Table 5 Regression analysis results on overall satisfaction with the organisation of out-of-hours care as dependent variable for both GP cooperatives separately (0 = not satisfied, 100 = very satisfied). Unstandardised coefficients Standardised coefficient B SD Beta t Significance Separate GP cooperative (R 2 = 0.35) n = 49 Constant 21.823 20.437 1.068 0.291 Age -0.122 0.366 -0.043 -0.333 0.741 Gender a -0.110 7.475 -0.002 -0.015 0.988 Gatekeeper function 0.956 0.201 0.697 4.755 <0.001 Out-of-hours care is an essential part of primary care -0.182 0.098 -0.273 -1.851 0.071 Integrated GP cooperative (R 2 = 0.28) n = 50 Constant 101.692 23.204 4.383 <0.001 Age 0.073 0.398 0.023 0.182 0.856 Gender a -9.375 7.335 -0.156 -1.278 0.208 Experienced workload -0.648 0.144 -0.551 -4.496 <0.001 a Gender: male = 0, female = 1 Gatekeeper function (0 = not guaranteed, 100 = highly guaranteed) Out-of-hours essential part (0 = not essential, 100 = highly essential) Experienced workload (0 = very low, 100 = very high) Adding the scale of experienced workload to the regression equation of the Separate GP cooperative in Table 5 will make variance explained in overall satisfaction only higher by 0.007 to 0.359 (F ratio of the change = 0.50 by 1 and 43 df., p = 0.482). Adding the scales of gatekeeper function and out-of-hours care as an essential part of primary care to the regression equation of the Integrated GP cooperative will make variance explained in overall satisfaction only higher by 0.036 to 0.317 (F ratio of the change = 1.13 by 2 and 43 df., p = 0.332). Discussion GPs in this study are generally satisfied with the way out-of-hours primary care is currently organised. However, GPs from the separated cooperative are more satisfied than GPs working at the integrated cooperative. Mainly three factors are related to overall satisfaction. These are: experienced workload, guarantee of the gatekeeper function, and attitude towards out-of-hours care as being an essential part in general practice. To our best knowledge, this is the first study to investigate GPs' satisfaction with out-of-hours care as organised in separated and integrated primary care cooperatives. One British study and one Dutch study have looked into GPs satisfaction with out-of-hours care[ 7 , 10 ]. However, these studies solely focused on separated out-of-hours care models. The Dutch study showed that 70% of the GPs were satisfied with cooperative based out-of-hours care [ 10 ], and the British study found that 92% of the GPs were satisfied with the way out-of-hours care was arranged [ 7 ]. Since these studies used different ways to measure satisfaction it is difficult to compare them with our results. The results of this study indicate a difference in satisfaction between GPs from the separated and integrated cooperative. A possible explanation for this difference could be the fact that the integrated cooperative has to deal with a larger number of patients compared to the separated cooperative [ 5 ]. In this study however, experienced workload of GPs from the integrated cooperative did not differ from that of the GPs of the separated one. Obviously workload is also dependent on staffing of the cooperative. We presume that the difference in satisfaction might well be explained by other factors, which we have not investigated in this study. At the time of the study the integrated model was still in its experimental phase; housing in the integrated cooperative was generally not considered to be optimal. In this phase of the experiment the waiting room was very small and quickly overcrowded. Also, the space in the doctor's offices was quite limited and contained only room for one bed and no desk. In addition, at this time also patients' and GPs' privacy were not as sufficiently guaranteed as in the separated model. These factors may have had an effect on GPs' overall satisfaction with out-of-hours services. Three opinions were found to be significantly related to GP satisfaction with the organisation of out-of-hours care. The two opinions that weighted most heavily on satisfaction were experienced workload and gatekeeper function. We found that the GPs' opinion on the gatekeeper function during out-of-hours was related to satisfaction with the organisation of out-of-hours care specifically for GPs of the separated GP cooperative. The fact that this opinion is not related to satisfaction with the organisation in the integrated cooperative is probably due to the fact that this is not an issue at this cooperative, because the GP's gatekeeper function is fully guaranteed; all patients entering the out-of-hours centre are screened by a GP and if necessary referred to a medical specialist. In the separated model however, the patient can still bypass the GP and attend the emergency department of the hospital without a GP's referral. GPs who feel to have too little grip on these self-referring patients appear to be less satisfied with their arrangements of out-of-hours care. We have not been able to investigate GPs' satisfaction prior to the reorganisation from practice based out-of-hours care to cooperative based out-of-hours care, whilst GPs' dissatisfaction with practice based out-of-hours care was one of the important reasons why primary care in the Netherlands was reorganised. Nevertheless, this study shows that GPs feel that current out-of-hours primary care is better organised compared to former practice-based out-of-hours care. These results are in line with previous research [ 10 ]. However, this is not surprising considering the effort that has gone into reorganising the out-of-hours services and the prior dissatisfaction. All those who were in favour of the change of the out-of-hours system will obviously be satisfied with the fact that out-of-hours care has been reorganised, and feel that the new system is better than formerly. A distinct feature of the integrated model is the close cooperation between primary and hospital emergency care. This offers possibilities to improve communication and to exchange expertise. This is reflected by the high satisfaction score of GPs from the integrated model with the cooperation with the medical specialists of the hospital. Because GPs and medical specialists now work at the same site, it is easier to consult each other. Furthermore, GPs who have referred a patient to one of the medical specialists have access to feedback, i.e. they can check on the patient a few minutes later to see if they were right in their diagnosis. Nevertheless, region-specific differences may also have accounted for this difference, because in the region with the integrated cooperative there is a longer tradition in cooperation between primary and secondary care. We investigated GPs' opinions on working at two contrasting models of out-of-hours primary care, i.e. a separated and an integrated GP cooperative, in order to gain insight in GPs' preferences for either one of these models. Until November 2001, the Maastricht GP cooperative for out-of-hours care was the only cooperative in the Netherlands that was integrated with a hospital A&E department. Consequently, the Maastricht out-of-hours care organisation was the only service at the time of the study that could be used as an example of integrated out-of-hours care. There are limitations to generalise the results of the study to other regions. First, results of the study reflect the opinions of GPs at only two cooperatives in the South of the Netherlands. Second, the integrated GP cooperative was still in its trial phase and may therefore have not been a good representative of a well-established GP cooperative. Nevertheless, this is the first study to address GP satisfaction with an integrated GP cooperative and may therefore, despite the limited generalisability, give some indication of relevant aspects of integrated out-of-hours care for further research and care development. Currently, three regions in the Netherlands are working according to an integrated out-of-hours care system. However, at the moment GPs in other regions consider adopting this organisational structure. Furthermore, the current Dutch minister of health care has stated to be in favour of an intensive collaboration between primary and emergency care, for this will probably reduce costs [ 13 ]. The results of this study can support the current discussion in the Netherlands on the organisation of out-of-hours primary care. Future research should focus on the economic efficiency of both models and patient preference with respect to the organisation of out-of-hours primary care, because these are important features to take into account when developing out-of-hours care. Conclusion GPs in this study appear to be generally satisfied with the organisation of GP cooperatives for out-of-hours care. Furthermore, GPs working at the separated cooperative seem to be more satisfied compared to GPs working at the integrated cooperative. Competing interests The author(s) declare that they have no competing interests. Authors' contributions CvU, FN, GV, RW, GW, and HC participated in the design of the study. CvU, FN and GV developed the questionnaire. CvU and FN performed statistical analysis. CvU drafted the manuscript. FN, GV, RW, GW, and HC provided critical edits to this manuscript. HC supervised the study. All authors have read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079837

Termination of the leprosy isolation policy in the US and Japan : Science, policy changes, and the garbage can model

Background In both the US and Japan, the patient isolation policy for leprosy /Hansen's disease (HD) was preserved along with the isolation facilities, long after it had been proven to be scientifically unnecessary. This delayed policy termination caused a deprivation of civil liberties of the involuntarily confined patients, the fostering of social stigmas attached to the disease, and an inefficient use of health resources. This article seeks to elucidate the political process which hindered timely policy changes congruent with scientific advances. Methods Examination of historical materials, supplemented by personal interviews. The role that science played in the process of policy making was scrutinized with particular reference to the Garbage Can model. Results From the vantage of history, science remained instrumental in all period in the sense that it was not the primary objective for which policy change was discussed or intended, nor was it the principal driving force for policy change. When the argument arose, scientific arguments were employed to justify the patient isolation policy. However, in the early post-WWII period, issues were foregrounded and agendas were set as the inadvertent result of administrative reforms. Subsequently, scientific developments were more or less ignored due to concern about adverse policy outcomes. Finally, in the 1980s and 1990s, scientific arguments were used instrumentally to argue against isolation and for the termination of residential care. Conclusion Contrary to public expectations, health policy is not always rational and scientifically justified. In the process of policy making, the role of science can be limited and instrumental. Policy change may require the opening of policy windows, as a result of convergence of the problem, policy, and political streams, by effective exercise of leadership. Scientists and policymakers should be attentive enough to the political context of policies.

Background Leprosy, or Hansen's disease (HD), is a chronic infectious disease caused by Mycobacterium leprae [ 1 ]. The disease has been known since ancient times: The origin of the word leprosy dates back to Greek and Latin[ 2 ]. Over a long time period, the disease can be disfiguring, and societies have stigmatized victims of the disease. This attribute is deeply discrediting since the stigmatized individual is disqualified from full social acceptance. Leprosy was thus dreaded, not because it killed, but because it left one alive with no hope[ 3 , 4 ]. After Armauer Hansen's discovery of the bacteria in 1873, the disease became feared as a contagion, and segregation was recommended for prevention. The advent of effective drugs in the 1940s drastically changed the course of disease, and in many countries, patient isolation was deemed unnecessary and terminated. However, some countries that practiced patient isolation experienced difficulties in changing their policies, abolishing leprosaria and reintegrating the patients into the community[ 5 ]. As a result, prolonged policies caused the institutionalization of patients for a period longer than scientific knowledge justified. Public policy termination is often perceived as the final outcome of a political, but highly rational, policy process. When a policy's objectives are reached and maintained, its relevance and applicability should be reconsidered and, if found redundant, outmoded, or dysfunctional, the policy should be terminated[ 6 ]. Termination might occur with either a bang or a whimper: public programs and policies may end suddenly, perhaps after a lengthy resistance, or they may end slowly after a long-term decline in the resources with which they are [ 7 , 8 ]sustained. In either case, when termination does not take place properly, the policy may cause harmful effects, either materially or ideologically, rather than beneficial ones[ 9 ]. Adopting a policy to terminate a law is always a struggle presenting a number of obstacles, especially. Those posed by agents with vested interests[ 10 ]. To overcome various political hurdles, a set of effective political strategies is sometimes required, as well as political maneuvering by those skilled at policy [ 11 - 13 ]termination. There seems to be a general expectation that the introduction, maintenance and termination of health and medical policies are deliberate, meaning both rational and scientifically justifiable. However, the relationship between science and policy is actually quite complex. It is rarely a linear and rational one in which science finds and establishes the facts and then policymakers incorporate them to solve social problems. This study examines the history of leprosy control policy in the US and Japan, focusing especially on the national legislative changes concerning patient isolation. It seeks to examine the functions of science in policy change, in this case the abolition of the long-standing patient isolation policy. Importance of favorable political context, along with skillful policy entrepreneurs, is then addressed. Methods Materials To examine the process of policy making concerning the isolation of patients with leprosy / HD, a comprehensive search and collection of historical documents were conducted both in the US and Japan. After a thorough search of archives of public records, both printed and on-line, using the keywords leprosy and Hansen's disease, relevant documents were exhaustively collected from libraries, leprosaria/sanatoria, offices of patients' organizations, and the museum of Hansen's disease (Carville and Tokyo) in each country. Sources include the Research Library of the National Archives and Record Administration (Washington)[ 14 , 15 ], the libraries at Louisiana State University (Baton Rouge) and Harvard University (Cambridge and Boston), and the National Diet Library (Tokyo). Databases of medical articles, such as MEDLINE[ 16 ] and ICHUSHI[ 17 ], were utilized to search for publications on leprosy/HD. Other types of documents and books, such as theses, essays and recollections, were also gathered. Supplementary interviews were then conducted during 2000–2004 with leprosaria directors and physicians, administrators, and patients, both past and present, in the US and Japan. The Garbage Can model of policy making The observation of politics involves making observations conform to sets of assumptions, which are called models. These models help delineate the relationships among conditions and patterns in political life. The Garbage Can model of policy making was proposed by Cohen et al[ 18 ], and further developed by Kingdon[ 19 ] as a contrast to linear, comprehensive, and rational models[ 20 ]. This model assumes that policy windows open only when the process streams of problems, policies and politics converge, separately and independent of each other. In such a convergence, problems are brought to the attention of people in and around government by systemic indicators, by focusing events like crisis and disasters, and by feedback on the operation of current programs. Alternate policy proposals are developed from the many possible ideas floated by those both in and out of government. The proposals that survive to achieve serious consideration must meet several criteria, including technical feasibility, a fit with dominant values and the current national mood, budgetary workability and political support. Political support may be affected by a variety of influences including swings of national mood, administrative or legislative turnover, interest group campaigns, and social movements. In this context, policy windows are the opportunities to push forward one's preferred proposal or conception of the problem. These windows of opportunity in policy making can open either as a result of happenings in the political stream or by the development of compelling problems. In the Garbage Can model, solutions and problems have equal status as separate streams in the system, and the popularity of a given solution at a given point in time often influences which problems are focused on. In other words, collections of choices are looking for problems, issues and feelings are looking for decision situations in which they might be aired, solutions are looking for issues to which they might be the answers, and decision makers are looking for work. Agendas are set by problems or politics and alternatives are generated in the policy stream. So, outcomes are heavily dependent on the coupling of the streams. Sometimes in this process, policy entrepreneurs – people who invest their resources in pushing their pet proposals or problems – are responsible, not only for prompting important people to pay attention, but also for coupling solutions to problems and for setting both problems and solutions in a functional political context. This Garbage Can model was sometimes employed to explain the dynamics of policy making in the areas other than health care, and proved to be a powerful analytical tool[ 21 ]. In this study, the courses of events were analyzed through this model, and important factors for and obstacles to linking science and policy were examined. With respect to leprosy policy, history after WWII is divided into three periods. Period I covers the time period of the late 1940s through 1950s, when the advent of new drugs could have opened the policy windows in both countries. Related events in Japan seem to have lagged about five years behind those in the US, presumably due to the delayed introduction of those drugs. Period II represents the time in the 1960s and 1970s, when true legislative policy change did not occur, but incremental administrative actions were taken. Finally, Period III encompasses the 1980s and 1990s, when the policy of patient isolation was legislatively abolished in both the US and Japan. Relevant facts preceding Period I are also succinctly presented. Results Isolation of leprosy patients in the United States US background In 1865, Hawaii established a policy for the quarantine and isolation of HD patients. A leper settlement was established at Kalaupapa on Molokai island, and the Kalihi Receiving Station was opened on Honolulu to diagnose cases and provide emergency treatment[ 22 ]. In 1873, when Mycobacterium leprae was discovered by Hansen and leprosy was thus proved to be a contagion, the seclusion of patients in their homes or in hospitals was advocated for disease prevention[ 23 ]. By 1894, Louisiana had established a leprosarium, and several other states, such as New York and California, had also introduced similar isolation policies for HD patients[ 24 ]. The First International Congress on Leprosy held in Berlin in 1897 recommended isolation as the appropriate policy measure against leprosy. In 1909, the U.S. government built another leper colony in the Philippines on the Island of Culion. After several national and state surveys of HD patients, the need for a national policy to control HD and the desirability of patient isolation were discussed repeatedly during the 1890s. The first legislation, passed in 1898, authorized a thorough investigation of leprosy. A commission was appointed, and based on its recommendation, legislation was introduced that allowed for the establishment of a national leprosarium for the segregation of lepers with the goal of preventing the spread of leprosy in the United States[ 25 ]. It is worth noting that scientists were never unanimous about the necessity of isolation. Scientific publications at the time described HD as a relatively non-contagious disease[ 26 ]. After succeeding reports and debates, legislation was passed in 1917 that provided for purchase of a site and receipt of any person afflicted with leprosy who presented himself or herself for care, detention, and treatment, or who might be apprehended under authority of the United States Quarantine Acts[ 27 ]. The American Academy of Medicine, the American Medical Association, and the American Dermatological Association expressed their support for the isolation policy, arguing that patient isolation serves for disease control and patient treatment, as well as protection of patients from social stigma and ostracism. The 1917 legislation also directed that public health employees assigned to the institution be paid one and one-half times the regular pay due to the disagreeable and dangerous nature of the service[ 28 ]. Having failed to locate a new site, in 1921 the U.S. government took over the Louisiana Leper Home in Carville, Louisiana, and the United States Public Health Service (PHS) opened the US Marine Hospital Number 66 as a National Leprosarium[ 29 ]. The rules "Regulations governing the care of lepers: regulations for the government of leprosaria and for the apprehension, detention, treatment, and release of lepers" were drawn up by the Surgeon General in 1922 to implement the 1917 legislation. The 1922 rules reiterated the admission options (voluntary or involuntary), prohibited patients from leaving the sanitarium grounds, and provided that patients shall not "hold communication" with patients of the opposite sex. The rules also outlined a procedure for discharge of patients, which included a period of one year of special observation for any signs of leprotic retrogression after medical tests showed no signs of the disease. After this year, the patients were requested to appear before a board of three or more medical officers who might pronounce the patient to be no longer a menace to public health[ 30 ]. The institution was gradually expanded in the year 1933, having 65 rooms and outpatient facilities in 1941. By 1945, there were 369 inpatients[ 31 ]. During those years, there was no really effective treatment of the disease. Most of those at the facility were involuntarily admitted (only 15% of admissions between 1930 and 1945 were reportedly voluntary)[ 32 ]. US period I (1940 through 1950)) In 1941, Dr. Guy Faget at the National Leprosarium started a clinical trial of Promin, a sulfone drug, for the treatment of HD. Its beneficial effects became rapidly known and were reported within a few years in leading medical journals, such as Public Health Reports and Journal of the American Medical Association (JAMA)[ 33 ]. Other forms of sulfone drugs, Diasone and Promazole, were also developed and replaced the traditional HD treatment of applying chaulmoogra oil[ 34 ]. The international academic community paid close attention, and the 1946 Conference on Leprosy in Rio de Janeiro reported significant advances from the introduction of the sulfone group of drugs[ 35 ]. The enactment of the Public Health Services Act of 1944[ 36 ] could have provided a forum for discussion and revision of the US HD policy. Parts D and G of the Act, however, literally duplicated the 1917 Act, apparently without discussion. As before, its Section 331 states – "that the Service (PHS) shall, in accordance with regulations, receive into any hospital of the Service suitable for his accommodation any person afflicted with leprosy who presents himself for care, detention, or treatment, or who may be apprehended under section 332 (regulation) or 361 (quarantine) of this Act, and any person afflicted with leprosy duly consigned to the care of the Service by the proper health authority of any State, Territory, or the District of Columbia. The Surgeon General is authorized, upon the request of any health authority, to send for any person within the jurisdiction of such authority who is afflicted with leprosy and to convey such person to the appropriate hospital for detention and treatment." Only a year later, in 1945, the American Public Health Association advised against isolating people with leprosy. It was by then well established that the majority of the population had a natural immunity to leprosy, and that it was only mildly contagious to the rest. Since the relative infectiousness of the two types of leprosy had been measured by epidemiological studies, which demonstrated the very low infectiousness of the tuberculoid type, experts insisted that only open (infectious) cases, if any, required isolation[ 37 ]. However, at that point, scientists were again not unanimous concerning patient isolation policy. Dr. Faget, who should have known the effects of sulfones better than anyone else, wrote in Public Health Reports that the only reliable means to eradicate leprosy is isolation. Dr. G. W. McCoy, an ex-official of the PHS and Dean of the Louisiana State University Medical School, on the other hand, recommended that since infectivity varies depending on clinical types, the universal isolation policy should be abandoned. He stressed that when there is room to question infectiousness, it should be used to serve patients (meaning to protect patients' liberty)[ 38 ]. In response to these debates, the Federal Security Agency (FSA) set up under the Surgeon General the National Advisory Council on Leprosy. The Council discussed the issues raised by the United Patients' Committee for Social Improvement and Rehabilitation – composed of the Patients' Federation, Veterans/Legionnaires, and the staff of The Star (a monthly periodical published by the HD patients) – which had expressed its objection to the Public Health Services Act. Abolition of forced isolation and detention and the broadening of outpatient treatment services were discussed, but only the latter was supported[ 39 ]. After a while, an experimental project for outpatient services was established in New Orleans. Efforts to revise the law began soon after the enactment of Public Health Services Act. In 1948, G. H. Rarey (US Army – retired Colonel, national vice president of the American Federation of the Physically Handicapped) and Paul Strachan (president, American Federation for the Disabled) drafted the National Leprosy Bill, and asked Congressman Charles J. Kersten (R-Wisconsin) to submit it to Congress[ 40 ]. In the following year, a similar bill, the National Leprosy Act was submitted by Senator Claude D. Pepper. Both of these draft legislation intended drastic changes in leprosy control policy, including: dissemination of pertinent facts concerning leprosy (to promote an enlightened public opinion, a new and more accurate understanding of leprosy); treatment of leprosy patients (to establish leprosy treatment principles, methods, and regulations for administering leprosy treatment more in harmony with the customs of society as applied to the care and treatment of persons afflicted with other diseases; to arrange treatment centers, both inpatient and outpatient services); the National Advisory Council on Leprosy; rehabilitation and reemployment of leprosy patients; financial assistance for leprosy patients and their dependents; compensation for disability incident to leprosy; and expansion of leprosy research. At the Congressional hearings on the bills, the experts' testimony took different positions[ 41 ]. As a drafter of the bill, Colonel Rarey, who had served in the Philippine Islands and encountered many leprosy patients, argued insistently against the segregation of leprosy patients, claiming that it was not effective, could destroy patients' social lives, and might hinder patients from seeking proper treatment. He stated, "Many cases of this disease are not communicable. The remainders are classified by leprologists as feebly communicable. The status and low degree of communicability of leprosy in the US does not justify continuance of the present compulsory segregation policies." He also criticized the PHS, arguing that the FSA's favoring of segregation erred on the ultraconservative side of this question. On the other side, J. Donald Kingsley, Acting Administrator of the FSA, presented opposition to the bill. Although he indicated sympathy with the basic objectives of the bill, the necessity for law revision was denied. He argued that most of the objectives of the bill could be more effectively achieved through intensification of activities already authorized by the existing law. The bill was criticized as defective in attempting to divide responsibility for the treatment and care of patients not hospitalized in PHS hospitals, between the PHS and the various federal, state, and local governmental agencies. Especially stressed was the point that the proposed Act does not meet squarely the problems of forced detention of leprosy patients: It was insisted that the existing authority of the PHS should not be repealed or cast in doubt without careful consideration and provision for alternative methods of meeting the problem, should need arise. The FSA, while recognizing the accumulating evidence showing the effectiveness of the sulfone drugs, thus retained a conservative position. The arguments of the FSA were accepted, and as a consequence, those bills, though submitted repeatedly through 1954, were never enacted. The only leprosy-related legislation passed in this period was a bill enacted in 1951, which dealt with transportation costs for released patients[ 42 , 43 ]. At the administrative level, however, a series of changes was introduced. In 1946, patients were given the right to vote. In 1947, the PHS removed HD from the list of quarantinable diseases which required a travel permit. In 1948, the barbed wire fence was removed from the facility, and the first active patient was released to the care of her private physician. In the same year, the PHS officially recognized the name Hansen's disease as a replacement for the term leprosy. In the 1950s, patients were allowed to marry. Twelve negative monthly tests were no longer required for discharge; instead those who qualified for discharge were required to be financially stable and assure authorities that there were no children living where they intended to go[ 44 ]. US period II (1950 until 1980) In the early 1950s, patient isolation was criticized more and more and eventually condemned at the International Congress on Leprosy and at other international conventions [ 45 ]. Following advice that the isolation of patients should be limited to infectious cases, the abolition of compulsory isolation was repeatedly recommended also by WHO[ 46 ] and UNICEF[ 47 ]. Definition of the cases suitable for temporary isolation gradually became delineated[ 48 ]. In 1952, a decision was made that no administrator should stay at the institution for more than three years. Accordingly in 1953, the government appointed a new director, Dr. Edward M. Gordon, a former director of the PHS hospital in Chicago. Soon after his arrival, he announced that those who did not need to be medically hospitalized should leave the institution[ 49 ]. His policy was that those able-bodied residents diagnosed as arrested should be discharged; that discharge should be recommended to those who were arrested and partially disabled; that those who were disabled (blind or physically handicapped) could stay in the institution, but could leave if their relatives or friends offered appropriate care. He also passed a rule prohibiting able-bodied residents from working in the institution. Fearful of losing their homes and jobs, some of the patients sent lawyers to Washington to lobby elected officials[ 50 ]. After three years, Dr. Gordon left for Fort Monroe in Virginia, and Dr. Edgar B. Johnwick was appointed as the new director. In 1956, on the third day of his duty, Dr. Johnwick declared before the patients that no one would be discharged from the hospital against his/her will and that no one would be kept in the hospital against his/her will[ 51 ]. During this period, scientists, while acknowledging the clinical effects of sulfones and the distinction between infectious types, were not unanimous in negating the utility of patient isolation. Dr. L. F. Badger of Leprosy Control Unit, the Communicable Disease Center (later, renamed as Center for Disease Control), stressed in 1956 the importance of isolation of infectious patients, as well as the need for patient followup and continued treatment of arrested cases[ 52 ]. Conversely, in 1958, Dr. Meyer, Medical Director at Carville, insisted that as disease control measure, isolation is not necessary in many cases and has serious defects[ 53 ]. No overt controversies were found after that in the US literature. Meanwhile in 1960, the World Health Organization advised against isolation and in 1963 at the 8th International Leprosy Congress in Rio de Janeiro, the International Leprosy Association recommended against isolation and furthermore recommended that leprosy needed no special care beyond that afforded other communicable diseases[ 54 ]. The last compulsory isolation was reportedly enforced in 1960, and Part 32 of the Federal Code of Regulations "Medical Care for Persons with Hansen's Disease and Other Persons in Emergencies" dropped the term "detention" in 1975[ 55 ]. It should be noted that all these policy changes were made through administrative action, not by changing statutory law. Law revision was not visible on the agenda as these policy changes proceeded. Even in 1974 when Hawaii ruled that all future new cases of the disease would be treated as outpatients, no debate took place on the federal level[ 56 ]. Compulsory isolation was terminated as the practice of involuntary admission and that of forced institutionalization ended. US period III (Post 1980 through 2000) The driving force to terminate the isolation policy legislatively and abolish the leprosarium came not from new medical findings but from arguments for economic efficiency and budget cutting. In 1981, the Reagan administration and Congress agreed that it no longer made sense for the government to run public health hospitals, since Medicare, Medicaid, and the general oversupply of hospitals made specialized hospitals obsolete. A congressional subcommittee, backed by the Omnibus Budget and Reconciliation Act, thus targeted the nation's nine public health hospitals, including the National Leprosarium at Carville[ 57 ]. However, lobbied by the several groups which had developed over the years to oppose termination – including the community of Carville, the patients, a society of Legionnaires and veterans -, Congressman Gillis Long fought for an exemption for Carville. Consequently, the leprosarium survived the challenge based on the argument that HD patients need special care and considerations (The institution was renamed as Gillis Long Hansen's Disease Center later in 1986)[ 58 ]. To the Congress, after 1981, Congressmen Henry A. Waxman and Gillis W. Long instead proposed a bill to change HD policy. Avoiding the issue of closing down the Carville institution, the bill was intended to permit treatment of HD outside PHS facilities and to eliminate additional pay for personnel treating HD. In the report attached to the bill[ 59 ], Richard Ashbaugh (Acting Director, Bureau of Medical Services, PHS) indicated the PHS's support for the bill. He stated, "Leprosy pay ... was instituted because of a mistaken belief that workers were at significant risk in contracting Hansen's disease ... Not only is leprosy pay an unjustifiable expense, it actually increases the stigma associated with Hansen's disease." He went on to say, "The PHS is strongly committed to the least restrictive possible treatment of Hansen's disease ... " A similar act was submitted in the subsequent year, but also not enacted at least in part because beneficiaries of the center's payroll, including the patients working at the facility, made a strong objection to the bill. Without legislative support, the PHS had been subsidizing ambulatory care centers for HD patients, outside the HD Center since 1981. The PHS National Outreach Program started 11 regional outpatient services nationwide[ 60 ]. When the PHS in 1983 did a formal review of the Center, it issued a report which highlighted the Center's economic inefficiency, making that the focal point of the discussion. The Center at that time housed only 200 patients but comprised 98 buildings on 337 acres. The staff included 317 civil service and PHS workers and 125 part-time patient employees. The PHS recommended that the Center continues its custodial care and research programs, but asked for more outpatient clinics and a study of the elimination of residential care. In the following year, the PHS set up a utilization review committee in Carville to review periodically the conditions of patients who had been hospitalized more than two years, for their possible discharge ability[ 61 ]. Finally, legislation was made. In 1985, PL99-117 was enacted which stated the PHS ... "shall provide care and treatment (including outpatient care) without charge ... to any person suffering from Hansen's Disease who needs and requests care and treatment for that disease"[ 62 , 63 ]. The term "detention" was deleted from the Act, though the provision of residential care was not completely negated. The hazardous duty salary supplements, while had been reduced to one and one-quarter time, were discontinued for new hires. In 1988, three years after the legislation, the PHS published its report "Strategic Plan, National Hansen's Disease Center (NHDC)[ 64 ]." The plan reiterated the situation of the NHDC, and evaluated whether it would be cost effective and feasible to contract out the patient care activities of the Center and transfer the research activities elsewhere. The report recommended expanding the Center's mission to include other nerve-desensitizing diseases, contracting out long-term patient care, and moving the Center's research facilities to Baton Rouge. It also argued the necessity of maintaining the Center so that patients who had lived at Carville for decades could be cared for compassionately in familiar surroundings. The report reached the definitive conclusion that there should be no new resident admitted to the Center, so that over time the population would dwindle to nothing and the entire facility could be closed. Accordingly in that same year, Dr. John Duffy, a new director, began the move of the Center's acute care, research and educational functions to Baton Rouge, while allowing the current residents to stay in Carville[ 65 ]. Thus, in practice, both voluntary and involuntary isolation and hospitalization ended. Nonetheless, despite these developments, closure of the leprosarium required more than a decade, thwarting both a 1990 plan to transform the institution into a geriatric prison and a 1996 plan to make it a federal prison for minimum security geriatric patients. The efficiency argument made by the PHS was always counterposed by the equity and civil rights arguments offered by the patients and their allies. The patients, though demanding that their basic human rights and freedom be restored, claimed a right to lifelong care by the government[ 66 ]. Only the research branch was moved in 1992 to a location at Louisiana State University in Baton Rouge. Then, agenda was set again by the economic efficiency argument, but this time with a new set of policy alternatives. Representative Richard H. Baker proposed that the site be used as a job training school, and that patients be given an annual living subsidy if they departed[ 67 ]. Baker, US District Judge Frank Polozola, and Jim Mitchell (PHS) visited Carville to discuss with patients and staff members the idea of closing the Center. This proposal was quashed once when Congressman Cleo Field, who represented the district from 1992 to 1996, objected. However, when the 1996 election returned the district of the Center (District 6 of Louisiana) to Baker, he resubmitted the bill with the proposal that the site be transferred to the State of Louisiana[ 68 ]. This time his plan was adopted. PL 105-78, "Relocation of Gillis W. Long Hansen's Disease Center" was signed into law in 1997[ 69 ]. The legislation returned the physical facility to the State of Louisiana without charge, though it specified that the Carville site must be used for health or educational purposes for 30 years. It offered a voluntary separation incentive payment to civil service employees. It directed that at or through the Center, the Secretary of the Department of Health and Human Services (DHHS) must provide short-term care and treatment without charge, including outpatient care, for Hansen's disease and related complications. The Secretary, however, was not permitted to provide long-term care for any disease or complication through the Center. For long-term-care patients, the Secretary was instructed to provide for long-term care without charge for the remainder of the life of the patient. The bill also directed the relocation of the patients at the Center to Baton Rouge within three years unless such relocation was not feasible, and it reassured the remainder that they could stay at Carville as long as they were able to live independently. It further offered a $33,000 annual stipend to any patient who chose to leave the institution, and this irrevocable option could be chosen at anytime by a patient. In August 1999, the federal government transferred the leprosarium to the State of Louisiana for use as a Job Corps training site. The Louisiana National Guard initiated its Youth Challenge program shortly after that[ 70 ]. Some patients left Carville going either to the Baton Rouge facility or to the other places, but quite a few remained. Today, the NHDC in Baton Rouge admits about 180 HD patients, and treats several hundred patients annually on an outpatient basis[ 71 ]. Isolation of leprosy patients in Japan Japan background Since ancient times, historical records indicate, many HD patients lacked permanent homes, leaving them to wander around living in both towns and rural areas, and sometimes creating their own colonies. Leprosy had been understood to be a hereditary disease, but since the mid 19th century, the view that it was the result of a contagion became known gradually, first among the medical experts, and then among the public[ 72 ]. The recommendation of the first International Conference on Leprosy held in Berlin in 1897 led Japanese experts to support patient segregation, although not always unanimously. A national survey was conducted about leprosy, and Law No. 11, "The Act on Leprosy Prevention," was passed in 1907. At that time, the Ministry of Internal Affairs (MIA) explained that despite the mild infectiousness of leprosy, Law No. 11 was required for the sake of vagrant lepers, those without means of support[ 73 ]. Five public leprosaria were established by local governments in 1909. Voluntary organizations and local governments launched the No Leprosy Movement in 1924, which tried to find all leprosy patients and send them to the leprosaria. Subsequently, in 1931, the first national leprosarium was opened, and at that time, Law No. 11 was revised to the Leprosy Prevention Law, which allowed all patients, whether they could be cared for at home or not, to be hospitalized without any financial burden levied on their families[ 74 ]. The Ministry of Health and Welfare (MHW), the successor of the MIA, nationalized all of the existing leprosaria so as to coordinate their activities. Ministerial officials issued manifests that leprosy should be eradicated from Japan through absolute segregation[ 75 ]. In accordance with the increasing institutional capacity of leprosaria, the number of patients sent to leprosaria increased considerably. Japan period I (1940 through 1950s) The sulfone drug Promin came to Japan in 1946 shortly after WWII ended. Its significantly beneficial effects were reported shortly at the Congress of the Japan Leprosy Association[ 76 ]. However, many physicians remained unconvinced of the real efficacy of the drug: They argued that the disease could relapse, even though it might show initial response[ 77 , 78 ]. The new drugs were difficult to obtain either by import or by domestic production, but patients were desperate to try them. They organized the Federation of National Leprosarium Patients (FNLP) and launched lobbying activities to acquire the drug. In response, the Diet (the legislative body in Japan) approved in 1950 a budget for sulfones. The drugs were delivered to leprosaria, and, a year later, the first 35 patients were officially discharged as a result of their improvement[ 79 ]. Meanwhile after WWII, during the late 1940s and early 1950s, a new government was established in Japan, and legal systems were thoroughly reviewed and revised in compliance with the new Constitution. As part of that process, leprosy wound up on the agenda in the Diet in the early 1950s during discussions of social security systems. Debate went on until the revision of the existing law of 1931. On February 15, 1950, leprosarium directors Drs. Kensuke Mitsuda, Yoshinobu Hayashi, and Ryoichi Yajima testified at the Health and Welfare Committee of the House of Representatives, along with the Director of the Medical Affairs Bureau, the MHW[ 80 ]. They insisted on the necessity of continued isolation of leprosy patients, argued for the desirability of the government subsidy for Promin, and recommended the expansion of leprosaria. On October 10, 1951, the Health and Welfare Committee of the House of Councilors heard testimony from five experts, including three leprosarium directors, Drs. Hayashi (then also President of the Japan Leprosy Association), Mitsuda and Miyazaki. Dr. Hayashi stressed the fact that there were approximately 9000 institutionalized patients, and 6000 outside the leprosaria who posed a threat of infection. He argued that leprosaria should be expanded to institutionalize all of the latter and that hazardous pay (about 5–10% at that time) should be increased as in the US. Dr. Mitsuda suggested that a more stringent law should be implemented to require isolation in order to prevent spread of the disease. He contended that though the necessity of isolation is sometimes refuted in the US, leprosy, regardless of its types, must be isolated to prevent infection and that sterilization should be recommended to patients for this purpose. He stressed that the new drugs, although promising in treatment, could not be expected to eradicate leprosy. Dr. Miyazaki essentially agreed with Dr. Mitsuda, adding that patients, many of them with deformities and disabilities, could not be released from leprosaria while they remained unaccepted by society and unable to live outside. He suggested that public enlightenment efforts would be advisable. The other experts argued for expansion of research into leprosy[ 81 ]. The bill to revise the Leprosy Prevention Law was submitted as a cabinet bill in 1953. The Minister of Health and Welfare explained to the House members that leprosy is hard to cure, that patient isolation is the only measure that prevents leprosy infection, and that the welfare of patients and their families should be protected by the government. Masayoshi Yamaguchi, Director of Public Health Bureau, the MHW, stated that patient isolation is the sole measure for leprosy prevention and protection of the public welfare, and that compulsory isolation should be warranted by law as a last resort. He suggested that patients might be discharged when leprosarium directors consider their isolation unnecessary and recommended that temporary leave also be allowed at the discretion of directors[ 82 ]. Facing the possibility of more stringent laws, the Patients' Federation expressed concern that the prospective law under consideration was focused too narrowly on social protection, while ignoring patients' rights[ 83 ]. The revised Leprosy Prevention Law of 1953 was in essence a reflection of the arguments of the experts and maintained a legal basis for compulsory isolation of patients proven to have bacilli and for prohibition of leave without permission. In response to patients' pleas, nine supplementary resolutions were adopted, including provisions for living stipends and patient work, improvement in living conditions, promotion of research, and installation of rehabilitation facilities. The MHW did initiate programs for patient rehabilitation, but the Ministry was still devising a plan to expand leprosaria capacity to hospitalize leprosy patients. The Notice of the Vice-Minister, issued soon after the promulgation of the Law, still described isolation as the only reliable means of prevention of the spread of the disease. A tentative standard for discharge was officially detailed by the MHW in 1956, but it was stated as not being intended to facilitate discharge[ 84 ]. In practice, about 500 patients were admitted or readmitted to leprosaria in 1956, while fewer than 100 were discharged. Japan period II (1960s through 1980s) In 1961, the legislature of the government of the Ryukyu Islands, then under the auspices of the US army, took a major step when it passed the Hansen's Disease Prevention Act. It provided that the Chief Executive may advise hospitalization and may also order an improved patient to leave the hospital[ 85 ]. Inspired by the policies of the Ryukyu Islands and acquainted with the international recommendation that most leprosy patients should be treated on an outpatient basis, thus abolishing compulsory isolation, the Patients' Federation (FNLP) repeatedly voiced support for revision of the Leprosy Prevention Law of 1953[ 86 ]. They repeatedly insisted that the Law lacked a scientific basis and violated human rights, and argued that they were victims of enforced segregation and of social stigmas fostered by the Law[ 87 ]. In 1963, the Patients' Federation filed its petition for the revision of the Law, sending more than 200 patients to pressure the Diet and the MHW. Despite these efforts, the Diet did not add revision of the Leprosy Prevention Law to its agenda. The MHW officials argued that the existing law legitimized compulsory isolation of leprosy patients, which in turn constituted the legal basis for the government's responsibility to provide them with care and a comfortable living environment. Revision of the Law might eliminate the leprosaria, making it impossible for the patients to live on public support. Overt transition of leprosy control to an outpatient basis could also lead to a similar outcome. It was also argued that the transformation of leprosaria from medical facilities to rehabilitation facilities would be difficult because of budget constraints[ 88 ]. Concerned about the future of leprosaria, patients did not have the unanimity necessary for law revision. A 1965 survey disclosed that only 16% of patients had an intention to be discharged in the future while the rest felt that they were unable or unwilling to leave their leprosaria[ 89 ]. Leprosarium directors in Japan gradually relaxed the implementation of the law. Patients became increasingly free to leave their leprosaria, and abscondments were not punished after the 1960s. In addition, treatment began to be offered outside leprosaria, although on an informal outpatient basis[ 90 ]. Concerned that law revision could eventually harm patients' welfare, patients, as well as the officials in charge, came to pay more attention to improving the leprosaria and providing other benefits for patients. The preservation of the Law was implicitly justified as a way to guarantee patients' welfare. In May 1972, the Ryukyu Islands, where leprosy treatment was based on outpatient services, were returned to Japan. This return again focused the attention of both patients and medical professionals on policy issues. In 1976, when the Federation of Leprosarium Directors drafted a revision proposal which incorporated specific discharge codes, and then submitted it to patients for their consideration, the Patients' Federation objected to the proposal based on the concern that the adoption of specific discharge codes could result in forced discharge[ 91 ]. As was seen in the 1960s, many patients remained hesitant to address the issue of law revision, and their movements focused more on compensation for low-wage patient labor and redressing the long-term compulsory segregation of the past. A decade later, in 1984, the Patients' Federation itself created a committee to examine revision of the Law, but continued to be concerned about the fate of their adopted homes, the leprosaria[ 92 ]. Consequently, the Federation of Leprosarium Directors only directed that leprosaria should not be abruptly discontinued as many of their residents were already too old to leave. The MHW endeavored to improve living conditions in national leprosaria in response to repeated pleas filed by patients, as well as discussions in the Diet. Medical staffing was increased, patient labor was gradually reduced or made more rewarding, and government allowances were increased for leprosaria and their patients[ 93 ]. Officials did warn patients of the possibility that the numbers and sizes of leprosaria could be reduced as part of administrative reform if revisions to the Law were implemented too quickly. Japan period III (1980s through 2000) When Fujio Otani became the Director General of the Tofu Kyokai Foundation, which had been established to serve patients with leprosy, the stalemate in policy change began to resolve. Otani was a medical officer in the MHW, and had been Chief of the Section of National Hospitals and Sanatoria in the 1970s, and Director General of Medical Affairs Bureau in the early 1980s. Recruited to the Foundation after his retirement, he also served as the Chairman of the Advisory Council on Public Health in the MHW. Since the recommendation in the mid 1980s of the UN Commission on Human Rights to improve psychiatric treatment in Japan, Otani had been inspired to act for the protection and restoration of human rights among patients[ 94 ]. In 1989, when the Patients' Federation consulted with him on its draft petition on the law, he began to commit himself to the issue of law revision. In 1992, encouraged by Otani, the Patients' Federation filed a petition with the Minister of Health and Welfare. Although many patients still worried about the fate of their leprosaria, Otani promised them that their homes would be maintained[ 95 ]. In the following year, the Tofu Kyokai Foundation was officially consulted by the Ministry on the prevention policy for leprosy. A committee was established, composed of leprologists, medical experts, lawyers, media representatives, bureau officials and patients, and presided over by Otani. He tried to appeal to public opinion by establishing the Museum of Hansen's Disease (MHD) and hosting a series of symposia about the policies on HD[ 96 ]. In 1994 at the 67th Congress of the Japan Leprosy Association (JLA), Otani gave a special lecture and stated that the existing Leprosy Prevention Law should be abolished since it was not scientifically justifiable and violated patients' human rights. Six months later, as a result, the Federation of Leprosarium Directors and the JLA publicly confirmed his opinion that the existing law should be abolished[ 97 ]. Upon the release of the report by the MHW committee in 1995, the MHW organized an internal panel of experts on the abolishment of the Leprosy Prevention Law. This panel, again chaired by Otani, subsequently submitted its report which recommended abolition of the Law, continued provision of public support for existing patients, and the use of the term "Hansen's disease" in place of "leprosy" in laws and regulations. Finally in 1996, the "Act to Abolish the Leprosy Prevention Law," drafted by the MHW, was passed. It abolished the 1953 Law and at the same time codified the government's responsibility to provide existing HD patients with continuous medical and other social services. It was also provided that patients could either leave the leprosaria or stay there as long as they wished, and that those who decided to come back to the leprosaria after their discharge could be readmitted. At the time of the abolition of the old law, there were 5,413 patients in leprosaria, whose average length of stay was more than 40 years and whose average age was 72 years old. Only six of these patients actually left their leprosaria in the following two years[ 98 ]. In response to lawsuits filed in the late 1990s, in 2001 the court awarded financial settlements to those who had been isolated under the earlier laws[ 99 ]. The awards amounted to between $65,000 and $114,000 per person depending on the degree of suffering. Additional pay to the workers at leprosaria was preserved. Discussion This study demonstrates that the isolation of leprosy patients was introduced and made rigorous in both countries around the time when it became known that the disease was contagious. Patient isolation policies and leprosaria were maintained long after it became known that isolation is not necessary in the majority of cases. Remarkable was the stagnation of policy change in the post-war period. The preservation of the isolation policy provided patients with some social support, but continuously deprived them of their civil liberties. Furthermore, the policy as an authoritative statement on the disease may have fostered the social stigma associated with a belief that the disease is a dreadful contagion, thereby maintaining a hurdle to patients' reintegration into society. Evidently, the policy's abolition was not easily accomplished nor was achieved solely by advances in scientific knowledge. Garbage Can model of policy making Kingdon's Garbage Can model is very useful in understanding the failure of 'appropriate' policy termination, as well as its delayed termination, observed in both countries: Any policy has an inertia and efforts to abolish the patient isolation policy did not bear fruit until three streams, i.e., problem, policy, and politics, converged after the mid-1980s. Science, although presumably a potent factor, was not in itself a major driving force in opening a policy window. In the early post-WWII period (late 1940s through 1950s) both in the US and Japan, scientific developments such as learning that the infectiousness of leprosy is usually feeble and uneven across disease types and the development of sulfone drugs effective against the disease might have been expected to be the major factor in reconsideration of the traditional isolation and institutional policy for leprosy management. In reality, however, the laws were revised, not as a result of the scientific developments, but as a spillover from other policy agendas: the enactment of the Public Health Services Act in the US, and the revision of social security systems in Japan, without substantial policy changes. Once legislated, they could hardly be challenged. Most actors then participating in the policy discussion were well aware of the new drugs, and patients were anxious to reap the fruits of these drugs and achieve the restoration of their civil liberties, sometimes participating in political mobilization for law revision. Scientific and medical advances could have altered the face of issues in the problem stream, helped generate alternative methods of disease management in the policy stream, and changed the opinion of the public and experts in the political stream. However, many influential experts, who had been engaged in the establishment and/or expansion of leprosaria were determined to keep the policy and the institutions and played on remaining public fear of leprosy[ 100 ]. Those conservative "elites", primarily bureaucratic agency representatives in the US and medical professionals in Japan, manifested opposition to the proposed law revision, and their arguments were not critically reviewed and more or less accepted by the other actors. Consequently, science and medicine were unable to play a major role in changing the political environment. For these reasons, a valuable opportunity for legislative change was lost in this period when patients were still young and had not become so dependent on their sanatoria in both countries. After the unsuccessful exploitation of these incomplete policy windows, the three streams of problems, policies and politics did not converge for several decades. By the late 1960s, it could be said that a near consensus had developed in the research community that isolation was an unreasonable solution. Moreover, the infringement of civil liberties was perceived to be a problem, a perception which increased in importance as academics and international health communities repeatedly recommended the abolition of isolation policy as scientifically unjustifiable. In the problem stream, focusing events were created by patients, administrators and medical professionals with the goal of encouraging the public and politicians to put the issue on the agenda. Most obviously, patients published periodicals, invited media persons to their meetings, petitioned bureaucracies, and lobbied legislators in both countries. In Japan, patients even conducted sit-ins around the Diet and the Ministerial buildings. The repeated recommendations of international organizations, and the occasional official reports recommending outsourcing and outpatient services, also served as focuses. In the political stream, there were some changes as well. As the public's fear of leprosy dwindled (or at least became latent as they forget about the disease), so did the associated stigma. Improved public attitudes toward the disease meant that the political climate was more favorable to returning the patients to society, terminating the isolation policy, and abolishing leprosaria. In both countries, there were periodic turnovers of leprosaria directors, sometimes recruited from outside the leprosaria. The appointment of leprosaria directors from other public hospitals and academic institutions, who had the intention to discharge the long-term residents, certainly could have opened a window. Every time the issue reached the agenda, however, the proposed policy alternatives raised concerns among patients. Treating leprosy as an ordinary disease and integrating treatment into general medical care and public health measures threatened the 'special' status of leprosy. Many patients, who had become dependent on the leprosaria, feared losing their homes and privileges. If from a scientific perspective the disease no longer required isolation, patients sought to justify their continued privileges either as government duty to provide disease victims with compassionate care or as government compensation for past erroneous policies. Such patients' claims were not easily accepted. Given the lack of a public consensus on the moral underpinnings for patient support, the belief that long-term residents are entitled to generous government "compensation" was a hurdle. To surmount that hurdle, administrators and medical professionals would have had to acknowledge their wrongdoing and determine when the isolation policy could and should have been judged as obsolete. Additionally, public opinion, and of course the views of elected officials, would have needed to be nurtured to recognize and sympathize with patients' adversaries, allowing for generous government payments. As a result, the position of the patients was not really unified, and therefore they could not fully mobilize themselves politically. Consequently, this policy community more or less shared the view that the patients' disadvantages caused by the law were a necessary cost of its even greater benefits, namely the continuation of leprosaria[ 101 ]. When it proved impossible to accomplish decisive legislative changes, straightforward efforts to discharge long-term and medically unwarranted residents were also reduced. Instead policy adjustments were made by incremental administrative actions. Slow, limited and sometimes informal efforts were made for patients' discharge and for the provision of outpatient services. As one might expect[ 102 , 103 ], these disjointed and informal increments of change had limited success in modifying the policy's original effect. Since the policy under the existing law was administered on the premise that leprosy requires confinement and special care, it was limited in its capacity to compel patients to leave the leprosaria. As many patients chose to remain in their leprosaria, isolation ostensibly continued in the form of long-term residential care. In this way, the main focus of leprosy prevention policy gradually changed from social protection and patient care to the mere provision of residential places for patients and ex-patients (and later, rehabilitative services). These bureaucratic satisficing behaviors, as reported in other cases[ 104 , 105 ], could not raise again the fundamental question of law revision, and as a consequence many engaged themselves in pork barrel politics to preserve and improve life in the leprosaria. For many years, policy (change) focused on discrete, short-term outputs rather than broader, long-range outcomes[ 106 ]. In the 1980s, broad policy revision became possible again though the agendas were set by factors again other than science, specifically the discussion on economic inefficiency in the US and the issue of human rights violation in Japan. In the former, large government expenditure was a big social and political issue, and there was an active search for potentials for spending cuts. In the latter, AIDS as a source of social stigmas and the forced institutionalization of psychiatric patients were both prominent in the media, calling for the protection of the human rights of the diseased. Furthermore, as Bardach suggested[ 107 ], increased political competition and Cabinet turnover at that time may also have increased the likelihood that elected officials would take up potential social issues to be remedied. In the US, Representatives Baker and Field fought for a House seat, while in Japan the two major parties competed vigorously for government office. An indispensable key to policy change was the development of policy alternatives. Interestingly, they were quite common in both countries. Evidently, policy change occurred only after the leprosaria were recognized as legitimate homes for some patients and outpatient services were officially established. Patients were assured of their residences, but had the option of leaving the institutions. The patient's privilege of remaining in a leprosarium was then justified as partial compensation for the past compulsory segregation. In both countries, hazard duty payments were also maintained for ongoing employees, who could have otherwise opposed to policy changes. The primary difference, as reflective of the key arguments against the old policy, i.e., economic inefficiency and human rights violation, was that in Japan a public apology was offered by the government for its long-term negligence, though the apology was later used by the patients to win further government compensation through lawsuits. Another point is that in the US, large stipends were awarded to patients as an economic incentive to reduce the number of institutional residents. The functions of science and scientists in the policy process From the perspective of the Garbage Can model, problems, policies and politics typically evolve in separate, unconnected streams. When science is considered as a fourth factor, it is seen to influence all the streams, changing perception of problems, helping develop policies, and transforming political environments[ 108 ]. Thus, science can be seen as approving or disapproving certain (existing) policies. However, it does not automatically create those effects by itself, nor in a political vacuum. Even when science indicates that an existing policy is no longer relevant, as this case study shows, people may make objections to its abolition for a variety of other reasons. In comparison with basic research, furthermore, applied research, including public health and epidemiological research, is more likely to follow an agenda driven by forces other than science[ 109 ]. This means that the imminence of an issue, or its agenda status, can affect the production and use of scientific knowledge. Furthermore, scientific disputes will not be always resolved during the time the scientific issues are considered relevant to policy discussions[ 110 ]. Looking back at Period I, Promin was first mentioned in Leprosy Review in 1945, but it was not until 1952 that sulfone therapy became accepted among academics[ 111 , 112 ]. Controversy on the effectiveness of sulfone drugs certainly still lingered at the time when the legislative changes were on the agenda, namely, around the time of the enactment of the Public Health Services Act and subsequent policy proposals in the US and at the time of the revision of Leprosy Prevention Law in Japan. The development and acceptance of scientific knowledge might have been slowed by the attitudes of authoritative and powerful experts in Japan. Also, in the US, quite a few experts continued to justify the isolation of infectious cases for many years. Arguments that did not support their convictions were sometimes screened out by disqualifying them. Authoritative positions of medical and administrative elites in society also hindered the public's critical appraisal of their arguments, as did the importance then attached to public health[ 113 ]. The strength of the conservatives' arguments possibly came also from the systemic bias in their position as defenders of status quo: they had to simply attack a proposer's case as insufficient[ 114 ]. In an adversarial process of rule-making, knowledge claims are sometimes deconstructed, exposing areas of weakness or uncertainty. These revealed weaknesses provide justification for political decision makers to assert that they have a right to engage in interpreting science, especially in areas that are controversial. Again, uncertainty within science itself is also a subject for negotiation, decision, and argument in policy process, since the quality and amount of knowledge are the objects of social negotiation[ 115 ]. This partial transfer of cognitive authority to the legal and political arena may be seen as the way of assuring that the interpretation of (indeterminate) facts reflects the public values embodied in the legislation as well as the norms of the scientific community[ 116 ]. The connections between given scientific data, expert interpretation of these data and policy content are like chains of linked arguments and beliefs[ 117 ]. This process might be termed co-evolution or the process of mutual validation between policy and science[ 118 ]. In a case where science and policy are co-constructed through processes which occur in tandem, it becomes difficult to explain the one by using the other[ 119 ]. From the vantage point of history, the very absence or scarcity of critical review among legislators and the public in reviewing scientists' statements on the risk of disease spread might reflect and indicate their own fear of the disease and indifference to patients' human rights. When scientific knowledge is used in policy development, scientists and/or policymakers may choose to err either on the side of public safety or on that of patients' liberty and dignity. For a long portion of Period II, incomplete knowledge was not necessarily the primary reason why science did not play a major role. Scientists, experts and legislators could have acted strategically as policy entrepreneurs to take advantage of occasional opportunities, provided by several focusing events, to open the windows of opportunity. In other words, scientific opposition, or dissensus if any, was not a major hindrance to their actions. Scientists, however, preferred not to encroach upon policy debates in an official and overt fashion, thus avoiding direct questions about justification and continuation of the institutions. In both countries during the 1960s and 1970s, there was a near consensus, albeit implicit among the interested parties, including scientists, physicians, bureaucrats, patients and politicians, that the leprosaria should be preserved or at least not immediately abolished. Though scientific assessment appeared conclusive and potent enough if effectively presented, consideration for the possible negative consequences of policy outcomes constrained its use. In the resultant incremental decision making, the demand for science was minimal. Later in period III, when development of policy alternatives was definitely the key to changing policies, the process was not fueled by (new) scientific knowledge or discussion, but rather propelled by political skills which crafted both the policies and the favorable environments. A scientific consensus had already existed before the issue was taken up at this point, which illustrates the secondary importance of science in the emergence of policy windows. At most, the known arguments were reiterated and pushed to the fore. Policy change was not really contested and disputed in terms of science, simply because it did not have to be once attractive policy alternatives were proposed. From this vantage in all the periods, science remained instrumental in the sense that scientific rationality was not the primary objective for which policy change was discussed or intended, and in the sense that scientific arguments were not the principal driving force for policy change. To review the course of events: Some initiating event, not directly associated science, leads to a policy issue that must be decided on; a debate ensues on the possible policy options and on possible scientific views of the issue; a scientific assessment of the policy issue yields a rationale for a policy choice[ 120 , 121 ]. Thus, science is used either to legitimate policies developed for nonscientific reasons or is ignored if the consensus contradicts policy or there is scientific "dissensus." To the extent which science can be regarded as instrumental, namely that final policy decision was rendered to lawmakers, the role and responsibility of science could decrease[ 122 ]. The scientific base and the political will to translate it to policy are ingredients necessary to induce policy changes[ 123 - 125 ]. Inertia and/or political considerations certainly exerted huge influence[ 126 ]. Policy entrepreneurs and leadership Although many contextual conditions could facilitate changes in health policies, they do not in themselves activate actions by either legislators or administrators[ 127 - 129 ]. As is the case with policy adoption[ 130 , 131 ], successful termination might need a political leader, a terminator, to trigger and manage effectively the process of policy termination. He must cultivate support from both influential interest groups and from the general public and choose the arena for policy discussions[ 132 - 134 ]. Then, a person skilled in policy termination should get the termination on the agenda, orchestrate advocacy coalitions, negate the survival tactics of anti-termination coalitions, and manage the administrative details[ 135 - 138 ]. With respect to HD policy, for a long period, intellectual reluctance, the tendency to institutional permanence, dynamic conservatism, efficacious anti-termination coalitions, and legal obstacles certainly posed difficulties for policy changes in both countries[ 139 , 140 ]. After a set of these contextual conditions were met, however, entrepreneurs had to accomplish their tasks. A crucial step for policy termination, namely the translation of ideas into action by coupling concrete and acceptable policy proposals with problem situations and political opportunities, was accomplished by a policy termination expert[ 141 , 142 ]. When things proceed as the Garbage Can model suggests, policy change requires a good luck and/or a skillful policy entrepreneur, who can induce the convergence of streams and open the windows of opportunity. This study highlighted an essential role assumed by a skillful terminator, Baker in the US and Otani in Japan. In the US, Representative Baker, along with the PHS officials, held meetings with the patients and staffs at the institution so that all interested parties could reach agreement on his proposals. The PHS publicized its own review of the institution and its activities highlighting their economic inefficiency. In this way, Baker and the PHS maneuvered the political process to successful legislation enactment. In Japan, Otani dramatized the evils of the old policy, stressing the benefits of termination, while mollifying the worried patients by reassuring of their continued residence in the sanatoria. He thus successfully engineered a consensus among key interest groups, publicizing a thorough justification for termination and devising an acceptable plan. Through political leadership and skill, the issue was finally brought to the forefront, alternatives supplied, obstacles cleared, and the policy abolished. Both Baker and Otani conducted a series of bandwagon activities making effective use of the media. Conclusion Quarantine measures aim to protect the health and safety of the public, and consequently individual liberty is subordinate to the common good[ 143 ]. Parens patriae, the obligation of the state to act as parent of the country in caring for those who cannot care for themselves, is the other side of the duty of the state to protect the rest of the community from infected individuals[ 144 ]. The human rights or civil liberties of patients must be weighed the effectiveness and efficacy of the isolation measure. It is inarguable that science could and should play a key part. The utilization of scientific knowledge contributes to increased rationality of health policy, which is desirable especially when incongruence could produce adverse effects. However, when policy changes occur as a result of the convergence of three largely independent streams of problems, policies and politics, we cannot assume a rational and linear process which automatically incorporates science into policy. As discussed, a variety of dynamics influence the utilization of scientific research in policymaking[ 145 - 147 ]. For policies to be reflective of science, these models suggest, the interactions among researchers and policy makers are critical. Research is more likely to be utilized in a significant way when effective networks and mechanisms are established at the interfaces among researchers and policy makers[ 148 ]. However, even that may not be sufficient. Events in the various streams should be made readily understandable and visible, either in hopes of a serendipitous convergence or so that policy entrepreneurs can assist in their convergence, in order to open the window of opportunity. Problems should be foregrounded, political factors should be elucidated, and feasible and attractive policy alternatives should be envisioned. Experts should be mobilized to assume authoritative roles in linking science and policy, though it should always be noted that they too could be biased in one way or another. Political empowerment of the people who suffer from the negative policy outcomes, and therefore might be most attentive to them, could also help serve the purpose. Policy is always developed both from scientific and nonscientific considerations, as this study indicated. The Possibilities for the instrumental use and/or disuse of science should be made explicit to the public. Mobilization of different social views and values, in comprehensive consideration of direct and indirect policy impacts, could lead to a more explicit and formal social process, which could eventually secure and propel the process of muddling through[ 149 ]. Public governance over these processes, along with public's enlightenment and mobilization, might facilitate, check and complement the functions fulfilled by the experts. Analysis of policy and politics is the indispensable key to these processes. Competing interests The author(s) declare that they have no competing interests. Authors' contributions All the authors (HS, JF) fully participated in the planning of research, the collection of historical materials, and the carrying out of interviews in both the US and Japan. After thorough discussion, HS drafted the article, and both authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079838

A HEV-restricted sulfotransferase is expressed in rheumatoid arthritis synovium and is induced by lymphotoxin-α/β and TNF-α in cultured endothelial cells

Background The recruitment of lymphocytes to secondary lymphoid organs relies on interactions of circulating cells with high endothelial venules (HEV). HEV are exclusive to these organs under physiological conditions, but they can develop in chronically-inflamed tissues. The interaction of L-selectin on lymphocytes with sulfated glycoprotein ligands on HEV results in lymphocyte rolling, which represents the initial step in lymphocyte homing. HEV expression of GlcNAc6ST-2 (also known as HEC-GlcNAc6ST, GST-3, LSST or CHST4), an HEV-restricted sulfotransferase, is essential for the elaboration of L-selectin functional ligands as well as a critical epitope recognized by MECA-79 mAb. Results We examined the expression of GlcNAc6ST-2 in relationship to the MECA-79 epitope in rheumatoid arthritis (RA) synovial vessels. Expression of GlcNAc6ST-2 was specific to RA synovial tissues as compared to osteoarthritis synovial tissues and localized to endothelial cells of HEV-like vessels and small flat-walled vessels. Double MECA-79 and GlcNAc6ST-2 staining showed colocalization of the MECA-79 epitope and GlcNAc6ST-2. We further found that both TNF-α and lymphotoxin-αβ induced GlcNAc6ST-2 mRNA and protein in cultured human umbilical vein endothelial cells. Conclusion These observations demonstrate that GlcNAc6ST-2 is induced in RA vessels and provide potential cytokine pathways for its induction. GlcNAc6ST-2 is a novel marker of activated vessels within RA ectopic lymphoid aggregates. This enzyme represents a potential therapeutic target for RA.

Background Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the synovial membrane that leads to the destruction of cartilage and bone tissues. The major cellular components of the inflammatory infiltrate in RA synovium are T cells, macrophages, B cells, and dendritic cells (DCs) [ 1 ]. Tissue-infiltrating lymphocytes are often arranged in follicles which exhibit variable degrees of germinal center (GC) reactions [ 2 - 4 ]. These highly organized structures are similar to secondary lymphoid organs, and the process is referred to as lymphoid neogenesis [ 5 , 6 ]. In RA, this ectopic formation of lymphoid structures correlates with the expression of lymphotoxin-β (LT-β) and homing chemokines such as BLC (CXCL13) and SLC (CCL21), recapitulating the developmental process of lymphoid organogenesis [ 7 ]. The recruitment of cellular elements to secondary lymphoid organs relies on interactions between circulating leukocytes and the specialized endothelium of high endothelial venules (HEV), which are exclusive to these organs under physiological conditions. The interaction of L-selectin on lymphocytes with sulfated glycoprotein ligands on HEV of lymph nodes results in lymphocyte rolling on endothelium which represents the initial step in lymphocyte homing [ 8 ]. HEV-ligands for L-selectin consist of a set of heavily O-glycosylated glycoproteins, which include GlyCAM-1 and CD34 in the mouse and podocalyxin and CD34 in the human [ 9 ]. Recognition of these ligands by L-selectin requires sialylation, fucosylation and sulfation of their mucin-like domains [ 10 ]. The minimal recognition epitope appears to be comprised of a capping group known as 6-sulfo sialyl Lewis x (6-sulfo sLex) in which the C-6 position of GlcNAc is esterified with sulfate. The MECA-79 mAb recognises a critical sulfation-dependent determinant on these ligands that overlaps with the L-selectin recognition epitope. Expression of GlcNAc6ST-2 (commonly referred to as HEC-GlcNAc6ST or LSST), an HEV-localized sulfotransferase, is essential for the elaboration of functional ligands within lymph nodes, as well as the generation of the MECA-79 epitope [ 11 - 13 ]. Another sulfotransferase known as GlcNAc6ST-1 also contributes to this epitope in HEV [ 14 ]. MECA-79 recognizes a set of sialomucins, including the aforementioned L-selectin ligands, together with another recently identified sialomucin called endomucin [ 15 , 16 ]. The complex is referred to as peripheral node addressin (PNAd) [ 13 ]. MECA-79 blocks the attachment of lymphocytes to peripheral lymph node (PN) HEV in vitro and inhibits lymphocyte homing to PN in vivo [ 17 ]. The role of GlcNAc6ST-2 in the generation of L-selectin ligands in lymphoid organs has been clearly established in mice genetically deficient in this enzyme [ 18 - 20 ]. Lymphocyte homing to PN is reduced in the GlcNAc6ST-2 -/- mice as reflected by a smaller size of the PN and a 60% decrease in the number of total lymphocytes within this organ. In addition to lymphoid organs, induction of the MECA-79 epitope has been identified in several chronically inflamed tissues where it is proposed to participate in leukocyte recruitment [ 21 ]. Inflamed synovium contains high-walled vessels, resembling HEV, which express the MECA-79 epitope and are capable of binding L-selectin+ lymphocytes in ex vivo assays [ 21 - 25 ]. Recently, the use of GlcNAc6ST-2 specific antibodies has demonstrated de novo induction of this enzyme in HEV-like vessels and its correlation with the presence of the MECA-79 epitope in several mouse models of chronic inflammation [ 26 ]. In this study using an antibody to human GlcNAc6ST-2, we analyse whether this enzyme is expressed in RA and in cultured endothelial cells and its correlation with the expression of MECA-79 epitope. Results Expression of MECA-79 epitope and GlcNAc6ST-2 in synovial tissues We produced an anti-human GlcNAc6ST-2 antibody and tested it on human lymph node and tonsil sections. A vascular pattern of labelling was observed which colocalized to MECA-79 positive vessels of HEV morphology (Figure 1a ). A few mononuclear cells were also labelled with the anti GlcNAc6ST-2 antibody. Expression of the MECA-79 epitope was detected in 7 of 14 RA and none of the 9 OA synovial tissues. Most MECA-79 + vessels showed a HEV morphology but in some cases, small vessels with a flat endothelium were also labelled. Five of the 7 MECA-79 + tissues contained abundant lymphocytic infiltrates, which in 4 cases displayed a follicular pattern, and in 2 cases, MECA-79 + vessels were observed in the absence of significant infiltration (Table 1 ). Three MECA-79 negative RA tissues contained diffuse lymphocytic infiltrates and in one case, a single follicular infiltrate. Expression of GlcNAc6ST-2 was not detected in any of the OA synovial tissues. In contrast, 10 of 14 RA tissues showed GlcNAc6ST-2 by immunoperoxidase staining (Figure 2 ). Staining was located to endothelial cells of HEV vessels in the sublining and in some samples, also in small vessels with flat endothelium. In some sections, a few perivascular mononuclear cells were also stained. Double MECA-79 and GlcNAc6ST-2 immunofluorescence showed that all MECA-79 positive vessels also displayed GlcNAc6ST-2 (Figure 1b ). However, in a low proportion of GlcNAc6ST-2 + vessels, MECA-79 was not detected. Specificity of the immunoperoxidase GlcNAc6ST-2 labelling was confirmed by substituting normal rabbit IgG for the specific antibody. There was no detectable staining (data not shown). Preincubation of the anti-GlcNAc6ST-2 antibody with the peptide immunogen abrogated the staining (Figure 2b ). We also immunostained with a polyclonal antibody raised against murine GlcNAc6ST-2 [ 26 ] and observed a similar pattern of staining in small vessels (Figure 2c ). This antibody reacted with smooth muscle cells in the media of larger vessels which we attribute to a spurious cross-reactivity (data not shown). Immunostaining with this antibody was also abrogated by preincubation with murine GlcNAc6ST-2 peptide (Figure 2d ). Expression of GlcNAc6ST-2 by cultured HUVEC To analyze the potential role of LT-αβ and TNF-α, two cytokines abundantly expressed in RA synovium and associated with lymphoid neogenesis and GlcNAc6ST-2 expression in transgenic models [ 3 - 7 , 26 , 28 ], we examined the effects of the exposure to these cytokines on GlcNAc6ST-2 expression in human endothelial cells lacking the HEC phenotype (HUVEC). By RT-PCR, treatment with LT-αβ or TNF-α for 8 h induced a significant increase in the expression of GlcNAc6ST-2 mRNA in HUVEC (Figure 3a ). A synergistic effect of treatment with both cytokines was not detected (data not shown). Treatment with an unrelated cytokine (interferon-γ) did not significantly modify GlcNAc6ST-2 mRNA expression levels. By western blot analysis with the anti-human GlcNAc6ST-2 antibody, we detected a single 50 kD band in HUVEC protein extracts, consistent with the expected MW for human GlcNAc6ST-2 [ 11 , 12 ]. GlcNAc6ST-2 protein expression was significantly increased after 24 h of TNF-α or LT-αβ treatment in HUVEC (Figure 3b ). Immunofluorescent labelling of HUVEC cultures with anti-human GlcNAc6ST-2 antibody showed a weak, but clearly detectable signal above the non-immune rabbit IgG control. Pretreatment with either TNF-α or LT-αβ induced a clear increase of immunofluorescence with a perinuclear distribution which is characteristic of the Golgi apparatus (Figure 3c ). Neither TNF-α nor LT-αβ treatment induced expression of the MECA-79 epitope in HUVEC (data not shown). Discussion The presence of MECA-79 + vessels has been previously reported in human inflammatory lesions, often associated with the development of lymphoid neogenesis [ 21 , 26 ]. In a variety of unrelated inflammatory conditions (rejecting heart and kidney allografts, peribronchial specimens from asthmatics, thyroiditis, psoriasis, and H. pylori infection) induction of MECA-79 + vessels in association with inflammatory infiltrates has been demonstrated [ 29 , 32 ]. In some cases, MECA-79 + vessels also display HEV-like morphology although flat endothelium of inflammatory vessels can also express the MECA-79 epitope. In chronic arthritis synovial tissues, the presence of HEV and MECA-79 + vessels has been previously demonstrated [ 22 , 23 ]. Recently, the pathophysiological significance of MECA-79 + vessels has been directly demonstrated in a sheep model of asthma. Pretreatment of asthmatic sheep with intravenously administered MECA-79 blunted both late-phase airway responses and airway hyperresponsiveness induced by airway allergen challenge [ 33 ]. In murine models of lymphoid neogenesis, including spontaneous models (AKR mouse and NOD mouse), and transgenic models (RIP-BLC and RIP-LT-αβ), expression of the MECA-79 epitope is closely associated with the induction of GlcNAc6ST-2 [ 12 , 26 , 28 ]. Taking advantage of a newly-generated antibody to human GlcNAc6ST-2, our present results are the first to show this association in a chronic inflammatory disease in humans. Several of our observations suggest that the induction of GlcNAc6ST-2 represents an early event in the process of lymphoid neogenesis within RA synovium. In the first place, GlcNAc6ST-2 was expressed in flat and high endothelial vessels which exceeded the number of MECA-79 + vessels in double-labelled sections. Secondly, GlcNAc6ST-2 and MECA-79 reactivity were detected in some vessels which were not associated with lymphoid aggregates, and in a few cases, even in the absence of significant mononuclear cell infiltration. The latter observation also suggests that MECA-79 + vessels may contribute to lymphoid recruitment, but additional factors are necessary. This is consistent with the multistep model of leukocyte transendothelial migration to lymphoid organs, where posting of chemokines on endothelium and integrin-mediated adhesion of rolling leukocytes are required [ 8 ]. Lack of expression of MECA-79 epitope and GlcNAc6ST-2 in OA samples suggests that the scanty infiltration of OA tissues is not sufficient to induce these features and that RA-associated factors are needed. The development of lymphoid neogenesis in RA synovium has been suggested to facilitate local autoimmune responses permitting the development of B cells and local production of autoantibodies and immune complex formation [ 34 - 36 ]. The recent development of B-cell depleting therapy demonstrates an important role for these cells in RA [ 37 ]]. The recruitment of L-selectin + dendritic cells, B-cells and CD45RA T-cells to RA synovium correlates with the expression of homing chemokines such as CXCL13, CCL19 and CCL21, and the latter partially colocalizes to MECA-79 positive HEV [ 3 , 38 , 39 ]. We have also detected expression of CXCL12, a homing chemokine abundantly present in RA tissues in HEV-like vessels [ 40 ]. Local expression of LT-β and TNF-α are important factors in the pathogenesis of RA, and LT-β expression correlates with the expression of homing chemokines and lymphoid neogenesis [ 1 , 3 ]. The role of LT-αβ and homing chemokines in the induction of HEV has been suggested by murine studies in RIP-LT-αβ and RIP-BLC (CXCL13) mice which spontaneously develop lymphoid aggregates and GlcNAc6ST-2 expression in MECA-79 + vessels [ 26 , 28 , 29 ]. The role of this enzyme in lymphoid recruitment has also been studied in RIP-BLC/ GlcNAc6ST-2 -/- mice which exhibit a reduction in lymphoid infiltration [ 26 ]. We demonstrate that de novo induction of GlcNAc6ST-2 expression also occurs upon LT-αβ or TNF-α stimulation in cultured HUVEC, further supporting the role of LT-αβ and TNF-α as major inducers of extranodal lymphogenesis. Enforced LT-αβ expression in murine models of lymphoid neogenesis induces the expression of homing chemokines, making it difficult to dissect the role of LT-αβ and chemokines in GlcNAc6ST-2 expression and HEV-development [ 41 ]. A role for LT-αβ, which is downstream of chemokines, has been observed in a transgenic model of BLC expression by genetic or functional targeting of LT-αβ [ 42 ]. In murine models of extranodal lymphoid neogenesis, a non redundant role for LT-αβ signaling through the alternative NF-κB pathway has been demonstrated, whereas the role for TNF-α seems dispensable and ectopic LT-α does not induce GlcNAc6ST-2 expression (13, 28). Our data suggest that in human cultured EC TNF-α may contribute to this process by inducing GlcNAc6ST-2 although its relevance in vivo remains to be determined. Therefore, our data suggest that both LT-αβ and TNF-α can directly induce GlcNAc6ST-2 in cultured endothelial cells, although additional factors seem to be required for the full development of the HEC phenotype in vitro . One such factor may be the core 1 extension enzyme, known as Core1-β 3GlcNAcT, which is required for the formation of the MECA-79 epitope on O-glycans [ 29 , 43 ]. Our results further establish carbohydrate modifying enzymes as an important group of transcriptional targets for cytokines during inflammation. Increased carbohydrate sulfation of several glycoproteins, including heparan sulfate proteoglycans, CD44 and airway mucins, has previously been demonstrated in response to TNF-α [ 44 - 46 ]. In RA, TNF-α plays a major and multifunctional role in the pathogenesis of the disease whereas the role of LT-αβ is currently being explored [ 47 ]. The potential contribution of these factors to the development of HEV expands the spectrum of possible pathogenetic roles for these cytokines. Conclusion MECA-79 defined glycoproteins serve as ligands for L-selectin during the process of lymphocyte homing to lymph nodes. This class of ligands is also critical for inflammatory leukocyte trafficking and the associated disease, as recently demonstrated in a sheep model of asthma. Previous experiments in the mouse have shown the requirement for GlcNAc6ST-2 in generating the MECA-79 epitope and the associated L-selectin ligand activity in HEV and HEV-like vessels. Here, we demonstrate that MECA-79 + vessels within human RA synovium also express GlcNAc6ST-2, strongly implying that this enzyme contributes to the ligands on these vessels. Establishing the direct participation of GlcNAc6ST-2 in the formation of L-selectin ligands on RA HEV would identify this enzyme as a potential therapeutic target for inhibiting leukocyte recruitment to the joints. In the same context, our finding that TNF-α induces GlcNAc6ST-2 in cultured endothelial cells may help to rationalize the efficacy of anti TNF-α therapy in the treatment of RA and other inflammatory diseases. Moreover, the activity of LT-αβ in inducing GlcNAc6ST-2 in HUVEC is likely related to the established requirement for this cytokine in the formation of HEV during lymphoid organ development. Many opportunities for future investigation are clearly indicated. Methods Preparation of anti-human GlcNAc6ST-2 antibody A polyclonal antibody against human GlcNAc6ST-2 was prepared following the same basic procedures used for mouse GlcNAc6ST-2 [ 26 ]. The peptide chosen, RGKGMGDHAFHTNC, differed from that of the corresponding mouse sequence by one amino acid (D replacing Q) with the C-terminal C added to facilitate conjugation. The peptide was coupled to KLH and injected into New Zealand White rabbits by Covance Research Products (Denver, PA). 500 μg of conjugated peptide was mixed with 0.5 ml complete Freund's adjuvant and injected intradermally, followed by three boosts, every three weeks, with subcutaneous injections of 250 μg conjugated peptide in 0.5 ml incomplete Freund's adjuvant. The subsequent antiserum was subjected to purification on the immunogen peptide coupled to agarose using the SulfoLink Kit (Pierce Biotechnology, Rockford, IL), following the manufacturer's recommendations. The affinity purified antibody was washed into Dulbecco's PBS on a Centricon 30 (Millipore, Billerica, MA) and concentrated to 0.125 mg/ml. GlcNAc6ST-2 and MECA-79 immunolabeling Synovial tissues were obtained from 14 RA and 9 control (OA) patients at the time of knee prosthetic replacement surgery. All patients gave informed consent, and the study was approved by the ethics committee of the Hospital 12 de Octubre. Tissues were snap frozen in OCT and stored at -80°C. Human tonsil and lymph nodes frozen sections were used as controls for MECA-79 and GlcNAc6ST-2 immunolabeling. Frozen sections (8 μm) were fixed for 10 minutes in cold acetone. Double GlcNAc6ST-2 and MECA-79 immunolabeling was performed by blocking for 30 minutes in 5% normal goat serum PBS, followed by incubation of sections with 1.4 μg/ml MECA-79 mAb (rat IgM) and 0.3 μg/ml rabbit anti-human GlcNAc6ST-2 at room temperature for 1 hour. After washing in PBS, a secondary biotinylated goat anti-rabbit IgG at 1.3 μg/ml was added (Jackson Immunoresearch Laboratories, West Grove, PA). Fluorescence was developed with 1.5 μg/ml Cy3-conjugated goat anti-rat IgM and 1.8 μg/ml Cy2-conjugated streptavidin (Jackson Immunoresearch Laboratories). Immunoperoxidase staining was performed by overnight incubation at 4°C with either anti-human or anti-mouse GlcNAc6ST-2. Endogenous peroxidase was blocked in 0.3 % H 2 O 2 in methanol. Immunoperoxidase detection was performed by ABC method according to the manufacturer (Vectastain ® , Vector Laboratories, Burlingame, CA). Peroxidase activity was developed by diaminobenzidine substrate and slides were counterstained in Gills hematoxylin. Controls with non-immune rabbit IgG at the same concentration as anti-human or anti-mouse GlcNAc6ST-2 antibodies were included. Additional controls were performed by preincubating sections with human or mouse GlcNAc6ST-2 peptide at 10 μg/ml. To ensure identical conditions, RA and OA sections were mounted on the same slide and all slides were processed in parallel. GlcNAc6ST-2 expression in HUVEC cultures Human umbilical vein endothelial cells (HUVEC) were prepared from umbilical cords by collagenase digestion and were propagated in medium 199 (Life Technologies, Paisley, Scotland) with 20% FCS. Cultured cells were characterized as EC by flow cytometry with anti-endothelial cell antibody P1H12 (Chemicon International, Temecula, CA). The presence of HEC in these cultures was excluded by flow cytometry with MECA-79 mAb. HUVEC cultures were stimulated with either 25 ng/ml of TNF-α, 20 ng/ml of LT-α1/β2, or 200 ng/ml of interferon-γ (R&D Systems, Inc., Abingdon, UK) for 8–24 h and total RNA or protein were extracted. For immunofluorescent detection of GlcNAc6ST-2, HUVEC were cultured on glass coverslips and similarly treated. Coverslips were fixed with 4% paraformaldehyde and immunofluorescent detection of GlcNAc6ST-2 was performed as described above for tissue sections. RT-PCR was performed on cDNA synthesized from 1 μg of total RNA of EC cultures using the following GlcNAc6ST-2 oligonucleotides: upstream 5'-GCAGCATGAGCA AAAACTCAAG-3' and downstream 5'-TCCAGGTAGACAGAAGATCCAG-3', and β-actin oligonucleotides upstream 5'-CTACCTCATGAAGATCCTCAC-3' and downstream 5'-GTCCACGTCACACTTCATGATG-3'. Real-time monitoring of PCR reactions was performed on a Roche LightCycler instrument, using SYBR Green I kit (Roche Diagnostics, Mannheim, Germany). The specificity of the amplicons was checked by electrophoresis which demonstrated single transcripts of 449 bp ( GlcNAc6ST-2 ) and 303 bp ( β-actin ) as expected. For relative quantification, we calculated the n-fold differential expression by the ΔC t method (Ct denoting the threshold cycle of PCR amplification at which product is first detected by fluorescence) that compares the amount of target gene amplification, normalized to the β-actin endogenous reference as previously described [ 27 ]. Western blot detection of GlcNAc6ST-2 was performed on HUVEC protein extracts (50 μg) electrophoresed on a 10% polyacrylamide gel and electrophoretically transferred to nitrocellulose filters. Membranes were incubated overnight with either rabbit anti-human GlcNAc6ST-2 or control anti-β actin (clone AC-15, Sigma-Aldrich Química, Spain) antibodies in 5% non-fat dried milk-TBST. The filters were washed and incubated for 1 h with secondary antibodies linked to peroxidase at 0.08 μg/ml (Santa Cruz Biotechnology, Santa Cruz, CA). Bands were visualized by an enhanced chemiluminiscence system (Pierce, Rockford, IL). Authors' contributions JLP carried out the immunodetection studies and participated in the study design and coordination. BS performed the studies in cultured endothelial cells. MG and GP participated in the selection of patients, and the collection and preparation of synovial tissues. DT and MSS prepared the antibodies and developed the immunodetection techniques. SDR participated in the design and coordination of the study. All authors contributed to drafting the manuscript and have read and approved the final version.

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1079839

Heterologous expression in Tritrichomonas foetus of functional Trichomonas vaginalis AP65 adhesin

Background Trichomonosis, caused by Trichomonas vaginalis , is the number one, nonviral sexually transmitted infection that has adverse consequences for the health of women and children. The interaction of T. vaginalis with vaginal epithelial cells (VECs), a step preparatory to infection, is mediated in part by the prominent surface protein AP65. The bovine trichomonad, Tritrichomonas foetus , adheres poorly to human VECs. Thus, we established a transfection system for heterologous expression of the T. vaginalis AP65 in T. foetus , as an alternative approach to confirm adhesin function for this virulence factor. Results In this study, we show stable transfection and expression of the T. vaginalis ap65 gene in T. foetus from an episomal pBS- ap65-neo plasmid. Expression of the gene and protein was confirmed by RT-PCR and immunoblots, respectively. AP65 in transformed T. foetus bound to host cells. Specific mAbs revealed episomally-expressed AP65 targeted to the parasite surface and hydrogenosome organelles. Importantly, surface-expression of AP65 in T. foetus paralleled increased levels of adherence of transfected bovine trichomonads to human VECs. Conclusion The T. vaginalis AP65 adhesin was stably expressed in T. foetus , and the data obtained using this heterologous system strongly supports the role of AP65 as a prominent adhesin for T. vaginalis . In addition, the heterologous expression in T. foetus of a T. vaginalis gene offers an important, new approach for confirming and characterizing virulence factors.

Background The colonization of the urogenital tract of humans by the protozoan parasite Trichomonas vaginalis is responsible for trichomonosis [ 1 ], the most prevalent, non-viral sexually transmitted infection worldwide. Despite an estimated 8 million new cases per year in the United States alone [ 2 ], this health disparities disease [ 3 ] remains poorly studied. T. vaginalis infection is associated with adverse health consequences to both men and women, including infertility [ 4 , 5 ], atypical pelvic inflammatory disease [ 6 ], and increased HIV transmission [ 7 , 8 ]. Trichomonosis is also associated with preterm birth, low birth weight infants [ 9 ], predisposition to development of cervical neoplasia [ 10 ] in women and non-gonococcal urethritis [ 11 ] and chronic prostatitis [ 12 ] in men. T. vaginalis adherence to host VECs, a step preparatory to infection [ 13 ], is complex and involves four surface protein adhesins. Three adhesins studied to date at the molecular level share identity with metabolic enzymes of the hydrogenosome organelle [ 14 , 15 ]. Thus, the proteins exhibit functional diversity based on cellular location. AP65 is a prominent T. vaginalis adhesin that is targeted both to the surface and hydrogenosomes, and this compartmentalization is in part modulated by iron [ 15 ]. Further, the surface placement of AP65 is increased upon contact with host cells [ 15 ]. Recently, silencing of ap65 gene expression in T. vaginalis with antisense reinforced the role of AP65 as a major adhesion [ 16 ]. The related Tritrichomonas foetus is a causal agent of bovine fetal wastage [ 17 , 18 ]. Adherence by T. foetus to bovine VECs appears equally complex as that seen for T. vaginalis except that in this case, adherence involves glycoconjugates [ 19 , 20 ]. Interestingly, the interaction between T. foetus organisms and VECs is both cell- and species-specific [ 21 , 22 ]. For example, T. foetus attaches to bovine VECs, MDCK and HeLa epithelial cells, which have been used previously for studies on the host-parasite interaction. However, the bovine trichomonads have low background levels of adherence to human VECs. This may suggest that the phylogenetically-related T. vaginalis and T. foetus trichomonads differ in mechanisms of host parasitism and cytopathogenicity. We hypothesized that, given the different mechanisms of host VEC adherence by T. foetus and T. vaginalis , the heterologous expression in T. foetus of the T. vaginalis AP65 could be an alternative approach to confirm the function of AP65 as an adhesin. Importantly, no cross-hybridization and immuno-crossreactivity with ap65 and mAbs to AP65, respectively, were evident in T. foetus . We now show the stable expression in T. foetus of a single copy of the T. vaginalis ap65 gene encoding an ~65-kDa protein [ 23 ]. The stable transfectants expressed functional AP65 on the T. foetus surface. These findings importantly illustrate the utility of a stable transfection system for heterologus expression in T. foetus of T. vaginalis virulence genes. Results Low levels of adherence to immortalized human MS-74 VECs and lack of ap65 by T. foetus We wanted to perform comparative binding experiments between T. vaginalis and T. foetus to epithelial cells used in adherence assays [ 15 ]. Given the fact that T. foetus exhibits host tissue- and cell-specificity [ 24 ], it was not surprising to find the lower levels of adherence by T. foetus to HeLa epithelial cells and immortalized human MS-74 VECs. As shown in representative experiment in Figure 1 , at best only 40% levels of adherence were seen for T. foetus compared to T. vaginalis . We next wanted to know if T. foetus had mRNA that hybridized to ap65 used as a probe. As shown in Figure 1B , no RNA band was detected by Northern analysis for T. foetus (lane labeled Tf). In contrast, a band of ~2-kb was readily hybridized by total RNA of T. vaginalis . MR100, the drug-resistant T. vaginalis that is unable to adhere to host cells as shown recently also had no detectable ap65 transcript, as a control [ 15 ]. The equivalent amounts of rRNA bands seen in Figure 1C illustrate that similar amounts of RNA were added to the lanes used in the Northern blots (Figure 1B ). Episomal expression in T. foetus of ap65 The expression plasmid pBS- ap65-neo contains the neomycin ( neo ) gene, and as done recently, the transfectants were selected for Geneticin resistance at 100μg/ml [ 16 ]. As shown in Figure 2A , the presence of plasmid in transfectants was verified by PCR amplification of the neo coding region. A band of 750-bp was detected in the transfectants corresponding to pBS- ap65-neo (lane3) and pBS- neo (lane 4). Not unexpectedly, no PCR products were obtained using T. vaginalis (lane 1) and T. foetus (lane 2). We then checked for the episomal gene expression by means of RT-PCR. The PCR products following reverse transcription were separated on 1% agarose gels. Figure 2B confirms the presence of an ap65 band corresponding to 580-bp amplified from the transfectants with pBS- ap65-neo (lane 3), confirming the expression of ap65 from the plasmid. The same band is also seen in T. vaginalis (lane 1). As an internal control in these experiments, the common band of 650-bp seen in all lanes corresponds to the a-tubulin gene. This control was important to show the similar amounts of RNA used for RT-PCR in all samples. These results demonstrate that the episomal plasmid with the T. vaginalis ap65 gene is successfully expressed in transfected T. foetus . T. vaginalis AP65 and AP65-HA are expressed in T. foetus , and episomal AP65 has function We next wanted to confirm that the episomal ap65 transcript seen in Figure 2 was translated. A 65-kDa band was readily detected by mAb 12G4 to AP65 in immunoblots of extracts of total proteins from T. foetus transfected with pBS neo-ap65 (Figure 3A , lane 2), and the protein had the same size as AP65 detected in total protein blots of T. vaginalis (lane 4). There were no detectable bands in either wild type T. foetus (lane 1) or T. foetus transfected with the control plasmid (lane 3). This result also confirms that the 12G4 mAb did not cross-react with any protein in blots of T. foetus . Importantly, we further tested for the ability of episomal AP65 in T. foetus to bind to host cells using a ligand assay [ 15 , 25 ]. Figure 3B (lane 4) shows a typical result of a ligand assay using T. vaginalis , and mAb 12G4 used as a probe detected AP65 on blots of proteins that bound MS-74 VECs that were solubilized for electrophoresis and blotting. Likewise, AP65 from transfected parasites bound to MS-74 VECs (lane 2) and gave a band with the same M r . There were no detectable protein bands for wild-type T. foetus (lane 1) and T. foetus transfected with control plasmid (lane 3) handled identically in the ligand assay. These results show the ability of T. foetus transfectants to stably express functional AP65. Further, the lack of any immuno-crossreactivity with mAb is consistent with the absence of transcript (Figure 1B ) and illustrates the absence of an AP65-like protein reactive with mAbs in T. foetus . AP65 expressed in T. foetus is compartmentalized to the surface and hydrogenosomes Next, we performed cell membrane fractionation experiments to determine whether episomally-expressed AP65 in T. foetus transfectants localized to the surface membranes. Fractions enriched for membranes and hydrogenosome organelles were obtained by differential centrifugation, and the fractions were used for monitoring the presence of AP65 using a ligand assay. Figure 4A illustrates AP65 detected by mAb 12G4 in blots of total proteins from membranes and hydrogenosomes of T. vaginalis (lanes 2 and 5) and T. foetus transfected with the pBS- ap65-neo plasmid (lanes 3 and 6). As above, no bands were visualized on blots with total proteins of wild type T. foetus (lanes 1 and 4). We then used these fractions to perform a ligand assay, and as seen in Figure. 4B , both membrane and hydrogenosome fractions of T. vaginalis (lanes 2 and 5) and transfected T. foetus (lanes 3 and 6) had AP65 that bound to MS-74 VECs. Wild type T. foetus had no protein detected by mAb from the ligand assay (lanes 1 and 4). Immunofluorescence detects AP65 in transfectants on the surface and in hydrogenosomes It was then important to examine the possible placement and accessibility of episomal AP65 on the surface of T. foetus . Figure 5 presents immunofluorescence data with T. vaginalis showing mAb 12G4 detecting surface AP65 (A4) and mAb F11 reacting with hydrogenosomal AP65 (decarboxylating malic enzyme) (B4), data consistent with that shown in a recent report by us [ 16 ]. Importantly, as with the fractionation experiments performed above, mAb 12G4 detected AP65 on the surface of non-permeabilized transfected T. foetus (Figure 5A2 ), and mAb F11 was reactive with AP65 within hydrogenosomes of permeabilized trichomonads (Figure 5B2 ). The absence of any fluorescence for wild type T. foetus (A1 and B1) and T. foetus with control plasmid (A3 and B3) reinforced the specific binding of mAbs with episomal AP65 and reaffirmed results from Figures 3 and 4 above. The brightfield pictures are included to show the integrity of the trichomonads in the fluorescence experiments. These data further confirm that episomal AP65 is sorted both to the parasite surface and to hydrogenosome organelles, suggesting that the trafficking signals are present within the AP65 open reading frame and that T. foetus also possesses the protein compartmentalization machinery similar to that of T. vaginalis . Episomal expression of fusion AP65-HA and sorting to the parasite surface Having established surface AP65 on transfected T. foetus , we next wanted to confirm surface placement using a different protein construct that would be detected by a distinct mAb. We, therefore, expressed the AP65-HA fusion protein, and immunoblot of total proteins of transfected T. foetus shows a band detected by the anti-HA mAb (Figure 6A , lane 5). As expected, the mAb 12G4 to AP65 detected a protein band in blots of total proteins of T. vaginalis (lane 1) and the pBS- ap65-HA-neo transfected T. foetus (lane 2). The M r of the fusion protein was higher than AP65, as expected. Also, no bands were apparent with any mAbs in T. foetus with control pBS- neo plasmid (lanes 3 and 6). Finally, as shown in Figure 6B , immunofluorescence microscopy gave a positive reaction with anti-HA mAb with AP65-HA protein on the surface using non-permeabilized and in hydrogenosomes with permeabilized transfected (Tf-pBS- ap65-neo ) trichomonads. The T. foetus transfected with the control plasmid (Tf-pBS- neo ) was unreactive with anti-HA mAb and gave results identical to those shown for the wild type T. foetus . Finally, in data not shown, no fluorescence with anti-HA mAb was obtained using T. vaginalis organisms as controls under identical experimental conditions. These data using a different mAb to the fusion protein construct independently demonstrated the surface placement of episomal AP65 and AP65-HA on T. foetus . Episomally-expressed AP65 in T. foetus is related to enhanced adherence to VECs Finally, we wanted to perform a functional adherence assay to determine if episomal, surface-expressed and host cell-binding AP65 on T. foetus , as shown collectively above, would enhance adherence of parasites to immortalized human MS-74 VECs. Figure 7 presents representative data showing that transfected parasites with pBS- ap65-neo were 40% more adherent to VECs when compared to the background level of adherence of wild type T. foetus (Tf). T. foetus with control plasmid gave lower adherence levels equal to wild type parasites (not shown). Importantly, the enhanced adherence was inhibited by 36 % in the presence of rabbit polyclonal anti-AP65 IgG antibody (striped bar), indicating specific AP65-mediated adherence, as before for T. vaginalis [ 15 , 16 ]. Control normal rabbit serum IgG did not inhibit adherence of the pBS- ap65-neo transfected T. foetus (shaded bar). Levels of adherence were compared with those of T. vaginalis (Tv) and normalized to 100%. Discussion We have shown that trichomonads possess surface adhesins with functional diversity [ 26 ]. The adhesins are also enzymes in hydrogenosome organelles involved in energy generation. Although complex and multifactorial, the process of T. vaginalis adherence to human VECs is mediated in part by AP65, the surface protein that plays a major role in adherence [ 15 , 16 ]. In this study, we established the utility of the T. foetus bovine trichomonad as a heterologous expression model system. We feel that among the noteworthy findings of this report are the following: 1) We show the stable episomal expression of the T. vaginalis prominent ap65 gene (Figure 2 ). 2) The transcript is translated to form functional AP65 adhesin (Figure 3 ). 3) Episomal AP65 and fusion AP65-HA are compartmentalized to the surface and hydrogenosomes and are accessible to recognition by specific anti-AP65 and anti-HA mAbs (Figures 4 through 6 ). 4) AP65 on transfected T. foetus increased adherence to VECs compared to parasites transfected with control plasmid (Figure 7 ), and the enhanced binding was reduced with anti-AP65 antibodies, showing specific AP65 mediated VEC attachment. The stable expression of T. vaginalis AP65 in transfected T. foetus allowed us to characterize via cell fractionation experiments the cellular location of the episomal protein. Remarkably, AP65 fractionated to the plasma membrane and hydrogenosomes (Figure 4 ). This fractionation data was reaffirmed by fluorescence experiments (Figure 5 ), and the ability to detect both surface AP65 and surface AP65-HA using distinct mAbs provides strong evidence for the trafficking of T. vaginalis proteins to distinct cellular compartments in the bovine trichomonad. These results indicate that machinery for recognition of the surface and organelle targeting sequences is similar among these phylogenetically-related trichomonad species. Importantly, the expression of AP65-HA now will permit future subclone analysis for identification of the protein region within AP65 directing surface channeling. That surface AP65 on transfected T. foetus elevated levels of adherence to human VECs (Figure 7 ) is significant because human VECs are not the natural host cell for T. foetus [ 27 , 28 ]. The net increase in binding levels above T. foetus wild type controls were specific, as evidenced by the fact that anti-AP65 IgG reduced adherence to original control values. It is not inconceivable that the experimental conditions used here would not increase adherence by transfected T. foetus to those seen by T. vaginalis . One possible explanation is the fact that efficient and optimal adherence to VECs requires at least three additional adhesins, something that has been experimentally verified [ 15 , 16 ]. Alternatively, the copy number of episomal AP65 on T. foetus may not be optimal for production of amounts of adhesin needed for elevated adherence levels. Such a difference in copy number between AP65 on T. vaginalis and T. foetus (Fig. 3 ) may not be obvious based solely on fluorescence intensity, as this is not quantitative. In this regard, it may be that the equivalent protein of T. foetus decarboxylating malic enzyme is also surface expressed thereby interfering with sequestration of sufficient molecules of episomal AP65 for adherence. The future availability of specific antibodies to the T. foetus decarboxylating malic enzyme will be useful for testing this possibility. It is noteworthy that the ap65 gene and mAbs to AP65 did not cross-hybridize and immunoreact, respectively, with the equivalent decarboxylating malic enzyme gene and protein known to reside in hydrogenosomes of T. foetus [ 29 , 30 ]. These data also now indicate that there is significant nucleotide and amino acid sequence divergence between T. vaginalis and T. foetus in these equivalent genes and proteins. Importantly, except for the N-terminal sequence submitted to the GenBank (Accession number AAK55143.1), to our knowledge the entire decarboxylating malic enzyme gene of T. foetus has not been characterized [ 31 ]. Finally, not all episomal, surface-expressed AP65 molecules may be in the proper orientation to have adhesive function. Nonetheless, the significant increase in adherence attributable to episomal AP65 by transfected T. foetus once again underscores the role of AP65 in adherence by T. vaginalis . This paper now provides evidence for the functional expression of a heterologous virulence factor in a related trichomonad species. This ability to express T. vaginalis or other foreign proteins using T. foetus as a model is particularly noteworthy, especially if an identical and/or equivalent virulence factor and property are not detectable within the bovine trichomonad. We feel that this approach is innovative and represents a significant advance for understanding the contribution of specific T. vaginalis virulence factors to overall mechanisms of trichomonal pathogenesis. Methods Parasite and cell cultures Trichomonas vaginalis isolate T016 and Tritrichomonas foetus (02–97) were grown in Trypticase-yeast extract-maltose (TYM) medium supplemented with 10% heat-inactivated horse serum [ 32 ]. Immortalized human MS-74 VECs were grown as detailed recently by us [ 15 , 16 ] in D-MEM supplemented with 10% fetal bovine serum. VECs were grown at 37°C in a 5% CO 2 atmosphere. Plasmid construction The plasmid pBS- ap65-neo was constructed by cloning the coding region of AP65-3 in pBS- FdHAHA-neo (forward primer 5' GTCCAGCATATGATGCTCGCATCTTCA GTC-3' and reverse primer 5'-GTCCACGGTACCTTAGTAGAGTTGCTCGTATTC-3'). The parent plasmid pBS- FdHAHA-neo [ 33 ] was used by us recently [ 16 ]. The parent plasmid was partially digested and the 1.7-kb ap65-3 gene was cloned into the NdeI and Asp718 sites giving rise to pBS- ap65-neo . In order to generate AP65 with the HA tag, the stop codon of AP65 was mutated and cloned into pBS- FdHAHA-neo resulting in pBS- ap65-HA-neo . The new plasmids were confirmed by sequencing. Plasmid DNA for transfection was purified using maxi prep columns (Qiagen, Inc., Valencia, CA). Stable transfection and selection for G418 resistance Transfection of T. foetus cells was carried out by electroporation [ 34 ]. Parasites at early logarithmic phase of growth were used for transfection. Briefly, 4 × 10 7 parasites were centrifuged at 1,800 rpm at 4°C, and the pellet was resuspended in 400 μl fresh TYM before transferring into a 4-mm gap cuvette (BTX ® , Genetronics, Inc., San Diego, CA) with 25 μg of plasmid DNA. Electroporation was performed at 320 V, 1000 microfarads and 725 ohms using the ECM 630 Electro cell manipulator (BTX ® ). Following the pulse, cells were placed on ice for 10 min and transferred into two T25 flasks with 50 ml of fresh TYM-serum medium. The cells were grown free of drug for 24 h followed by the addition of Geneticin (Invitrogen-Life Technologies, Carlsbad, CA) at 100 μg ml -1 . Single cells were cloned using soft-agar plates [ 35 ]. The DNA was isolated from single cell cultures using DNAZol (Invitrogen) and further purified by phenol-chloroform extraction. The presence of plasmid in single cell clones was confirmed by PCR amplification of the neo gene. The sense 5' GATCGGTACCATGATTGATTGAACA AGATGGATTG-3', and antisense 5'-CTTTAGACCAAGTTCGTGTCAGAAGAACT CGTCAAG-3' primers were used in the PCR reaction. RNA isolation and RT-PCR analysis Total RNA was isolated using the Trizol reagent (Invitrogen). Briefly, 1 μg of total RNA was reverse transcribed using SuperScript II RNase H - Reverse Transcriptase (Invitrogen) in a reaction volume of 20 μl. One μl of the reverse transcribed cDNA was used as template for the PCR amplification. The ap65 primer sequences used for amplifications were ap65 -sense 5'-CAGTCAGTCGACCAGTTAGATATGGGTACAGAC-3' and ap65 -antisense 5'-GTGACAGGATCCCGCTCGCAGTTAGCGCATGTAG-3'. The α-tubulin gene primers were sense 5'-ACTCTGCTGCCTCGAGCACGGTATC-3' and antisense 5'-GAAATGACTGGTGCATAAGAGC-3'. The common annealing temperature for the primers of both genes allowed for the ap65 and α-tubulin primers to be included in the same PCR reaction. Immunoblot detection of episomally-expressed AP65 Total protein from parasites was obtained as before using trichloroacetic acid (TCA) precipitation [ 36 ] and separated on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [ 37 ] prior to blotting onto Hybond-P membranes (Amersham Pharmacia Biotech, Pisscataway, NJ) for immunoblot detection with mAb 12G4 to AP65 and anti-HA mAb. The mAbs and epitope reactivity were described recently [ 15 ]. Following blot reactivity with the mAbs, the bands were visualized by the chemiluminescence assay using horseradish peroxidase as the color developer (BioRad Laboratories, Hercules, CA). Sub-cellular fractionation The hydrogenosomes were isolated as recently detailed [ 15 ]. Briefly, 1 × 10 8 trichomonads were homogenized in buffer (250 mM sucrose, 20 mM KCL, 10 mM KH 2 PO 4 , 5 mM MgCl 2 and 20 mM Tris-HCl, pH 7.0). The homogenate was centrifuged to sediment nuclei and costae at 1,500 g for 10 min at 4°C. The supernatant was then transferred to a new tube and centrifuged at 12,000 × g for 10 min. The sediment was then washed once more with homogenization buffer and was highly enriched for hydrogenosomes, which was confirmed biochemically as recently described [ 16 ]. The sediment was resuspended in the buffer and stored at -70°C. Membrane isolation was also carried out as described recently [ 38 ]. T. foetus (1 × 10 8 ) were resuspended in phosphate-buffered saline (PBS), sonicated, and subjected to low-speed centrifugation at 500 × g for 15 min at 4°C. The resulting supernatant was further fractionated by sequential differential centrifugations of 1,500 × g, 10,000 × g, and 100,000 × g for 30 min at 4°C. The supernatant was used for protein analysis. Ligand assay to determine the amounts of adhesins on the surface The ligand assay to detect adhesins that bind to host cell surfaces was carried out as before [ 25 ]. Briefly, after fixation of the MS-74 VECs with glutaraldehyde, 10 6 epithelial cells were incubated with a trichomonal detergent extract derived from 2 × 10 7 solubilized parasites. After incubation, cells were vigorously washed to remove unbound and loosely-associated trichomonad proteins. Cells were boiled in electrophoresis dissolving buffer to elute the proteins bound to the MS-74 VEC followed by SDS-PAGE. The gels were further stained with Coomassie Brilliant blue for visualization, and duplicate gels were blotted onto Hybond-P membranes for immunoblot analysis using the mAbs as probes. Immunoprecipitation studies AP65 from the membranes and hydrogenosomes derived from the fractionation experiments was immunoprecipitated using mAb DM116 [ 15 ]. A standard immunoprecipitation protocol was used [ 39 ]. Briefly, the fractions (0.5 mg) prepared as mentioned above, were mixed with 5.0 μg of mAb DM116 for 4 h at 4°C. The extract and mAb were mixed on a rotating platform for 3 h with Protein A-Sepharose beads. The beads were washed three times with PBS, and samples were eluted by boiling for 10 min in SDS dissolving buffer. Eluted proteins were resolved by SDS-PAGE, and immunoblot analyses and ligand assays were performed as described above. Immunofluorescence detection of AP65 on the surface Immunofluorescence of AP65 on the surface and in hydrogenosomes of trichomonads was carried out using a modification of a recently-described procedure [ 15 ]. Briefly, 1 × 10 6 logarithmic-phase organisms were washed twice with cold PBS and fixed with 4 % paraformaldehyde for 10 min at RT. Fixed cells were washed in PBS and permeabilized with 1% NP-40 for 45 min at RT. Trichomonads were then blocked with 5% BSA for 1 h at RT prior to incubation for an additional 1 h at RT with hybridoma supernatants of mAb 12G4 (1:100) and anti-HA mAb F11 (1:1000). Parasites were washed with PBS and incubated for 1 h at 37°C with fluorescein isothiocyanate-conjugated anti-mouse IgG (Sigma) diluted 1:100. Finally, parasites were washed twice with PBS and observed under 1000× magnification using the Olympus BX41 microscope. Adherence assay Immortalized human MS-74 VECs [ 40 ] were used for the adherence assay as recently described [ 15 ]. Briefly, 2 × 10 4 MS-74 VECs were seeded onto individual wells of 96-well Costar flat bottom plates (Corning Inc., Corning, NY) and grown for 24 h in DMEM supplemented with 10 % fetal bovine serum (FBS). VECs were then washed twice with a medium mixture of DMEM:TYM (2:1; v/v) without serum. Trichomonads were labeled with [ 3 H] thymidine for 18 h, washed three times with DMEM-TYM and resuspended. Then, 4 × 10 5 tritium-labeled parasites were added to the individual wells of the 96-well plate with a confluent MS-74 VEC monolayer and incubated for 30 min at 37°C. Cells were then washed thoroughly with the DMEM-TYM. Individual wells were placed in mini-scintillation vials and radioactivity was measured. The assay was performed with quadruplicate samples. Reproducibility of experiments Unless otherwise stated in the text, all experiments were performed numerous times and no less than on four different occasions. Abbreviations AP65, adhesin protein Mr 65-kDa; DMEM, Dulbecco's minimum essential medium; HA, hemagglutinin; kb, kilobase; M r , relative molecular weight; pBS- ap65-neo , plasmid construct with the ap65 and neomycin genes; Tf, Tritrichomonas foetus ; Tv, Trichomonas vaginalis ; TYM, trypticase-yeast extract-maltose; VECs, vaginal epithelial cells. Authors' contributions ASK carried out the design of the study, performed transfections, Western blots, immunofluorescence, adherence assays and drafted the manuscript. VM constructed the plasmids, performed the Northern analysis, RT-PCR and wrote parts of the manuscript. JFA participated in the design of the experiments, offered suggestions during the experiments, and helped to draft the manuscript. All authors read and approved the final manuscript.

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1079840

Cloning and transcriptional analysis of the mouse receptor activity modifying protein-1 gene promoter

Background Receptor activity modifying protein-1 (RAMP-1) is a single transmembrane-domain protein required for the functional expression of calcitonin gene-related peptide (CGRP) receptors. To date, little is known about the molecular mechanism(s) that activate/inhibit RAMP-1 gene expression. Such mechanism(s) are likely to play a major role in modulating the responsiveness of tissues to CGRP. Results To initiate studies on the transcriptional regulation of the mouse RAMP-1 gene, RAMP-1 transcriptional initiation sites were mapped in a variety of tissues. Analysis of RAMP-1 expression in C2C12 myoblasts demonstrated that RAMP-1 mRNA is expressed at greatest levels in confluent myoblasts verses non-confluent and fused myoblasts. Transfection of confluent C2C12 myoblasts and NIH 3T3 cells with RAMP-1 promoter/luciferase deletion constructs revealed that 4.7 kb of RAMP-1 5' flanking region demonstrated optimal promoter activity while 343 bp of 5' flanking region was defined as a minimal RAMP-1 promoter. In non-RAMP-1 expressing HEK293 cells, constructs containing 4.7 kb to 782 bp of RAMP-1 5' flanking region were transcriptionally inactive. However, deletion of sequences -782 to -343 activated RAMP-1 promoter activity. Conclusion These findings suggest that tissue specificity of RAMP-1 gene expression is mediated by a negative acting transcription factor that represses RAMP-1 gene expression in non-RAMP-1 expressing tissues. This transcription factor is therefore likely to play an important role in modulating the responsiveness of tissues to CGRP.

Background The calcitonin gene-related peptide (CGRP) belongs to a family of related peptides that includes calcitonin (CT), adrenomedullin (AM) and amylin (AMY) [ 1 , 2 ]. To date, CGRP is one of the most potent endogenous vasodilatory peptides discovered. CGRP mediates sensory neurotransmission and inhibits insulin action on carbohydrate metabolism [ 2 ]. CGRP has been shown to modulate immune function by inhibiting the proliferation of T cells and synthesis of T cell-derived cytokines IL-2 and IFN-γ [ 3 - 6 ]. In the lung, CGRP mediates multiple effects some of which have potential implications in airway homeostasis [ 7 ]. CGRP has also been shown to have cardioprotective effects in rats and humans [ 8 , 9 ]. In skeletal muscle, CGRP potentiates muscle contraction [ 10 ], increases the numbers of acetylcholine receptors (AchR) [ 11 - 13 ] and their rate of desensitisation [ 14 ]. In addition, CGRP locally increases the rate of blood flow following muscle contraction [ 15 - 17 ]. The effects of CGRP are mediated by CGRP receptors that are generated by a complex of proteins [ 18 ]. CGRP receptors are formed by the interaction of two separate proteins. The first protein component is the calcitonin receptor like (CL) receptor. The CL receptor is a seven transmembrane-domain receptor but is inactive when expressed in cells alone [ 19 ]. The second protein component required for CGRP receptor function is receptor activity modifying protein-1 (RAMP-1). RAMP-1 acts as a molecular chaperone and is required for the transportation of the CL receptor to the cell surface in addition to pharmacologic specificity [ 18 ]. RAMPs are a recently identified group of single transmembrane-domain accessory proteins. To date, three members of the RAMP family have been identified (RAMP-1, RAMP-2 and RAMP-3) [ 18 ]. All share 30% sequence identity, differ in their tissue distributions and are comprised of approximately 160 amino acids that make up a large extracellular N-terminal domain, a single membrane-spanning domain and a short cytoplasmic domain [ 18 , 20 ]. Recently, Christopoulos et al. [ 21 ] demonstrated that RAMPS interact with a number of Class II G protein-coupled receptors (GPCRs) in addition to the CL receptor. These include the vasoactive intestinal polypeptide/pituitary adenylate cyclase activating peptide receptor (VPAC1R), the glucagon and parathyroid hormone receptors (PTH1 and PTH2). VPAC1R/RAMP-2 heterodimers display a significant enhancement of agonist-mediated phosphoinositide hydrolysis compared with VPAC1R alone. This suggests that RAMPs may play a more general role in modulating cell signalling through other GPCRs than previously thought. Despite the crucial role RAMP-1 plays in the generation of CGRP receptors, little is known about the molecular mechanism(s) regulating RAMP-1 gene expression. It is likely that such mechanism(s) play an important role in modulating the responsiveness of specific tissues to CGRP. To initiate studies on RAMP-1 gene regulation, we cloned and characterised the mouse RAMP-1 gene promoter. Analysis in three different RAMP-1 positive mouse tissues revealed multiple start sites of transcription. RT-PCR analysis of RAMP-1 mRNA in the C2C12 myoblast cell line demonstrated that endogenous RAMP-1 gene expression was greatest in confluent cultures compared to non-confluent or fused cells. Through the use of RAMP-1 promoter/luciferase constructs transfected into C2C12 myoblast cells and NIH 3T3 cells, we studied the effects RAMP-1 promoter deletions had on RAMP-1 transcriptional activity. We further demonstrated that the RAMP-1 promoter activity was tissue-specific and not expressed in the RAMP-1 negative cell line HEK293. Finally, we identified a repressor element in the RAMP-1 promoter which when deleted activates RAMP-1 promoter activity in HEK293 cells. This suggests the RAMP-1 gene is negatively regulated in non-RAMP-1 expressing cells. Results Determination of RAMP-1 transcriptional initiation sites and PCR cloning the mouse RAMP-1 promoter To date, the transcriptional initiation sites for any mouse RAMP transcript have not yet been determined. We therefore performed 5' rapid amplification of cDNA ends (RACE) analysis to map RAMP-1 transcriptional initiation sites. For these experiments, RNA from a number of known RAMP-1 positive mouse tissues (heart, brain and skeletal muscle) was employed. Sequence analysis of 8–10 5' RACE clones from each tissue demonstrated multiple start sites of transcription (Table 1 ). All transcriptional start sites in mouse heart, brain and skeletal muscle occurred within a 50 bp region directly 5' to RAMP-1's start site of translation. As demonstrated by the DNA sequence analysis above, this region is highly GC rich (78%). These results suggest that RAMP-1 transcriptional initiation sites do not significantly vary between the different RAMP-1 positive tissues tested. Having identified RAMP-1's major transcriptional initiation sites, we next wished to clone and characterise the mouse RAMP-1 gene promoter. Since extensive sequence data both 5' and 3' to RAMP-1's transcriptional start sites is available on the mouse genome data base (Accsession no. NT_039173.2) we employed RAMP-1 specific primers and PCR on mouse genomic DNA to generate a DNA fragment containing 4.7 kb of RAMP-1 5' flanking region. Promoter sequence motifs in the proximal 2.5 kb of 5' flanking region corresponding to consensus binding sites for a variety of transcription factors are present (Fig. 1 ). These include binding sites for the transcription factors NFκB, PBX1, AML1, OCT1, MITF, TEF1 and Sp1. Of note is a 147 bp region (-2188 to -2336) containing multiple GAAA and GGAA repeat sequences and a highly GC rich (78%) region of 144 bp region immediately 5' of the start site of translation (ATG). This GC rich region contains a potential binding site for the transcription factor Sp1. RAMP-1 gene expression in C2C12 myoblasts and myotubes Northern blot analysis has demonstrated that the RAMP-1 gene is expressed in mouse skeletal muscle [ 20 ]. Since a variety of muscle cell lines are widely available and are easily transfected with DNA constructs, we decided to employ such cell lines for our studies on the transcriptional regulation of the mouse RAMP-1 gene. Before undertaking transfection experiments in a muscle cell line, we first studied endogenous RAMP-1 gene expression in the myoblast cell line C2C12. Upon incubation with reduced serum levels and high cell density, C2C12 myoblasts fuse and form myotubes. Pittner et al. [ 22 ] previously demonstrated that incubation of this cell line with CGRP induces an increase in intracellular levels of cAMP making it highly likely this cell line expresses RAMP-1 mRNA. We therefore assayed RAMP-1 gene expression in this cell line by semi-quantitative RT-PCR analysis on total RNA from non-confluent, confluent and fused C2C12 myoblasts. Southern blot analysis of RT-PCR products from these cells demonstrated the presence of RAMP-1 mRNA under all culture conditions (Fig. 2 ). However, RAMP-1 mRNA levels appeared to be highest in confluent versus non-confluent and fused C2C12 myoblasts. Densitomitry demonstrated that RAMP-1 mRNA expression was at least 6 times higher in confluent vs. non-confluent myoblasts. RAMP-1 promoter deletional analysis in transiently transfected C2C12 myoblasts To delineate potential transcriptional regulatory elements in the 4.7 kb RAMP-1 promoter, a series of 5' RAMP-1 promoter/luciferase constructs were generated. Luciferase analysis on transfected confluent C2C12 myoblasts revealed that a RAMP-1 promoter/luciferase construct with approximately 4.7 kb of 5' flanking region (relative to RAMP-1's start site of translation) had the greatest levels of luciferase activity (30 fold over the background plasmid pGL3 basic). Deletion of approximately 700 bp of 5' flanking region (to -4102) caused up to a 55% drop in expression levels. Deletions to -343 of RAMP-1 5' region did not cause additional decreases in promoter activity. However, deletion to -164 reduced RAMP-1 promoter activity to 7-fold above background levels (Fig. 3 ). Similar results were obtained when RAMP-1 promoter/lucifersase constructs were transfected into RAMP-1 positive mouse NIH 3T3 cells. To determine if the RAMP-1 promoter demonstrates tissue specificity in transfected cell lines, we compared the transcriptional activity of the RAMP-1 promoter in transfected C2C12 myoblasts and a cell line known to not express RAMP-1 mRNA (HEK293). Luciferase analysis demonstrated that constructs containing 4.7 kb to 782 bp of RAMP-1 5' flanking region were transcriptionally inactive in HEK293 cells. A construct containing 470 bp of RAMP-1 5' flanking region demonstrated modest transcriptional activity while a construct containing 343 bp of 5' flanking region demonstrated significant promoter activity. The results of these experiments suggest that the RAMP-1 promoter region between -782 and -343 contains a regulatory element that represses RAMP-1 promoter activity in a non-RAMP-1 expressing cell line. Silencer activity of the RAMP-1 repressor element We wished to determine if the RAMP-1 repressor element could repress RAMP-1 promoter activity in a position and orientation independent manner (silencer function). We therefore employed PCR to generate a fragment (-343 to -782 of the RAMP-1 promoter) containing the RAMP-1 repressor element. The fragment was then cloned in two orientations 3' to the luciferase gene in the -343 RAMP-1 promoter/luciferase construct. The expression of these constructs was compared to activity from the -343, -470 and -4724 RAMP-1 promoter/luciferase constructs in transfected HEK293 cells. Luciferase assays demonstrated that the repressor element reduced expression from the -343 RAMP-1/luciferase construct by approximately 50% (see Figure 4 ). An unpaired Student's t-test revealed that these differences were statistically significant (p < .002 and p < .004 for 5'-3' and 3'-5' silencer constructs, respectively). However, these constructs demonstrated significantly higher levels of activity than the -470 RAMP-1/luciferase construct (p < .006 and p < .003 for 5'-3' and 3'-5' silencer constructs, respectively). These results suggest that the RAMP-1 repressor element located between -343 and -782 bp of RAMP-1 5' flanking region has modest but statistically significant silencer activity and functions best in its native position. Discussion Despite the important role RAMP-1 plays in CGRP cell signalling little is known about the transcriptional regulation of RAMP-1 gene. To date, no RAMP-1 gene promoter has been identified/cloned and RAMP-1 transcriptional initiation sites have not been determined in any RAMP-1 positive tissues. In the present study, we cloned the mouse RAMP-1 gene promoter and mapped multiple RAMP-1 transcriptional initiation sites in a variety of RAMP-1 positive tissues. Our data demonstrates that RAMP-1 transcriptional initiation occurs at similar sites and all within a 50 bp region in different RAMP-1 mRNA positive tissues. To initiate studies on the transcriptional regulation of the mouse RAMP-1 gene during muscle differentiation, we examined RAMP-1 gene expression in C2C12 mouse myoblasts by semi-quantitative RT-PCR. Our results suggest that RAMP-1 gene expression is present in non-confluent, confluent and fused C2C12 cells. However RAMP-1 mRNA expression appears to be increased in confluent and fused C2C12 cells. Chakravarty et al. [ 23 ] demonstrated that a strong correlation exists between the level of RAMP-1 mRNA expression and CGRP binding. This suggests that the transcriptional activity of the RAMP-1 gene may control the responsiveness of tissues to CGRP. It will be important to determine if increased RAMP-1 mRNA expression in confluent and fused C2C12 cells results in significantly higher levels of CGRP receptor expression and increases in intracellular cAMP after treatment with CGRP. The role increased RAMP-1 mRNA gene expression plays in the process of C2C12 induced muscle differentiation is not yet clear. Nobel et al. [ 24 ] showed that CGRP causes increased creatine kinase activity in myoblast cultures. Creatine kinase activity is a well established marker of fused myoblasts (myotubes). It is reasonable to hypothesise that increased RAMP-1 gene expression in confluent C2C12s makes these cells more responsive to CGRP and thus may facilitate myoblast fusion. Deletional mapping of the RAMP-1 gene promoter revealed that a 4.7 kb promoter fragment generates the highest levels of RAMP-1 promoter activity. However, a RAMP-1 promoter construct with only 343 bp of 5' region contains nearly 40% percent activity of the entire promoter. The delineation of such a small functional RAMP-1 promoter should facilitate the identification of DNA regulatory elements and the transcription factors they interact that activate RAMP-1 promoter activity in C2C12 cells. Transfection of RAMP-1 promoter/luciferase constructs into RAMP-1 negative HEK293 cells demonstrated that a transcriptional control element present between -782 and -343 in the RAMP-1 promoter represses RAMP-1 promoter activity in the RAMP-1 negative-expressing cell line HEK293 cells. While this element appears to have modest silencing activity, our results suggest that full repressor activity of this element requires it to be in its native position within the RAMP-1 promoter. We hypothesise that negative-regulatory transcription factors, present in non-RAMP-1 expressing cells, bind to this element and repress RAMP-1 gene expression. Computer analysis of the 480 bp RAMP-1 repressor element has revealed potential binding sites for a variety of positive acting transcription factors such as MyoD, c-Myb, NF1 and Elk-1. Importantly, binding sites for two transcriptional repressors have also been identified: mammalian transcriptional repressor RBPJkappa/CBF (RBPJK) and bZIP domain, transcriptional repressor (E4BP4). Studies on the roles these transcription factors play in the repression of RAMP-1 gene expression are now underway. Conclusion Promoter deletional analysis demonstrated that both positive and negative acting transcription factors regulate the tissue specificity of the RAMP-1 gene. DNase 1 protection and gel-shift experiments with extracts from C2C12 and HEK293 cells are underway to identify specific DNA sequences and the potential transcription factors they bind that mediate the activity of the RAMP-1 gene in RAMP-1 expressing and non- expressing cells. Once specific sequences/factors binding to the RAMP-1 repressor element are isolated, the effects that mutating these sequences has on RAMP-1 promoter activity in transgenic mice will be tested. These experiments will enable us to test the role such sequences/factors play in regulating the tissue-specific expression and activity levels of the RAMP-1 gene in vivo . Such transcriptional mechanism(s) are likely to play an important role in modulating the responsiveness of tissues to CGRP and may prove useful targets for future drug development aimed at regulating CGRP receptor gene expression. Methods 5' RACE analysis 5' RACE on total RNA from mouse heart, skeletal muscle and brain was performed employing a First Choice RLM-RACE kit (Ambion) according to manufacturers instructions. Briefly, 10 μg of total RNA from each tissue was treated with Calf Intestine Alkaline Phosphatase (CIP) for one hour at 37°C followed by extraction with phenol:chloroform and ethanol precipitation. The RNA was re-suspended in 11 μl of H 2 0. 5 μl of this RNA was then treated with tobacco acid pyrophosphate (TAP) for one-hour at 37°C. A 5' RACE adapter (provided in kit) was ligated to 5 μl of the TAP treated RNA. 2 μl of ligated RNA was subjected to reverse transcription (RT). RT products (1 μl) were subjected to two rounds of nested PCR according to manufacturers instructions. The first round of PCR employed an outer primer binding to the 5' RACE adapter (5'GCTGATGGCGATGAATGAACACTG 3') and an outer RAMP-1 gene specific primer (5' CGGGACCCTGACTATGGGAC 3'). The second round of PCR employed an inner primer binding to the 5' RACE adapter (5'GCTGATGGCGATGAATGAACACTG 3') and an inner RAMP-1 gene specific primer (5' TCTTCATGGTCACTGCCTGC 3'). Following the second round of PCR, PCR products were purified on an agarose gel and ligated into the PCR cloning vector pGEM-T Easy (Promega). A total of 8–10 clones representing each tissue were purified and subjected to DNA sequencing. Generation of RAMP-1 promoter/luciferase expression constructs To generate RAMP-1 promoter/luciferase deletion constructs, PCR utilising an antisense primer corresponding to sequences -26 to -43 (see Fig. 1 ) and sense primers corresponding to -145 to -164; -324 to -343; -470 to -450; -760 to -782; -935 to -948; -1220 to -1243; -1574 to -1593; -1827 to -1846; -2032 to -2093; -2342 to -2365; -2533 to -2511; -4102 to -4079 and -4724 to -4702 was carried out as described in Jagger et al. [ 25 ]. The PCR fragments were cloned into the vector pGEM-T Easy (Promega) and sequenced. The plasmids were then cut with Not 1 and the released RAMP-1 promoter fragment filled in with Klenow, purified and cloned into the luciferase expression vector pGL3 basic (Promega). To generate a construct containing the RAMP-1 repressor element cloned at the 3' end of the luciferase gene, the RAMP-1 promoter/luciferase construct containing 343 bp of 5' flanking region was cut with Bam H1. A region between -343 and -782 of the mouse RAMP-1 promoter was generated by PCR as above and cloned into the Bam H1 cut -343 RAMP-1 promoter/luciferase construct in two orientations. Tissue culture and RAMP-1/luciferase transfections C2C12, HEK293 and NIH 3T3 cells were maintained in DMEM supplemented with 10% foetal calf serum, 100 μg/ml penicillin and 100 U/ml of streptomycin. To generate fused C2C12 myotubes, cells were allowed to become confluent and growth media was replaced with media containing only 1% foetal calf serum. For transient transfections in all the above cell lines, cells were transfected with 1.0–1.5 μg of specific RAMP-1/luciferase construct, 0.2 μg of a co-transfection control plasmid pRLTK (Promega) and Fugene 6 (Roche) according to manufacturers' instructions. Luciferase assays were performed employing a Dual Luciferase Reporter Assay System (Promega) and a Turner Design TD-20/20 luminometer. Semi-quantitative RT-PCR on RAMP-1 mRNA in differentiating C2C12 myoblasts Total RNA from non-confluent, confluent and fused C2C12 myoblasts was isolated as above and reverse transcriptase (RT) reactions performed as previously described [ 25 ]. PCR amplification employing mouse RAMP-1 sense (5' CTATGGGGAGCTCACTTACTGC 3') and antisense (5'GCAGTCTTCCTTGGAGTTCAGAAT 3') oligos; mouse actin sense (5'GTTAACCAACTGGGACGACATGG 3') and anti-sense (5'GATCTTGATCTTCATGGTGC 3') mouse actin primers was performed as previously described [ 26 ]. A total of 30, 35 and 40 cycles were employed. The PCR products were run on a 1% agarose gel and Southern blotted to nylon membranes. The blots were probed with an end-labelled 32 P actin probe (5' CACACTGTGCCCATCTACGA3') or RAMP-1 probe (5'GCCCAATCCGGAAGTGGACA 3') followed by autoradiography and densitomitry. Abbreviations CGRP, calcitonin gene related peptide; CTR, calcitonin receptor; RAMP, receptor activity modifying protein; GPCR, G protein coupled receptor; CL, calcitonin receptor like Authors' contributions RM performed the 5' RACE analysis and semi-quantitative RT-PCR. All other experiments were conducted by MP.

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1079841

First molecular screening of deafness in the Altai Republic population

Background We studied the molecular basis of NSHL in Republic of Altai (South Siberia, Russia). The Altaians are the indigenous Asian population of the Altai Mountain region considered as a melting-pot and a dispersion center for world-wide human expansions in the past. Methods A total of 76 patients of Altaian, Russian or mixed ethnicity and 130 Altaian controls were analyzed by PCR-DHPLC and sequencing in the GJB2 gene. The GJB6 deletion and the common non-syndromic deafness-causing mitochondrial mutations were also tested when appropriate. Results 8.3% of the Altaian chromosomes were carrying GJB2 mutations versus 46.9% of the Russian chromosomes. The 235delC mutation was predominant among Altaians, whereas the 35delG mutation was most prevalent among Russian patients. Conclusion We found an Asian-specific GJB2 diversity among Altaians, and different GJB2 contribution for deafness in the Altaian and Russian patients. The high carrier frequency of 235delC in Altaians (4.6%) is probably defined by gene drift/founder effect in a particular group. The question whether the Altai region could be one of founder sources for the 235delC mutation widespread in Asia is open.

Background Non-syndromic hearing loss (NSHL) accounts for approximately 80% of cases of hereditary deafness and variety of genes are involved in NSHL [ 1 ]. Mutations in the GJB2 gene, encoding the connexin 26 gap-junction protein, account for a significant proportion of NSHL [ 2 ]. To date, the substantial contribution of several nuclear genes [ 3 , 4 ] and the pathogenic mitochondrial mutations in NSHL was established in some populations [ 5 , 6 ]. Different population distributions of more than 70 GJB2 mutations have been described and prevalence of some GJB2 mutations is known to depend on population ethnic origin [ 2 , 7 ]. The high frequencies of the 35delG mutation in the Caucasians, the 167delT in Ashkenazi Jews, and the 235delC mutation among east Asian populations have been shown to be the results of founder effects [ 8 - 12 ]. The underlying mechanisms accounting for the high prevalence of certain founder GJB2 alleles are unknown. The data concerning the molecular basis of deafness in the populations of Russia are scarce and mostly limited to the European part of Russia and to the screening of the sole mutation 35delG [ 13 - 16 ]. Such data are yet unknown for Siberia (that is an expansive Asian part of Russia) populated by different ethnic groups. The investigation presented here was performed on individuals with HI and subjects with normal hearing who are living in the Republic of Altai, situated in South Siberia and bordering Mongolia, China and Kazakhstan. Because of location, the Altai have been a particular region of great evolutionary importance. The archaeological, anthropological, and recent genetic data, mainly based on Y-chromosome and mitochondrial DNA studies [ 17 , 18 ] suggest that for thousand of years the Altai-Sayan Mountains with surrounding territories have been a zone of the first contacts between paleo-Caucasoids and Mongoloids; repeatedly this region was a peculiar "hybrid" area closely connected with Central Asia, and a source of subsequent human migrations. Indigenous inhabitants of the Altai Republic are the Altaians originated from several ancient Turkic-speaking tribes [ 19 ]. The Altaians mainly belong to the Central Asian type of the North Asian race. The Altai Republic population accounts for about 200,000 including Altaians, Russians, Kazakhs and other nationalities. The total number of Altaians is about 60,000. The rural population constitutes 3/4 of the Altai Republic people whereas approximately 50,000 persons live in the city of Gorno-Altaisk. The Altaians currently living in small settlements in the distant regions of the Altai Republic have mainly retained their native language, ethnic identity, and traditional marriage structure (patrilineal clan exogamy) [ 20 , 21 ]. On the contrary, the population of the town Gorno-Altaisk is characterized by higher rates of interethnic marriages among Altaians, Russians and other ethnicities. As a whole, the Altaian population was shown to be genetic heterogeneous resulted from its territorial subdivision and the specific Altaian clan compositions in different localities [ 20 - 22 ]. Recently, the first epidemiological data in Altai Republic revealed the spectrum of hereditary pathology in the Altai Republic population with different prevalence rates among the urban and the rural populations, and among main ethnic groups of population (Russians, Altaians, Kazakhs) [ 23 ]. We report here the results of a study based on patients presenting mainly prelingual deafness, with the mutation analysis of the GJB2 gene, the screening of the GJB6 -D13S1830 deletion, and the study of five mitochondrial mutations involved in NSHI. Moreover, the GJB2 screening was performed in 130 unrelated control subjects of Altaian ethnicity. Methods Patients This study was approved by the Altai Republic Ministry of Health. Data on individuals with hearing impairment were obtained from the Republican medical institutions, Association of Deaf People, Specialized School for Deaf Children of the Republic of Altai (the town Gorno-Altaisk), and local settlements on the territory of the Republic. We ascertained 76 patients with HI (37 males and 39 females) of different ethnic affiliation (Altaians, n = 40; Russians, n = 17; Kazakhs, n = 3; mixed or other ethnicity, n = 16) representing a total of 51 unrelated families. Their ages ranged from 3 to 80 years old (mean 30.2). Based on family histories, 19 patients were defined as sporadic cases, while 57 patients were issued from 32 unrelated families, presumably showing an autosomal recessive (20 families), autosomal dominant or maternal (8 families), and ambiguous (4 families) mode of inheritance. In addition to the patients, 58 DNA samples of family members with normal hearing were included. Informed consent was obtained from all adult participants and from parents of the children studied. Bilateral (mainly symmetrical) and prelingual/early onset HL was defined in most of the patients. Among the 76 patients 44 could be otoscopically examined, and pure tone audiometry was performed in the special medical service (of the town Gorno-Altaisk). As the other patients were mostly living in the small settlements distant from the town Gorno-Altaisk, the data was collected from the local unspecialized medical services and by direct interview with the patients and their relatives. Among the clinically documented patients (n = 44), HL was severe to profound in 32 patients, moderate in 10, and mild in 2 patients. Sensorineural type of HI was precisely defined for 38 out of 76 patients, and the remaining patients presented mixed (conductive-sensorineural, n = 6) or uncertain type of HL. It should be noted that the chronic otitis media, widespread in Altaian population, was frequently registered in the medical histories of these patients. We have analyzed the cohort of patients with HI without the possibility of excluding cases of environmental exposures (infections, prenatal or postnatal ototoxicity etc) or unknown etiology. Controls 130 unrelated subjects of Altaian origin with no familial history of hearing problems were used as controls. Most of DNA samples were obtained from the Southern Altaian population of the Ust'-Kan administrative region of the Altai Republic and the others from Northern Altaians living in the Turochak administrative region. All DNA samples studied were anonymized. Mutation analysis DNA was extracted from peripheral blood using standard protocols. GJB2 analysis was performed for the non-coding exon E1 and coding exon E2 and their flanking sequences by PCR-DHPLC as previously described [ 24 ]. Any abnormal profile observed by DHPLC was consequently analyzed by sequencing (ABI 310 sequencer, Applied Biosystems). The patients without any mutations in the coding exon E2 were analyzed in the non-coding exon E1 and tested for the mutation Δ( GJB6 -D13S1830) that includes the deletion of most of the GJB6 gene [ 24 ]. The screening of five known mitochondrial mutations (m.A1555G, m.7445A>G, m.7472insC, m.7510T>C, m.7511T>C) was performed in the GJB2 negative patients that were compatible with a maternal inheritance of HI. The A1555G mutation was analyzed as previously described [ 5 ] whereas the four others were analyzed by direct sequencing (position 7321–7620 of the mtDNA). Statistical analysis Differences between groups were analyzed by chi square statistics. P -values were considered to be significant when <0.05. Results GJB2 gene GJB2 mutations were detected in 23.7% of the patients (18 out of 76) (Table 1 ). We identified five different mutations, all of them lying in the coding exon: three known as recessive mutations (35delG, 235delC and 312del14), one as a dominant mutation (R75Q), the fifth mutation is a newly identified missense mutation, W172C. All the patients with GJB2 mutations were familial cases presenting prelingual profound HL except one patient, genotyped 35delG/235delC, who had moderate-severe HL. Mutations were present as homozygous or compound heterozygous state, with the exception of the R75Q that was identified as single in two members of family 11. Among all the families studied, family 11 deserves particular interest because dominant and pseudo-dominant deafness is observed with a complex segregation of different mutations, as shown on Fig. 1 (see in Discussion). Table 2 shows the distribution of GJB2 sequence variations in the two predominant ethnic groups (Altaians and Russians) of patients as well as in Altaian controls. Of the 60 unrelated Altaian patient alleles, only 8.3% were carrying GJB2 mutations with the 235delC representing 6.67% of the alleles and the W172C, 1.67%. In contrast, 46.9% (15 out of 32) of the unrelated Russian patient chromosomes carried GJB2 mutations with the 35delG accounting for the most frequent mutation (34.4%). The other mutations 312del14, R75Q and 235delC were detected in 6.3%, 3.1% and 3.1% of the Russian alleles, respectively. The polymorphism V27I and E114G were observed in Altaian patients as well as in Altaian controls (Tables 1 and 2 ). None of the Russian patients had both variants. Because the E114G variant was never identified as single in our study but also in others [ 25 , 26 ] we assumed that the E114G and V27I variants are in cis -configuration. The estimated allele frequencies of V27I and [V27I; E114G] are respectively 16.7% and 11.7% among 60 unrelated Altaian patient chromosomes, versus 13.1% and 6.5% in the Altaian controls (260 chromosomes). Furthermore, three other known sequence variations were identified in Altaian controls: the 235delC mutation (2.3%), the F191L variation with unknown pathogenic consequences (0.4%), and the non-pathogenic V153I variant (0.4%) as shown on Table 2 . A new sequence variation, IVS1+27G>C, was identified in one Altaian patient with severe-profound HL who had genotype IVS1+27G>C / [V27I; E114G] (Table 1 ) and in his non-affected mother (genotyped IVS1+27G>C/+). The IVS1+27G>C variation was not previously described, but splice site prediction programs would suggest its non-pathogenic consequences [ 27 ]. Finally, the 765C>T polymorphism (referred to as SNP1 [ 9 ]) located in the 3'UTR of the GJB2 gene was analyzed in patients. All alleles carrying the 35delG mutation were associated with the 765T variant; that is in accordance with recent data on the French patients [ 24 ]. Δ( GJB6 -D13S1830) and mitochondrial mutations Screening of the Δ( GJB6 -D13S1830) and mitochondrial mutations m.1555A>G, m.7445A>G, m.7472insC, m.7510T>C, m.7511T>C was negative in patients who had no GJB2 mutations. Discussion Specific GJB2 mutation spectrums and different GJB2 contributions for deafness were observed for the two main ethnic groups studied here. Prevalence of GJB2 mutations in Russian patients GJB2 mutations are responsible for deafness in 54% (7 out of 13) of the independent Russian families tested and the 35delG mutation was carried by 34.4% of the Russian alleles. These findings correlate with earlier reported data concerning Russian subjects with HI [ 13 - 16 ] and confirm the main contribution of the 35delG mutation in deafness among Caucasian populations [ 28 , 29 ]. GJB2 mutations with dominant and pseudo-dominant transmission We studied the segregation of different GJB2 mutations in a three-generation family (family 11; Fig. 1 ). All affected members presented prelingual profound HL and were using sign language to communicate. The dominant mutation R75Q (224G>A) was found in five members, as the sole mutation or in trans of another mutation (235delC or 312del14). R75Q was previously identified as a non-syndromic dominant mutation [ 7 ] and as a syndromic mutation [ 30 ] in a four-generation Turkish family with autosomal dominant inherited HI and congenital diffuse palmoplantar keratoderma. The age of onset and progression of HL were variable among the affected members of this family [ 30 ]. It should be noted that a previously described dominant mutation located at the same codon 75 (R75W) was found to have a variable penetrance regarding skin disease symptoms (even to their absence) [ 31 , 32 ]. Preliminary dermatological examination performed in all individuals of family 11 carrying the R75Q mutation did not reveal any skin disease in patients I-2, II-2 and II-4 though mild manifestation of probable keratosis was detected in patients III-2 and III-3. However, additional investigations need to be performed for elucidation of non-syndromic and/or syndromic status of the R75Q mutation as other factors can modulate the expression of a particular phenotype. The new missense mutation W172C (516G>C) was identified in trans of the 235delC allele in the Altaian patient I-1. This patient had both parents and a brother with normal hearing but an affected sister (not shown). A G>A substitution at position 516, generating a stop codon at position 172 (W172X) had been previously described [ 7 ]; moreover, the tryptophan at position 172 is conserved in many species (human, mouse, rat, cow, sheep). These data tend to imply that this GJB2 sequence variation is a severe recessive mutation although de novo appearance of the W172C mutation cannot be excluded in this case. GJB2 contribution in Altaian patients and controls This study has revealed an Asian-specific diversity and the prevalence of GJB2 sequence variations in the Altaians. High rates of the V27I and [V27I; E114G] alleles were detected in Altaian patients and Altaian controls with no significant differences between both groups (p > 0.05). These results, once more, confirmed that both sequence variations correspond to non-pathogenic polymorphisms and (that their high rates) represent a distinctive feature of particular Asian populations [ 10 - 12 , 25 , 33 - 36 ]. GJB2 gene contribution in deafness among Altaians (8.3%) is mainly defined by the recessive mutation 235delC (6.67%). This mutation is specific to some east Asian populations (Japan, China, Korea, Taiwan) with an allelic frequency up to 20% among deaf patients and a carrier frequency of 0% to 2.8% in normal populations and it represents the most prevalent GBJ2 pathogenic mutation as it accounts for up to 80% of the pathogenic alleles in patients [ 10 - 12 , 33 - 37 ]. The 235delC mutation, found in 4/60 unrelated chromosomes in Altaian patients, was also detected in 6/130 Altaian controls resulting in a carrier frequency of 4.6%, higher than in any other Asian populations. This high frequency could be attributed to the gene drift/founder effects in a certain subpopulation. The Altaian control group studied here consists mainly of the inhabitants of one administrative region of the Altai Republic. Earlier, it was shown that the marriage migrations among this Altaian subpopulation were, in their majority, restricted to the territory of this region [ 21 ]. Comparatively low GJB2 contribution among the Altaian patients (8.3%) could be explained by the fact that a few patients presented acquired hearing loss. Moreover, a specific marriage structure (patrilineal clan exogamy) practiced by the Altaians for a long time to avoid close inbreeding, could influence apparent scarcity of affected GJB2 homozygotes by restriction of random marriages of Altaians belonged to certain clans. Finally, some other deafness-causing genes, not considered in this study, could be involved in deafness among Altaians. The additional study of the GJB2 gene diversity in other Altaian groups will be informative for verification of the 235delC prevalence pattern among the Altaians. Recent studies had established that the 235delC mutation among east Asian populations was derived from a common ancestral founder [ 10 - 12 ]. Yan et al. [ 12 ] roughly estimated the age of the 235delC mutation to be about 11500 years old and speculated that the 235delC mutation might have arisen in the Lake Baikal area and then spread to Mongolia, China, and Japan through subsequent migrations. Taking into account that the territories of South Siberia, including the Altai region and the Lake Baikal area were melting-pots and dispersion centers for world-wide human expansions in the past, we suggest that the Altai region could be one of the founder sources for the 235delC mutation widespread in Asia. To elucidate this hypothesis, the 235delC prevalence will be tested among indigenous people living in other regions of Altai and surrounding territories and SNP analyses will be performed to verify the common origin of the 235delC alleles. Conclusion In this study we found an Asian-specific GJB2 diversity among the Altaians and different GJB2 contribution for deafness in the Altaian and Russian patients. Additional investigations remain necessary to elucidate all genetic origins of HL in Altaian patients. The high carrier frequency of the 235delC mutation in normal hearing Altaians (4.6%) is probably defined by gene drift/founder effect in a particular Altaian group. The question whether the Altai region could be one of the founder sources for the 235delC mutation widespread in Asia is open. Finally, we presented new evidence that the CX26 R75Q mutation, earlier described as syndromic mutation (autosomal dominant inherited hearing impairment with congenital diffuse palmoplantar keratoderma), could be associated with a high variable expression of the skin symptoms. Competing interests The author(s) declare that they have no competing interests. Authors' contributions VT and OP collected the family data and recruited patients and controls for the study; LP provided a part of control blood samples; OP and NPR carried out the molecular studies; AFR supervised the whole study in the laboratory of MC. AFR and OP wrote the manuscript. All authors read and approved the final version of the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079842

The GABBR1 locus and the G1465A variant is not associated with temporal lobe epilepsy preceded by febrile seizures

Background Polymorphism G1465A in the GABBR1 gene has been suggested as a risk factor for non-lesional temporal lobe epilepsy (TLE); however, this genetic association study has not been independently replicated. We attempted to replicate this study in our cohort of patients with TLE. Furthermore, we also analyzed the coding sequence of this gene and searched for disease-causing mutations. Methods We included 120 unrelated individuals with TLE that was preceded by febrile seizures (FS) who did not have any evidence of structural lesions suggesting secondary epilepsy. 66 individuals had positive family history of TLE epilepsy and 54 were sporadic. Each patient was genotyped for the presence of G1465A polymorphism. All exons of the GABBR1 gene were screened by single strand confirmation polymorphism method. Genotypes were compared with two independent matched control groups. Results We detected two A alleles of the G1465A polymorphism in one homozygous control subject (0.87% of all alleles) and one A allele in a patient with TLE (0.45%, not significant). Other detected polymorphisms in coding regions had similar frequencies in epilepsy patients and control groups. No disease causing mutations in the GABBR1 gene were detected in patients with sporadic or familial TLE. Conclusion Our results indicate that TLE preceded by FS is not associated with the polymorphisms or mutations in the GABBR1 gene, including the G1465A polymorphism. The proportion of TLE patients with FS in the original study, reporting this positive association, did not differ between allele A negative and positive cases. Thus, our failure to reproduce this result is likely applicable to all non-lesional TLE epilepsies.

Background Temporal lobe epilepsy (TLE) preceded by febrile seizures (FS) was historically considered to be an acquired disorder. However, recent studies have confirmed the existence of distinct genetic syndromes encompassing both FS and TLE [ 1 , 2 ]. Additionally, a positive history of FS in families with temporal lobe epilepsy is more common than expected by chance; this suggests that FS may be an initial manifestation of some temporal lobe epileptic syndromes [ 3 - 6 ]. The genetic etiology of TLE preceded by FS remains unknown. Two loci for this condition have been mapped, however, the causative genes have not been identified [ 1 , 2 ]. γ-aminobutyric acid (GABA) is the main inhibitory neurotransmitter system in the brain. Mutations in the GABA-A receptor subunit genes (GABR A ), encoding products that form a heteropentameric chloride channel, have been identified as rare causes of idiopathic generalized epilepsies showing a Mendelian inheritance pattern [ 7 ]. However, mutations in the GABR A genes have thus far not appeared to be a major cause of TLE or other focal epilepsies [ 8 ]. The second, or B-subtype of GABA receptors (GABR B ) function by acting through G-protein coupled signaling systems. Alteration in this GABA system during epileptogenesis is suggested from experimental data [ 9 , 10 ]. Furthermore, a previously described variant (G1465A) in the GABBR1 locus, encoding the GABR B β1 subunit has been reported in association with TLE, including TLE preceded by FS [ 11 ]. Genetic association studies are fraught with many difficulties and independent replication is important to establish the validity of any reported association [ 12 ]. We report genetic analysis of GABBR1 gene by thorough screening of GABBR1 exons for disease-causing mutations, and attempted replication of association at the G1465A polymorphism in a cohort of our patients exhibiting TLE preceded by FS. Methods Subjects This study included 120 unrelated patients carrying a diagnosis of TLE preceded by FS. Informed consent, approved by the Institutional Review Board was obtained from every subject enrolled. A TLE diagnosis was made based on a history obtained from the patient and witnesses, with particular attention to descriptions of aura and complex partial seizure semiology, suggesting temporal lobe origin. Diagnoses were supported by electroencephalograph (EEG) recordings and MRI neuroimaging; some cases involved EEG-video recordings. MRI examination included 3D gradient echo imaging (Spoiled Gradient Recalled Acquisition) performed in the coronal plane with the slice thickness 1 mm, FLAIR and T2-weighted coronal sections; all coronal sections were perpendicular to the long axis of the hippocampus. Additional MRI sequences included T2-weighted axial and T1-weighted sagittal brain images. Only subjects with a reliable clinical classification were included in these studies. Data collection involved age and duration at first FS, presence of any focal ictal or postictal features, total number of FS, and age at FS remission. Information was obtained from parents and/or older siblings. FS were classified as complex if they lasted longer than 15 minutes, had focal ictal or postictal features, or occurred more than once during the same day. Detailed family histories were obtained from all patients, including relatives with a history of epilepsy, FS, or both. Information was obtained from all first-degree relatives (mother, father, brothers, sisters, and children); information regarding second-degree relatives involved questions related to grandparents, grandchildren, nieces, nephews, aunts and uncles. Genetic analysis DNA was isolated from peripheral blood from all subjects and relevant affected and unaffected family members enrolled in this study using standard methods. The current study focused solely on affected probands. The GABBR1 G1465A polymorphism was analyzed as previously described [ 13 , 14 ]. The PCR product was digested overnight with EagI restriction enzyme following the manufacturer's protocol (New England Biolabs; Beverly, MA). Products were analyzed by electrophoresis on 2% agarose gels and subsequent visualization of resolved fragments. PCR primers flanking all exons were designed from published sequence and public genomic assemblies covering the locus (primer sequences are available upon request) [ 15 ]. Exon screening was performed using single strand confirmation polymorphism (SSCP) analysis; abnormal mobility shifts were sequenced. All polymorphisms identified, including G1465A, were analyzed in two distinct ethnically- (Caucasians) and gender-matched control samples. The first comparison group consisted of DNA samples from 118 unrelated, healthy subjects obtained from the Vanderbilt Center for Human Genetics Research DNA Resource Core. The individuals from this group were absent any family history of epilepsy. The second control group corresponded to the Caucasian panel of 100 DNA samples from healthy individuals purchased from the Coriell Cell Repositories [ 16 ]. Statistical analysis Allele frequencies comparisons between patient and control groups was determined using a chi-square test. Mann-Whitney U test was used to compare demographic and clinical characteristics between patients with epilepsy and control subjects. Results The 120 case subjects enrolled in this study included 49 men and 71 women (average age 34.8 ± 14.9 years) with a positive history of TLE, in which FS preceded the onset of epilepsy. Average age of TLE onset was 22.81 ± 10.08 years, with an average syndromic duration of 12.65 ± 8.12 years. Positive family history of epilepsy was reported in 66 individuals, while 54 were apparently sporadic cases. These two subgroups did not significantly differ in their demographic composition; however, familial cases had a significantly earlier age of epilepsy onset compared to sporadic cases (13.64 ± 8.23 and 27.44 ± 11.78, p < 0.01). All familial cases had at least one TLE affected relative in either previous or successive generations, consistent with vertical transmission of this trait. Medically refractory epilepsy was present in 23 patients (19%). Genotype analysis of the G1465A variant detected a single A allele in one subject with TLE, who exhibited poor seizure control in spite of optimal clinical management. This patient had a positive family history of TLE and FS on one parent's side of the family. Genotype analysis of both parents indicated the A allele was present in the unaffected parent from the other side of the family, and thus did not segregate with disease. Two alleles of the same variant were detected in the control group in one control subject who was homozygous for this polymorphism. Allele frequencies for case and control groups corresponded to 0.45% or 0.87%, respectively; p = 0.988). Genotype accuracy for G1465A was confirmed by combined sequence and SSCP analyses that showed distinct and unambiguous mobility shift patterns for both heterozygous and homozygous states. SSCP analysis of all coding exons did not reveal any apparent disease-causing mutations in GABBR1 . We did, however, identify one previously described nonsynonymous variant (C59T) in exon 1, resulting in an alanine to valine substitution in one patient. This known single nucleotide polymorphism (SNP) is present in 1–3% of normal subjects, and it has been reported in a homozygous state in one affected individual [ 13 ]. Given the known normative frequency of the C59T SNP (rs#1805056), control samples were not screened. Several SNPs were detected in exon 11, which also contains the G1465A, variant, previously reported to be in exon 7 [ 11 ]. We detected three previously reported synonymous (silent) sequence changes – T1473C (rs#N/A), T1476C (rs#N/A), and T1485C (rs#29225) [ 14 ]. They appeared to be present on the same haplotype, based on segregation analysis of carrier relatives with this allele and constant SSCP mobility shift patterns. The frequency of this three-SNPs haplotype was determined to be 10.83% (26/240, including one homozygous individual); there was no difference between sporadic and familial cases. Analysis of controls (236 chromosomes) initially detected an allele infrequency of 4.23%, suggesting a significant trend (p < 0.05). Subsequent analysis of the second control sample identified a frequency very similar to the TLE patient sample (18/200, 9%, p = 0.63). Finally, we identified several novel intronic SNPs not predicted to alter splicing, and their frequency did not differ significantly between the TLE and control samples (data not shown). Discussion The 1465A allele at GABBR1 was proposed as a potential risk factor for TLE, particularly in patients with medically refractory seizures [ 11 ]. We were unable to replicate this result, and no association of this allele with TLE was detected in our cohort of patients. Furthermore, systematic screening of all GABBR1 exons did not identify any obvious disease-related variants or novel SNPs or haplotypes significantly associated with the development of TLE. The conflicting results between our study and that reported by Gambardella et al, [ 11 ] are unlikely due to clinical differences in the subject population or insufficient power. The sample sizes were similar (120 in our study and 141 in the original report [ 11 ]) in both studies. Our cohort had a history of antecedent FS, due to our ongoing effort to characterize this TLE subtype. The samples analyzed by Gambardella et al.[ 11 ] were mixed in this regard, and included patients with and without FS. Only 14.8% of their patients had a positive history of febrile convulsions, but the presence or absence of an FS history did not correlate with carrier status of the 1465A allele (16.7% with vs. 14.5% without 1465A) [ 11 ]. Our patient sample was more heavily loaded towards those with positive family history (55%) when compared with the study by Gambardella and colleagues [ 11 ] whose patient samples comprised of 36.2% of patients with a family history of epilepsy. The control 1465A allele frequency in the current study was somewhat higher than in the previous study (0.87% vs. 0.26%, respectively), however, the difference between these two populations is not significant. This is consistent with other studies that found <1% frequency for the 1457A variant with an exception of one report, which identified 10% frequency of this allele in a cohort of 50 patients [ 11 , 13 , 14 ]. Our results support the conclusion that G1465A is a rare polymorphism. We also verified the presence of G1465A polymorphism by SSCP and sequencing and we had a complete agreement between different methods, arguing against incorrect genotyping as an explanation of our negative finding. A systemic screening of all exons at the GABBR1 locus identified alleles at three known SNPs in exon 11 that also contains the 1465A variant, thus defining a unique haplotype [ 14 ]. These sequence changes are silent without any amino acid change in the protein. The comparison of the haplotypic frequency with our first control group suggested a potential increase, although a plausible functionality is currently unclear. The nonreplication in the second control group leaves the initial trend in doubt, and compels a conservative interpretation. Spurious association that cannot be subsequently replicated can be caused by several factors [ 17 ]. Other than an inadequate sample size, selection bias is probably the most likely explanation of discrepancies between association studies. Unrecognized population stratification has been detected even in studies with otherwise proper design [ 18 ]. The problem of population stratification despite a careful selection is also demonstrated by the differences between the frequencies of the 1465A variant between our two control groups. It has been suggested that two control groups should be used in association studies, one population-based and one family based [ 19 ]. We did not have a family-based control group available but our conflicting data between two independent population-based control groups suggest that this approach is also useful in confirmation of true genetic association. Conclusion Our data suggest that variants in GABBR1 are not significantly involved in the pathogenesis of TLE preceded by FS. The proportion of TLE patients with FS in the original study, which reported this positive association did not differ between allele A negative and positive cases, and thus our failure to reproduce this result is likely applicable to all non-lesional TLE epilepsies. Competing interests The author(s) declare that they have no competing interests. Authors' contributions SM carried out the molecular genetic studies, participated in the results interpretation, and helped to draft the manuscript. BA-K participated in the design of the study and its coordination, and helped to draft the manuscript. JSS participated in the design of the study and helped to draft the manuscript. JLH participated in the design of the study and helped to draft the manuscript. PH conceived of the study, participated in its design and coordination, performed statistical analysis, and drafted the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079843

Polymorphism screening and haplotype analysis of the tryptophan hydroxylase gene (TPH1) and association with bipolar affective disorder in Taiwan

Background Disturbances in serotonin neurotransmission are implicated in the etiology of many psychiatric disorders, including bipolar affective disorder (BPD). The tryptophan hydroxylase gene ( TPH ), which codes for the enzyme catalyzing the rate-limiting step in serotonin biosynthetic pathway, is one of the leading candidate genes for psychiatric and behavioral disorders. In a preliminary study, we found that TPH1 intron7 A218C polymorphism was associated with BPD. This study was designed to investigate sequence variants of the TPH1 gene in Taiwanese and to test whether the TPH1 gene is a susceptibility factor for the BPD. Methods Using a systematic approach, we have searched the exons and promoter region of the TPH1 gene for sequence variants in Taiwanese Han and have identified five variants, A-1067G, G-347T, T3804A, C27224T, and A27237G. These five variants plus another five taken from the literature and a public database were examined for an association in 108 BPD patients and 103 controls; no association was detected for any of the 10 variants. Results Haplotype constructions using these 10 SNPs showed that the 3 most common haplotypes in both patients and controls were identical. One of the fourth common haplotype in the patient group (i.e. GGGAGACCCA) was unique and showed a trend of significance with the disease (P = 0.028). However, the significance was abolished after Bonferroni correction thus suggesting the association is weak. In addition, three haplotype-tagged SNPs (htSNPs) were selected to represent all haplotypes with frequencies larger than 2% in the Taiwanese Han population. The defined TPH1 htSNPs significantly reduce the marker number for haplotype analysis thus provides useful information for future association studies in our population. Conclusion Results of this study did not support the role of TPH1 gene in BPD etiology. As the current studies found the TPH1 gene under investigation belongs to the peripheral serotonin system and may link to a cardiac dysfunction phenotype, a second TPH gene that functions predominantly in the brain (i.e., nTPH or TPH2) should be the target for the future association study.

Background Bipolar affective disorder (BPD) is a chronic, severe mood disorder characterized by recurrent episodes of mania and depression. It often interferes with the patient's ability to cope with his daily routine and has a high mortality rate from suicide. BPD is estimated to have a lifetime prevalence of 0.1% to 1% in Taiwanese Han [ 1 ]. The high prevalence, together with the high frequency of hospitalization, psychosocial impairment, suicide, and substance abuse has made the disease a major public health concern. Although previous studies in epidemiology strongly suggested genetic factors in the etiology of affective disorder [ 2 ], establishing the mode of transmission and searching for predisposing genes using linkage analyses have not been successful. Association studies have been useful in mapping genes for complex diseases and are now being applied to many psychiatric traits [ 3 ]. However, the success of this approach requires the markers of the candidate gene being used are either the causative variant(s) or in linkage disequilibrium with the causative variant(s) [ 4 ]. Results from these studies suggest the involvement of several genes, each of which has a minor effect on the pathogenesis of BPD [ 5 ]. Serotonin (also known as 5-hydroxytryptamine) is a major neurotransmitter in the central nervous system (CNS) and is involved in various physiological events, such as mood control, sleep, thermoregulation, learning, and memory [ 6 ]. Its role in psychiatric disorders is well documented [ 7 ]. Disruption of serotonergic function has been implicated in the pathogenesis of many psychiatric disorders, including BPD [ 8 , 9 ], making genes involved in serotonin transmission and its metabolic pathway good candidates for involvement in BPD pathogenesis. Tryptophan hydroxylase (TPH) catalyzes the biopterin-dependent monooxygenation of tryptophan to 5-hydroxytryptophan, which is subsequently decarboxylated to form the neurotransmitter, serotonin [ 10 ]. Two tryptophan hydroxylase isoforms have been identified [ 11 - 13 ] and the expression of TPH1 and TPH2 were found to be mainly in the pineal gland [ 14 ] as well as in the peripheral tissues (duodenum, kidney or lung; [ 15 ]) and in the brain stem [ 12 , 15 ], respectively. The TPH1 gene has been the subject of intensive study regarding its possible involvement in many psychiatric and behavioral traits [ 16 ]; And an increasing number of investigations have studied the TPH2 gene effect in association with several psychiatric and behavioral traits including antidepressant response [ 17 ] and major depression [ 18 ]. The TPH1 gene, which has been mapped to human chromosome 11p15.3-p14 [ 13 ], spans a region of 29 kilobases and consists of 11 exons [ 19 ]. Numerous mRNA species are transcribed from a single promoter by alternative splicing of three exons (1A, 1B, and 1C) in the 5'- untranslated region (UTR) [ 19 , 20 ] and two exons (11A and 11B) in the 3'UTR [ 19 , 20 ]. The structure of the gene and the sequence of the coding region are very similar to those for the genes for human tyrosine hydroxylase and phenylalanine hydroxylase, which belong to the super-family of aromatic amine acid hydroxylases. Polymorphisms within intron 7 of the TPH1 gene have been implicated to be associated with several psychiatric conditions such as BPD [ 21 ], suicidal behavior and alcoholism [ 22 ], and aggression-related traits [ 23 ]. Furthermore, the TPH1 intron 7 polymorphism was associated with the 5-HIAA level in both impulsive Finnish alcoholics [ 24 ] and healthy men [ 25 ] thus suggesting that TPH1 genotypes may participate in the regulation of serotonin turnover rate in the central nervous system. However, such associations were not found in other studies involving suicidal behavior [ 26 ], BPD and mood disorders [ 27 , 28 ], and attention-deficit hyperactivity disorder [ 29 ]. These studies indicate that the role of TPH1 in predisposing different psychiatric traits varies and could be modified by other factors like ethnicity or gene-to-gene interactions [ 30 ]. Preliminary study in our laboratory has found an allelic association (P = 0.03) between the TPH1 intron 7 A218C polymorphism and BPD in 42 Taiwanese bipolar patients and 70 matched healthy controls [ 31 ]. Results from these studies suggest that TPH1 may play a role in regulating serotonin metabolism. More recently, sertraline, a selective serotonin reuptake inhibitor and an effective antidepressant, has been shown to up-regulate TPH1 expression and serotonin synthesis [ 32 ], suggesting one of the mechanism for its pharmacological action in antidepressive therapy. To provide information of the TPH1 gene for future genetic analysis of disorders involved the disturbances in the serotonergic system in Taiwan, we have systematically screened all 11 exons and the promoter region of the human TPH1 gene for common sequence polymorphisms and have identified 5 single nucleotide polymorphisms (SNPs). In addition, we carried out a case-control study of 108 BPD type I patients and 103 normal controls looking for an association between BPD and these 5 SNPs plus 5 others taken from the literatures [ 33 , 34 ] and a public SNP database [ 35 ], tested both as individual SNPs and as the haplotypes. Methods Subjects Since 1998, probands were recruited from bipolar outpatients in the Chung Shan Medical University Hospital and the Taichung Rehabilitation Hospital in Taiwan. Controls were subjects from local volunteer blood donors who have no family or personal history of major affective disorder and other psychiatric disorders were matched to cases on the basis of ethnic or geographic origin, sex, and age. The gender ratios are identical in both case and control groups (male to female is 53% to 47 %) and the age of populations are 38 ± 13.7 and 26.4 ± 7.9 in case and control groups, respectively. The age of onset is 28.4 ± 12.7 in all patients and is 30.3 ± 14.5 and 26.4 ± 10.2 in male and female patients, respectively. Clinical interviews were conducted by experienced psychiatrists (leading by Dr. Lai) for all subjects after the study procedure had been fully explained and information on general demographic data, such as age, sex, and ethnicity, was obtained. Patients assessment was purely by direct clinical interview by the treating clinician according the procedure described in the DSM-IV diagnoses of lifetime Major depressive disorder and bipolar I. Additional information required to reach diagnosis was also collected from all clinical and hospital records where available but the comorbidity with other psychiatric/neurological disorders or medical problems were not considered as an inclusion/exclusion criteria. This study was approved by the University Ethics Committee and written informed consent was obtained from all participants. In total, 108 BPD and 103 controls (all Taiwanese Han) were recruited for this study. DNA preparation Approximately 10 ml of peripheral blood was collected from the recruits using EDTA anticoagulant venous blood tubes, and DNA prepared using a BIO-RAD InstaGene™ Whole Blood Kit (Bio-Rad Laboratory, Hercules, CA) according to the manufacturer's protocol. Primer design for searching for, and typing of, TPH1 gene polymorphisms All primers used were designed using the Primer3 program [ 36 ] or OLIGO 5 Primer Analysis Software (Molecular Biology Insights, Inc., cascade. CO., USA). The primers used to screen the TPH1 gene (Table 1 ) were designed using the published sequence (GenBank accession number: AC005728). The primers used in exon-wide SNP scanning were designed from the intronic sequence roughly 50 bp upstream and downstream of each exon to amplify the entire exon sequences. In addition, overlapping fragments covering from 1,151 bp upstream of the 5' promoter region to 1,572 bp downstream of the 3'UTR were also amplified to screen for polymorphisms within these regions. The forward and reverse primers for base excision sequence scanning (BESS) product amplification were labeled, respectively, with fluorescent 6-FAM and HEX to facilitate variant detection. SNP identification in the coding and regulatory regions of the TPH1 gene In order to identify all SNPs in the coding and regulatory regions of the TPH1 gene, we designed primers to generate PCR products for use in SNP identification using the BESS Base Reader Kit (Epicentre Technologies, Madison, WI) according to the manufacturer's protocol. Each identified SNP was sequenced to confirm the sequence variant. The screening panel included 50 unrelated subjects with or without BPD. The BESS T & G Base Reader Kit, which identifies all types of point mutation, deletion, insertion, repeat expansion, and frameshift mutation at sites involving thymine or guanine, was used to systematically search for sequence variants in pooled samples. DNA sequence variants are detected by cleavage of the amplification products at modified nucleotides, generating a defined series of fragments which can be easily separated on a standard sequencing gel and detected using a fluorescent dye detection system. Briefly, PCR products were generated using FAM-labeled forward primers and HEX-labeled reverse primers. PCR amplification was performed in a 25 μl volume containing 1 unit of Taq polymerase, 1× PCR buffer, 0.2 μM of each labeled primer, an appropriate concentration of MgCl 2 , 200 μM BESS T/G Scan dNTP Mix, and 50 ng of genomic DNA. Thermal cycling conditions were a pre-denaturation of 3 min at 94°C; 35 cycles of 30 sec at 94°C, 30 sec at the appropriate annealing temperature indicated in Table 1 , and 30 sec of extension at 72°C; and a final extension at 72°C for 5 min. For the excision reaction, 5 μl of the amplification reaction was mixed with 1 μl of the BESS T/G-Scan Excision Enzyme Mix and 1 μl of 10X BESS T/G-Scan Excision Enzyme Buffer, the mixture incubated for 30 minutes at 37°C, and the reaction stopped by adding 5 μl of Stop/Loading Buffer. One microliter of the excision reaction products was mixed with gel loading solution containing 12 μl of formamide and 8 μl of GENESCAN™-500 size standards (Applied Biosystems, Forster City, CA, USA), then the mixture was denatured for 5 min at 95°C, loaded onto a capillary polymer of the ABI 310 Genetic Analyzer, and run for 30 min for size separation. Analysis was performed using the GENESCAN 672 program (Applied Biosystems). To reduce the cost and speed up the process, a pooling methodology was used. A preliminary test indicated that the sensitivity of BESS T/G-Scan analysis allowed the detection of alleles with a frequency in the population greater than 4.5 % (data not shown), i.e., The assay can recognize 1 heterozygous individual in a DNA pool from this individual and 9 homozygotes (equivalent to a minor allele with a frequency of 5%). DNA from 10 individuals was therefore pooled (10 ng/μl of DNA from each individual) and five such pools, representing 100 chromosomes, were prepared from 50 randomly selected subjects for polymorphism identification. Selection of SNP markers for the TPH1 gene In addition to the SNPs identified in the present study, five SNP markers within and flanking the human TPH1 gene were also selected to test the association with BPD. Based on the literature [ 34 ] and a public SNP database [ 35 ], SNP markers roughly 10 kb apart and with a relatively high minor allele frequency were selected. The TPH1 intron 7 A218C polymorphism, which has been suggested to be associated with Taiwanese BPD in our previously work, also has been included in the present study (i.e. in7SNP1; A20004C). The genomic localizations of the 10 SNPs examined relative to the transcription start site are given in Table 1 . SNP genotyping Except for the intron 7 A218C polymorphism, which was genotyped using a modified A mplification R efractory M utation S ystem (ARMS), all other SNPs of the TPH1 gene were genotyped using a multiple SNP genotyping system which involves multiplex PCR and multiple single base extensions (MSBE). Briefly, two multiplex PCR reactions were performed. One was in a 20 μl volume containing 70 ng of genomic DNA, 2.4 μl of primer mix (0.1 μM of the primer pairs for 5'flankingSNP3, in1SNP1, in2SNP1, and in6SNP1, and 0.2 μM of the primer-pair for in3SNP1), 400 μM dNTPs, 1× PCR buffer, 1.5 mM MgCl 2 , and 1 U of Taq polymerase. The conditions used were an initial denaturation step of 5 min at 95°C, 30 cycles of amplification (30 s at 95°C, 60 s at 51°C, 90 s at 72°C), and a final extension step of 10 min at 72°C. The other multiplex PCR was performed under the same conditions, but using 0.8 μl of primer mix containing 0.1 μM of the primer pairs for 5'flankingSNP1, 5'flankingSNP2, 3'UTRSNP1, and 3'UTRSNP2. A 4 μl aliquot of the PCR products was treated with 5 U of shrimp alkaline phosphatase (SAP) and 0.1 U of exonuclease I in a total volume of 10 μl to remove primers and unincorporated dNTPs; the reaction was carried out at 37°C for 1 hour, then terminated by incubation at 72°C for 15 minutes to inactivate the enzymes. The multiplex SBE reaction was carried out using SNP-specific primers and fluorescent-labeled terminators (the ABI PRISM SNaPshot Multiplex Kit). The short-extension reaction was performed on a thermal cycling machine for 25 cycles of 10 s at 96°C, 5 s at 50°C, and 30 s at 60°C. After short-extension, excess ddNTPs were removed from the SBE products by addition of 0.5 U of SAP to the reaction mixture and incubation at 37°C for 1 hour. The purified SBE products were electrophoresed on a ABI PRISM 310 Genetic Analyzer and analyzed using GeneScan software (ABI PRISM). Statistical analysis The chi-squared test for allelic and genotypic distributions between patients and controls was performed using the CROSSTAB program implemented by SPSS. The Hardy-Weinberg equilibrium was analyzed using the HWE program, version 2.33 [ 37 ]. Pairwise LD coefficients D' [ 38 ] among the 10 SNPs were estimated and statistical significances were determined by using the SNP Alyze ® program (SNP and Disease Association Analysis software; Dynacom Co., Ltd. Kanagawa, Japan). In addition, the PHASE 2.0 program [ 39 , 40 ] was used to construct haplotypes and perform a case-control permutation test, then the Fisher's exact test was applied to test differences in haplotype frequencies between cases and controls. All Fisher's exact tests (two tails) and estimation of the odds ratio of BPD associated with a particular haplotype were performed using the PROC FREQ program implemented by SAS package (SAS Institute Inc., Cary, NC, USA). Haplotype-tag SNPs were selected using SNPtagger software [ 41 ]. To consider the multiple comparisons, a Bonferroni correction was applied in this study thus the p value for reaching significance is 0.005 for 10 SNPs. In addition, the potential confounders such as personality disorders, substance abuses or organic disorders were not considered in the statistic analyses. Results Detection of TPH1 gene polymorphisms in the Taiwanese Han population To detect SNPs in the human TPH1 gene, we used the enzymatic cleavage approach to screen all 11 exons, parts of neighboring introns, and the promoter of the gene in 30 unrelated healthy and 20 BPD probands from a previously recruited population [ 31 ]. No sequence variation in the coding region of the TPH1 gene was identified. However, screening for nucleotide variants up to -1,151 bases in the promoter region and down to 1,572 bases in the 3'UTR identified five SNPs. Two, A-1067G (5'flankingSNP2) and G-347T (5'flankingSNP3), were found in the promoter region. Of the remaining 3 SNPs, T3804A (in1SNP1) was located in intron 1/exon 1c, while C27224T (3'UTRSNP1) and A27237G (3'UTRSNP2) were located in the 3'UTR of exon 11. SNP genotyping and single locus association analysis For further association analysis, all 5 SNPs identified in this study, plus one polymorphism in the promoter region (5'flankingSNP1;[ 33 ]) and four selected from the dbSNP database (in2SNP1, in3SNP1, in6SNP1, and in7SNP1), were genotyped with MSBE or ARMS (Table 1 ). All genotypes were in Hardy-Weinberg equilibrium (HWE), except for the G-347 T marker in the promoter region that had departure from the HWE in both controls (p = 0.024) and patients (p = 0.024). Genotype coverage was generally good and about 90% of the subjects were successfully typed. All 10 SNPs were genotyped and have been analyzed by Fisher's exact test between the case and control groups. No association can be obtained between any of the TPH1 gene polymorphisms and BPD. The genotypic and allelic distributions of each marker are listed in Table 2 . Pairwise linkage disequilibrium (LD) measurement and haplotype analyses The size of the human TPH1 gene is about 29 kb and the LD between all pairs of 10 SNP markers in all subjects was estimated using the SNP Alyze ® program (Table 3 ). The data above and below the diagonal line in Table 3 show, respectively, the results of the pairwise LD analysis, as indicated by the D' [ 38 ]; a higher value indicating a higher degree of LD), and the p value for the chi-squared test. As shown in Table 3 , the D' values ranges from 0.032 to 1 and the majority of the D' value is greater than 0.5. These data suggested that sequence variants within the TPH1 gene are in strong disequilibrium (the upper diagonal of the Table 3 ). Furthermore, as indicated by the lower diagonal of Table 3 , statistically significant LD was detected for almost all marker pairs among these SNPs. Few exceptions were observed for marker pairs T3840A / A-1067G, A27237 / G-347T, and A27237 / GA12517C in which the D' values are 0.076, 0.032, and 0.039, respectively. Haplotypes constructed using the PHASE 2.0 program [ 39 ] revealed a total of 70 haplotypes in combined controls and patients (data not shown). Among these haplotypes, the majority is in very low frequency but the four major haplotypes with frequency greater than 2% are found in 59.8% of the total population. Estimation of haplotype distribution and the Fisher's exact test values for testing the significance of differences in individual haplotype frequencies between case and control groups for the haplotypes with frequency > 2% in at least one group were listed in Table 4 . Additional test was performed to assess overall haplotype frequency profile differences, rather than frequency differences for a specific haplotype, and p value estimated from 1000 permutation tests was also obtained (Table 4 ). Although a trend of significance was detected for the GGGAGACCCA haplotype between the case and control groups (p = 0.028) and few haplotypes showed slightly increased risks in which the value of odds ratios are larger than 2, the significances were abolished after Bonferroni correction for multiple tests thus suggested the effect is minor (Table 4 ). Furthermore, the overall comparison for all haplotypes between two groups was not significant (p = 0.082) and the p value from 1000 time permutation test was also not significant (p = 0.602). Haplotype tag selection Selecting haplotype-tag SNPs (ht-SNPs) can systematically and efficiently refine the process of haplotype construction and reduce the marker number for genotyping. In this study, three htSNPs (5'flankingSNP1, in2SNP1, in6SNP1) were selected to represent all haplotypes with frequencies larger than 2% using SNPtagger software [ 41 ] (Table 5 ). Results from the htSNP analysis suggest that the information obtained by examining three SNPs (instead of 10) should be sufficient for genetic analysis, since these can be used to construct and represent all common haplotypes with frequency >2%. Five haplotypes were constructed using these 3 htSNPs and statistical analyses detected no differences in haplotype distributions between cases and controls (Table 5 ). Discussion The involvement of the TPH1 gene in the pathogenesis of affective disorder is supported by several lines of evidence. Previous studies have reported a significant association between TPH1 intron 7 A218C polymorphism and BPD in the French population [ 21 ]. In addition, two current meta-analyses confirmed the significant association of suicide-related behavior with TPH1 A218 polymorphism [ 42 , 43 ] and suggested that the A allele has a dose-dependent effect on the risk of suicidal behavior [ 43 ]. Since these results suggested that TPH1 could be a strong candidate for involvement in BPD, we have systematically searched all the exons and promoter region of the gene for SNPs. Five polymorphisms were identified, one of which, 3'UTRSNP2, is a novel polymorphism not previously reported. The only sequence variant found in all TPH1 exons was in the exon 1c/intron1 region (T3804A, dbSNP ID: rs623580), but the polymorphism is within the 5'-UTR and therefore does not result in an amino acid substitution. This finding is consistent with a previous report in which no coding sequence variant of TPH1 was detected in Americans (Indians or Caucasians), Italians, and Finns [ 44 ]. These results demonstrate the highly conserved nature of the human TPH1 gene. The exon 1c/intron 1 T3804A polymorphism has been previously reported and has an estimated minor allele frequency of 0.001 in the Swiss population [ 34 , 45 ]. The five identified SNPs, together with five additional SNPs taken from the literature and a public database, were tested for an association with BPD in Taiwanese patients. In general, the alleles in the TPH1 gene were common, with minor allele frequencies ranging from 21% to 49% and from 22% to 49% in cases and controls, respectively. The genotypic frequencies of all markers, except the G-347T (5'flankingSNP3) marker in the promoter region, showed Hardy-Weinberg equilibrium (HWE) in both populations. The departure from HWE of 5'flankingSNP3 was observed in both controls (p = 0.024) and patients (p = 0.024). A separate study in our lab indicated that the location of the G-347T polymorphism is on the transcriptional repressor GATA1 binding site [ 46 ] and alteration of alleles indeed change promoter activity in a luciferase reporter gene system [ 47 ]. The departure from HWE could be explained by the negative selection of the homozygous TT individuals. And the presence of very low frequency of TT genotype in the population may be the reason for the impossibility of detecting any association. Despite the strong functional role of the TPH1 G-347T polymorphism, no association was detected between any of the polymorphisms and BPD in Taiwanese. The extent and distribution of LD in humans has been a hot topic, especially for gene mapping of complex diseases. In this study, significant LD, as measured by the D' and P values using the SNP Alyze ® program, could be detected between T-1721G and C27224T polymorphisms of the TPH1 gene, which are separated by roughly 29 kb (Table 3 ). Because LD-induced association between multiple loci that harbor disease-predisposing alleles can be identified by haplotype-based analyses [ 48 ], haplotypes were constructed and their frequencies were compared between cases and controls. Haplotype distributions among 10 TPH1 SNPs were estimated and twelve haplotypes with frequency larger than 2 % in at least one group were listed (Table 4 ). The three most common haplotypes were identical in both groups and were found in 58 % and 56.7 % of the patients and controls, respectively. Although significant difference in haplotype distribution was seen in one comparison, the association was weak and was lost after the Bonferroni correction for multiple tests. Additional permutation tests for haplotype distributions in case and control groups were performed and result indicated no differences in haplotype profile between two groups (p value = 0.602). The data suggest that both BPD patients and controls are actually from the same population thus it implies the TPH1 gene may not be related to BPD etiologies. Positive association between TPH1 intron 7 polymorphism and BPD was identified in our previous study [ 31 ], but the replication with extended samples has failed to confirm this association in both single-locus and haplotype analyses. One possible reason for this discrepancy could be at the sampling bias as the age of controls in the extended population is much younger than the cases. Since mood disorders may occur late in life, the sampling bias represents a major limitation of this study. Another possible explanation may be that the TPH1 gene has only a minor effect on BPD etiology, this effect being missed when heterogeneous samples are used. Alternatively, the positive association we obtained initially could be a false positive outcome from a very small sample size being used. Recently, studies of Tph1 (the original Tph) knockout mice were found to express normal amounts of serotonin in brain, but not in the periphery [ 11 ], and resulted in a cardiac dysfunction phenotype [ 49 ]. Follow up studies found that the second tryptophan hydroxylase (TPH2, also known as nTPH) gene is predominantly expressed in the brain stem, while the classical TPH1 is expressed in the pineal gland [ 14 ] and peripheral tissues (duodenum, kidney or lung;[ 15 ]. The amount of TPH2 mRNA expression in individual raphe cells was estimated to be approximately 2.5-fold greater than the level of TPH1 expression in pinealocytes [ 14 ]. These findings have changed the consideration of linking polymorphism of TPH1 gene with various psychiatric diseases [ 12 ] and perhaps, could explain the lack of association between the TPH1 gene and BPD obtained in this study. Whether these two paralog proteins are regulated independently or if they have distinct functions in the brain are still under investigation; studies to establish connections between TPH2 gene and various psychiatric diseases including BPD are on going and should provide more insights regarding the TPH2 function in the brain. Studies on sequence variations in the human genome have revealed that the human genome can be parsed objectively into haplotype blocks, with limited diversity within each block [ 50 , 51 ]. Johnson et al. [ 52 ] determined the extended haplotype at any given locus in a population to identify the SNPs in a gene or a LD region, information that is essential for association studies. The so-called "haplotype tag SNPs (htSNPs)" capture the majority of haplotype diversity within a region and thus represent the minimal number of markers that need to be typed to define the common haplotypes (higher than 5% frequency in the population). In this study, we used a publicly program to define htSNPs that represent all haplotypes of the TPH1 locus with a frequency > 2% [ 41 ]. Three htSNPs, corresponding to all common haplotypes were generated using SNPtagger software [53] (Table 5 ). Although further analysis using haplotypes constructed with htSNPs revealed no differences in frequency distributions between cases and controls (Table 5 ), the htSNPs selected for human TPH1 gene significantly reduce the number of markers required for genotype analysis (in this case from 10 to 3 SNPs), which may be useful in other studies on the Taiwanese Han population. Conclusion Bipolar disorder is a complex genetic disorder with a spectrum of phenotype is associated with bipolar susceptibility genes. Since the subject assessment in this study was only by direct clinical interview according to the DSM-IV but not supported by any other standard tools like the SCID, it is worth noting the potential weakness of the study. The simple assessment procedure without dimensional measures of other psychopathological features also limits the possibility to detect association with particular attribute related to the disease. In summary, results from both single-locus and haplotype analyses did not support the role of TPH1 gene in BPD etiology. Furthermore, our data indicated significant LD within this 29 Kb interval of the TPH1 gene in Taiwanese population. Since haplotype frequencies and LD often differ between racial/ethnic groups, the three htSNPs identified in this study should be beneficial in future application for genetic study in Taiwanese population. Competing interests The author(s) declare that they have no competing interests. Authors' contributions TJL was in charge of clinical examination and provided clinical samples. CYW designed polymorphism screening methods and identified all TPH1 polymorphisms in Taiwanese. HWT conducted genotyping experiments and provided statistical analysis. YJL contributed to genotyping data analysis. HSS provided overall study design, analysis, and drafted the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079844

The effects of cholesterol lowering with simvastatin on cause-specific mortality and on cancer incidence in 20,536 high-risk people: a randomised placebo-controlled trial [ISRCTN48489393]

Background There have been concerns that low blood cholesterol concentrations may cause non-vascular mortality and morbidity. Randomisation of large numbers of people to receive a large, and prolonged, reduction in cholesterol concentrations provides an opportunity to address such concerns reliably. Methods 20,536 UK adults (aged 40–80 years) with vascular disease or diabetes were randomly allocated to receive 40 mg simvastatin daily or matching placebo. Prespecified safety analyses were of cause-specific mortality, and of total and site-specific cancer incidence. Comparisons between all simvastatin-allocated versus all placebo-allocated participants (ie, "intention-to-treat") involved an average difference in blood total cholesterol concentration of 1.2 mmol/L (46 mg/dL) during the scheduled 5-year treatment period. Results There was a highly significant 17% (95% CI 9–25) proportional reduction in vascular deaths, along with a non-significant reduction in all non-vascular deaths, which translated into a significant reduction in all-cause mortality (p = 0.0003). The proportional reduction in the vascular mortality rate was about one-sixth in each subcategory of participant studied, including: men and women; under and over 70 years at entry; and total cholesterol below 5.0 mmol/L or LDL cholesterol below 3.0 mmol/L. No significant excess of non-vascular mortality was observed in any subcategory of participant (including the elderly and those with pretreatment total cholesterol below 5.0 mmol/L), and there was no significant excess in any particular cause of non-vascular mortality. Cancer incidence rates were similar in the two groups, both overall and in particular subcategories of participant, as well as at particular primary sites. There was no suggestion that any adverse trends in non-vascular mortality or morbidity were beginning to emerge with more prolonged treatment. Conclusion These findings, which are based on large numbers of deaths and non-fatal cancers, provide considerable reassurance that lowering total cholesterol concentrations by more than 1 mmol/L for an average of 5 years does not produce adverse effects on non-vascular mortality or cancer incidence. Moreover, among the many different types of high-risk individual studied, simvastatin 40 mg daily consistently produced substantial reductions in vascular (and, hence, all-cause) mortality, as well as in the rates of non-fatal heart attacks, strokes and revascularisation procedures.

Background Observational studies have found blood total cholesterol concentrations below about 4.0 mmol/L (155 mg/dL) to be associated with higher rates of mortality and morbidity from certain non-vascular causes (in particular, cancer of lung, liver, pancreas and blood; chronic obstructive pulmonary disease; cirrhosis; suicide; and haemorrhagic stroke) [ 1 , 2 ]. Excluding events within the first few years after the baseline measurement of cholesterol attenuates some, but not all, of these associations. It remains unclear, however, whether these inverse associations are causal (with low cholesterol actually causing certain diseases), or instead due to confounding or reverse causation (with certain habits or conditions independently causing both lower cholesterol and disease) [ 3 , 4 ]. Randomised trials are not subject to such biases, but the trials of cholesterol-lowering interventions before the statins were unable to assess causation reliably, chiefly because they involved too few non-vascular outcomes (even in combination) and assessed only modest cholesterol reductions from high pretreatment concentrations [ 5 , 6 ]. Some slight excesses of non-vascular deaths or cancers in particular trials provoked much comment over the years, contributing to the uncertainty that surrounded cholesterol-lowering [ 7 - 11 ]. More recently, in previous trials of statin treatment, LDL cholesterol was typically reduced by about 1.0 mmol/L (38 mg/dl) and major vascular events reduced by about 25% [ 12 - 19 ]. Although meta-analyses of those trials indicate that statin therapy reduces vascular mortality [ 20 - 23 ], the secondary prevention trials involved few deaths from non-vascular causes [ 12 - 15 ], and the primary prevention trials involved relatively few deaths from any cause [ 16 - 19 ]. Moreover, these trials provided relatively little information about the effects of lowering total cholesterol to the low concentrations (e.g. around 4.0 mmol/L) that had previously raised concerns. By contrast, the Heart Protection Study (HPS) involved large numbers of deaths from both vascular and non-vascular causes among people presenting with below-average cholesterol levels who were randomly allocated to have their cholesterol lowered substantially for several years [ 24 ]. Consequently, it allows outstanding concerns about the potential hazards of lowering cholesterol to be addressed much more reliably than has previously been possible. The present report provides detailed information about the effects of lowering cholesterol on cause-specific mortality, site-specific cancer incidence and other major morbidity in a range of different circumstances. Methods Details of HPS have been reported previously [ 24 - 29 ] (see also ) and are summarised below. Recruitment and follow-up Men and women aged about 40 to 80 years with non-fasting blood total cholesterol concentrations of at least 3.5 mmol/L (135 mg/dL) were eligible provided they had a medical history of: occlusive arterial disease; diabetes mellitus; or treated hypertension (if also male and aged at least 65 years). People were ineligible if their own doctor considered statin therapy to be clearly indicated or contraindicated, or if they had a past history of: stroke, myocardial infarction or angina hospitalisation within the previous 6 months; chronic liver disease or evidence of abnormal liver function; severe renal disease or evidence of substantially impaired renal function; inflammatory muscle disease or evidence of muscle problems; concurrent treatment with cyclosporin, fibrates or high-dose niacin; child-bearing potential; severe heart failure; life-threatening conditions other than vascular disease or diabetes (including any cancer except non-melanoma skin cancer); or any other condition that might limit long-term compliance. Eligible and consenting patients entered a pre-randomisation "run-in" treatment phase, which involved 4 weeks of placebo followed by 4–6 weeks of 40 mg simvastatin daily. Compliant individuals who were still not considered by their own doctors to have a clear indication for, or contraindication to, statin therapy were then randomly allocated to receive 40 mg simvastatin daily or matching placebo tablets for about 5 years (and separately, using a 2 × 2 factorial design, to receive antioxidant vitamins or matching placebo capsules [ 29 ]). Randomised participants were to be seen at 4, 8 and 12 months, and then 6-monthly (with follow-up by telephone for individuals who did not attend or, alternatively, via their general practitioner). Compliance with study treatment was assessed by questioning participants and reviewing the calendar-packed tablets remaining. Blood samples were taken at each follow-up visit for central laboratory assay of alanine transaminase to monitor liver function, and of creatine kinase in any participant reporting unexplained muscle symptoms or concomitant use of a non-study statin. To assess the effects of the treatment allocation on the lipid profile, assays were performed on non-fasting blood collected from a sample of participants due for follow-up at about the same time each year, and from all participants attending follow-up at an average of 4.6 years. Differences in average blood lipid concentrations were based on comparisons between all those allocated simvastatin and all those allocated placebo, irrespective of whether or not they were still compliant. Information was recorded at each follow-up about any suspected myocardial infarction, stroke, vascular procedure, cancer or other serious adverse experience, and about the main reasons for all other hospital admissions. UK national registries provided information about the sites of any registered cancers and the certified causes of death. Further details were sought from general practitioners (and, if considered necessary, hospital records) about all reports that might relate to major vascular events, cancers (e.g. investigations) or deaths. All such information was coded according to prespecified criteria by coordinating centre clinical staff, who were unaware of the participants' study treatment allocation. Analyses were based on confirmed plus unrefuted reports of events. Cancers were classified according to their primary anatomical site rather than their histology (except that skin cancers were subclassified as melanoma or non-melanoma), with definite confirmation for 98% of the included cancers. Mortality follow-up was available for 99.7% of participants, with certified causes for 98.2% of deaths. Statistical analyses The main comparisons involved logrank analyses of the first occurrence of particular events during the scheduled treatment period among all those allocated simvastatin versus all those allocated matching placebo tablets (i.e. "intention-to-treat"). These logrank analyses yielded both the event rate ratio (RR) and the test of statistical significance (two-sided probability value). The prespecified primary comparisons were of the effects of allocation to simvastatin on deaths from all causes and, separately, on deaths from all coronary causes and from all non-coronary causes. But since simvastatin appeared to reduce the risk of death not only from coronary causes but also from other vascular causes (as well as preventing non-fatal vascular events) [ 24 ], the present analyses are of all vascular deaths and of all non-vascular deaths. Prespecified secondary comparisons included the effects of simvastatin allocation on specific vascular and non-vascular causes of death [ 25 ], with due allowance made in their interpretation for multiple hypothesis testing, for the effects observed on relevant non-fatal events, and for evidence from other studies. It was not anticipated that there would be adequate statistical power to assess the effects of study treatment on vascular or total mortality directly in different circumstances. Instead, the prespecified comparisons involved assessment of the effects on major coronary events (defined as non-fatal myocardial infarction or coronary death), and on the even larger numbers of major vascular events (i.e. major coronary event, stroke or arterial revascularisation), in the first two years and in the later years of scheduled treatment (in order to determine whether any protective effect increases with time), and in various prespecified subcategories determined at study entry, including: sex; age (<65; ≥65<70; ≥70 years); and pretreatment plasma concentrations of total cholesterol (<5.0; ≥5.0<6.0; ≥6.0 mmol/L) and of LDL cholesterol (<3.0; ≥3.0<3.5; ≥3.5 mmol/L). Pre-specified tertiary comparisons included the effects on vascular mortality separately during years 1–2 and years 3+ of follow-up, on cause-specific mortality in the prespecified subcategories of pretreatment total cholesterol, and on site-specific cancer incidence and cerebral haemorrhage. Tests for heterogeneity or, if more appropriate, trend were to be used to assess whether the proportional effects observed on particular outcomes in specific subcategories differed clearly from the overall effect (after due allowance for multiple comparisons). Role of the funding sources The investigators were responsible for the study design, data collection, data analysis, data interpretation, and writing of the report, independently of all funding sources. Results Patient enrolment A total of 20,536 individuals were randomised between July 1994 and May 1997. Mean age at study entry was 64.0 years (SD 8.4), with 5806 aged at least 70 years. Prior to any statin treatment being started, participants who were subsequently randomised had mean non-fasting plasma concentrations of total cholesterol of 5.9 mmol/L (SD 1.0), directly-measured LDL cholesterol of 3.4 mmol/L (0.8), HDL cholesterol of 1.06 mmol/L (0.3) and triglycerides of 2.1 mmol/L (1.4). There were 4072 randomised participants whose pretreatment measurements of total cholesterol were below 5.0 mmol/L (193 mg/dL) and 6793 with pretreatment LDL cholesterol below 3.0 mmol/L (116 mg/dL). The large size of the study (and the use of minimised randomisation [ 30 ]) produced good balance between the treatment groups for the main prognostic features that were measured (see subcategory figures below), and should have done likewise for those that were not. Compliance and effects on blood lipids The mean duration of follow-up was 5.0 years for all randomised participants: 5.3 years for those who survived to the scheduled end of study treatment and about half that for those who did not (yielding 51,121 person years of follow-up for all those allocated simvastatin and 50,664 for all those allocated placebo). Compliance at each follow-up was defined as at least 80% of the scheduled simvastatin or placebo tablets having been taken since the previous follow-up. Among the participants allocated 40 mg simvastatin daily, average statin use during the scheduled treatment period was 85% (with 82% compliant with their allocated simvastatin, 3% on non-study statin alone and 2% on both). By contrast, among those allocated placebo, 4% at the end of the first year of follow-up, but 32% at the end of the fifth year, were taking non-study statin therapy, yielding an average of 17%. Non-study statin use in the placebo group was more common among those who already had diagnosed coronary disease at entry, were younger or had higher pretreatment total or LDL cholesterol concentrations [ 24 ]. In each subcategory studied, however, the average difference in total cholesterol was about 1.2 mmol/L (range: 1.1–1.3 mmol/L: table 1 ) and in LDL cholesterol was about 1.0 mmol/L (range: 0.9–1.1 mmol/L). In particular, among the 4072 participants whose pretreatment measurements of total cholesterol were below 5.0 mmol/L (193 mg/dL), the average total cholesterol concentration during the trial was 3.5 mmol/L in the simvastatin group compared to 4.6 mmol/L in the placebo group. Table 1 Average plasma total and LDL cholesterol concentrations during follow-up Baseline characteristic Plasma total cholesterol (mmol/l) Plasma LDL cholesterol (mmol/l) Simvastatin Placebo Difference* Simvastatin Placebo Difference* Sex Male 4.1 5.3 -1.2 2.2 3.2 -1.0 Female 4.6 5.8 -1.2 2.5 3.4 -0.9 Age (years) <65 4.3 5.4 -1.1 2.4 3.2 -0.9 ≥65 <70 4.1 5.4 -1.3 2.2 3.3 -1.0 ≥70 4.2 5.5 -1.3 2.2 3.3 -1.1 Total cholesterol (mmol/L) <5.0 3.5 4.6 -1.1 1.8 2.6 -0.9 ≥5.0 <6.0 4.0 5.2 -1.2 2.1 3.1 -1.0 ≥6.0 4.8 6.0 -1.2 2.7 3.7 -1.0 LDL cholesterol (mmol/L) <3.0 3.7 4.9 -1.1 1.8 2.7 -0.9 ≥3.0 <3.5 4.1 5.3 -1.2 2.2 3.2 -1.0 ≥3.5 4.7 5.9 -1.2 2.7 3.7 -1.0 ALL PATIENTS 4.2 5.4 -1.2 2.3 3.3 -1.0 * Intention-to-treat comparisons with missing data imputed from initial pre-treatment screening values. The absolute difference in LDL-cholesterol that would be produced by full compliance with 40 mg simvastatin daily can be estimated as the ratio of the absolute difference to the estimated compliance. For example, -1.0/67% = -1.5 mmol/L. Effects on vascular mortality and morbidity Overall, allocation to simvastatin produced a highly significant 17% (SE 4; p < 0.0001) proportional reduction in vascular mortality during the 5 years of the study (table 2 ). This reflected a definite 18% (SE 5; p = 0.0005) reduction in deaths due to coronary causes, together with a non-significant 20% (SE 12; p = 0.1) reduction in fatal strokes and 12% (SE 13; p = 0.3) reduction in deaths from other vascular causes, with no significant difference between the effects observed on these different vascular causes (heterogeneity p = 0.9). Among the coronary causes there were also no statistically significant differences among the effects of statin allocation on deaths attributed to acute myocardial infarction, sudden death, heart failure secondary to coronary disease, or other coronary causes (heterogeneity p = 0.5). There was no apparent difference between the treatment groups in the small number of deaths attributed to cerebral haemorrhage, whereas there was a non-significant reduction in fatal strokes due to ischaemic (or unknown) causes. Other vascular causes comprised deaths from peripheral vascular disease (which included ruptured aortic aneurysm) and from a variety of other cardiac conditions (including a few attributed to heart failure), again with no significant heterogeneity in the effects of simvastatin allocation on these different causes of death (heterogeneity p = 0.7). Table 2 Effect of simvastatin allocation on vascular and non-vascular causes of death Cause of death Simvastatin-allocated (10,269) Placebo- allocated (10,267) Death rate ratio (& 95% CI) P-value Coronary Acute MI 141 (1.4%) 191 (1.9%) 0.73 (0.59 – 0.91) Sudden death 147 (1.4%) 154 (1.5%) 0.95 (0.75 – 1.19) Heart failure* 65 (0.6%) 78 (0.8%) 0.82 (0.59 – 1.15) Other coronary 234 (2.3%) 284 (2.8%) 0.82 (0.69 – 0.97) Subtotal: Coronary 587 (5.7%) 707 (6.9%) 0.82 (0.74 – 0.92) 0.0005 Stroke Haemorrhagic 23 (0.2%) 24 (0.2%) 0.95 (0.54 – 1.68) Ischaemic (or unknown) 73 (0.7%) 95 (0.9%) 0.76 (0.56 – 1.03) Subtotal: Stroke 96 (0.9%) 119 (1.2%) 0.80 (0.61 – 1.05) 0.1 Other vascular Peripheral vascular 58 (0.6%) 63 (0.6%) 0.91 (0.64 – 1.30) Other cardiac* 40 (0.4%) 48 (0.5%) 0.83 (0.54 – 1.25) Subtotal: Other vascular 98 (1.0%) 111 (1.1%) 0.88 (0.67 – 1.15) 0.3 VASCULAR 781 (7.6%) 937 (9.1%) 0.83 (0.75 – 0.91) <0.0001 Neoplastic Respiratory 127 (1.2%) 133 (1.3%) 0.94 (0.74 – 1.20) Gastrointestinal 112 (1.1%) 103 (1.0%) 1.08 (0.82 – 1.41) Genitourinary 47 (0.5%) 46 (0.4%) 1.01 (0.67 – 1.52) All others 73 (0.7%) 62 (0.6%) 1.17 (0.83 – 1.63) Subtotal: Neoplastic 359 (3.5%) 345 (3.4%) 1.03 (0.89 – 1.19) 0.7 Other non-vascular Respiratory 90 (0.9%) 114 (1.1%) 0.78 (0.59 – 1.03) Gastrointestinal 35 (0.3%) 41 (0.4%) 0.85 (0.54 – 1.33) Other medical † 47 (0.5%) 49 (0.5%) 0.95 (0.64 – 1.42) Non-medical 16 (0.2%) 21 (0.2%) 0.75 (0.40 – 1.44) NON-VASCULAR 547 (5.3%) 570 (5.6%) 0.95 (0.85 – 1.07) 0.4 ALL DEATHS 1328 (12.9%) 1507 (14.7%) 0.87 (0.81 – 0.94) 0.0003 * Heart failure deaths were subdivided into those considered to be due to coronary disease and those (5 versus 8) due to other vascular (or unknown) causes. † Includes renal, infectious, metabolic, neurological and unknown deaths. The apparent reductions in different types of vascular death with allocation to simvastatin are reinforced by more definite effects on the larger numbers of non-fatal and fatal vascular events considered together. For example, the very definite 27% (SE 4; p < 0.0001: figure 1 ) reduction in first non-fatal myocardial infarction or coronary death (i.e. "major coronary events") following randomisation reinforces the observed effect on coronary death [ 24 ]. Similarly, the definite 25% (SE 5; p < 0.0001) reduction in first non-fatal or fatal stroke following randomisation (444 [4.3%] simvastatin versus 585 [5.7%] placebo) indicates that the trend toward fewer fatal strokes with simvastatin is likely to be real [ 28 ]. Despite concerns about possible adverse effects of statin therapy on heart failure [ 31 ], the non-significant trend toward fewer heart failure deaths due to any cause (70 [0.7%] vs 86 [0.8%]; RR 0.81 [0.59–1.10]; p = 0.2) is supported by a marginally significant reduction in first hospital admission for worsening heart failure or heart failure death (354 [3.4%] vs 405 [3.9%]; RR 0.86 [0.75–1.00]; p = 0.05). Figure 1 Effect of simvastatin allocation on major coronary events by year of follow-up . Rate ratios (RR: black squares with area proportional to the amount of "statistical information" in each subdivision) comparing outcome among patients allocated simvastatin to that among those allocated placebo are plotted, along with their 95% confidence intervals (CI: horizontal lines). For relevant subtotals, the result and its 95% CI is represented by a diamond, with the overall proportional reduction and statistical significance given alongside. Squares or diamonds to the left of the solid vertical line indicate benefit with simvastatin (with nominal significance of at least two-sided P < 0.05 when the horizontal line or diamond does not overlap the vertical line). The broken vertical lines indicate the overall rate ratios. In this high-risk population with occlusive arterial disease or diabetes, about 1.5% of placebo-allocated patients died from vascular causes during each year of follow-up. A highly significant 24% (95% CI 10–35; p = 0.002) proportional reduction in vascular mortality emerged during the first two years after the initiation of simvastatin treatment (figure 2 ), which is reinforced by the prespecified analyses of major coronary events (figure 1 ) and of major vascular events (figure 3 ). Further reductions in vascular mortality were observed during the subsequent years of the scheduled treatment period, and again these are reinforced by the observed effects on vascular events. About one third of the placebo-allocated participants were taking a statin by the end of year 5, and this may account for the somewhat smaller reductions in vascular deaths (and vascular events) observed during the later years. Even so, the continuing reductions in vascular mortality (and morbidity) during each period resulted in increasing absolute benefits with more prolonged treatment and follow-up (figure 4 ). As a consequence, whereas 9.1% of the placebo-allocated patients died of vascular causes during an average of 5 years of follow-up, only 7.6% of those allocated simvastatin did so. Hence, lowering LDL cholesterol by an average of 1.0 mmol/L for 5 years was associated with the prevention of 14 (SE 5) vascular deaths per 1000 participants. Figure 2 Effect of simvastatin allocation on vascular, non-vascular and all-cause mortality by period of follow-up . Symbols and conventions as in Figure 1. Figure 3 Effect of simvastatin allocation on major vascular events by year of follow-up . Symbols and conventions as in Figure 1. Figure 4 Life-table plot of effects of simvastatin allocation on vascular and non-vascular death . See figure 2 for numbers of participants dying during follow-up. Figure 5 indicates that the proportional reduction in the rate of vascular death was about one sixth in various different circumstances, and this pattern is also reinforced by the prespecified subgroup analyses of the much larger numbers of major vascular events (figure 6 ). For example, there was a 17% (SE 5; p = 0.0004) reduction in vascular mortality among the 15,454 men and a 19% (SE 11; p = 0.08) reduction among the 5082 women (heterogeneity p = 0.9 between effect in men vs women), which is reinforced by the highly significant reductions in major vascular events both among men and among women. Similarly, the proportional reductions in vascular mortality appeared to be about the same among younger and older participants (heterogeneity p = 0.9), as was also the case for major vascular events. But since the older participants were at higher absolute risk of vascular death, these similar proportional effects translated into larger absolute benefits at older ages during the 5 year treatment period. The reduction in vascular mortality also appeared largely independent of the pretreatment lipid concentrations, and this pattern is again reinforced by the parallel analyses of major vascular events. Figure 5 Effect of simvastatin allocation on vascular death in participants subdivided by presenting features . Symbols and conventions as in Figure 1. P-values for chi-squared tests for heterogeneity across different subgroups are given on the right. Figure 6 Effect of simvastatin allocation on first major vascular event in participants subdivided by presenting features . Symbols and conventions as in Figures 1 & 5. Effects on non-vascular mortality and morbidity Overall, there was no evidence that reducing total cholesterol by an average of 1.2 mmol/L for 5 years produced any adverse effect on the aggregate of all non-vascular deaths (RR 0.95 [95% CI 0.85–1.07]; p = 0.4: table 2 ). More than half of these non-vascular deaths were due to cancer, and there were no significant differences between the treatment groups in the numbers of deaths from all cancers or from cancers at particular sites (or in the numbers of incident cancers: see below). Among participants allocated simvastatin there were non-significantly fewer deaths from all respiratory causes, including those due to chronic obstructive pulmonary disease (26 [0.3%] simvastatin vs 39 [0.4%] placebo; RR 0.66 [0.41–1.08]; p = 0.1). This apparent lack of any adverse effect on respiratory death is reinforced by the similar numbers of participants in the two treatment groups who either died from, or were admitted to hospital for, any respiratory illness (811 [7.9%] simvastatin vs 820 [8.0%] placebo; RR 0.98 [0.89–1.08]; p = 0.7) or for chronic obstructive pulmonary disease (88 [0.9%] vs 110 [1.1%]; RR 0.79 [0.60–1.05]; p = 0.1). Moreover, lung function assessed by spirometry in all those attending the final visit showed no differences between the treatment groups: forced expiratory volume in 1 second (FEV 1 ) of 2.06 L simvastatin vs 2.05 L placebo (difference 0.01 L [SE 0.01]; p = 0.5); and forced vital capacity (FVC) of 2.82 L vs 2.82 L (difference 0.00 L [SE 0.01]; p = 0.9). This was the case even among participants with pretreatment total cholesterol measurements below 5.0 mmol/L: FEV 1 of 2.16 L vs 2.15 L (difference 0.00 L [SE 0.03]; p = 0.9); and FVC of 2.94 L vs 2.95 L (difference 0.00 [SE 0.03]; p > 0.9). There were similar numbers of deaths in the two treatment groups from gastrointestinal causes, which included a small number attributed to liver disease (5 simvastatin vs 3 placebo). Few patients reported developing cirrhosis (4 vs 4), but there was no apparent adverse effect on the much larger number with any non-fatal or fatal liver-related serious adverse event, either overall (197 [1.9%] vs 200 [1.9%]; RR 0.98 [0.80–1.19]; p = 0.8) or among those with pretreatment total cholesterol measurements below 5.0 mmol/L (48 [2.4%] vs 41 [2.0%]; RR 1.18 [0.78–1.78]; p = 0.4). There was also no suggestion of any adverse effect of cholesterol-lowering therapy on other medical causes of death (table 2 ), which included small numbers due to infections (7 vs 17) and renal disease (10 vs 10). This apparent lack of effect on renal death is reinforced by the similar numbers in the two treatment groups who developed renal failure or died from renal causes (71 [0.7%] vs 63 [0.6%]; RR 1.12 [0.80–1.57]; p = 0.5). Relatively few non-medical deaths occurred, with the majority being due to accidents and injuries (12 vs 15) and the remainder due to complications of medical or surgical procedures (4 vs 5) or suicide (0 vs 1). There was no apparent excess in the numbers who reported attempted suicide (14 [0.1%] vs 11 [0.1%]; RR 1.26 [0.58–2.76]; p = 0.6), development of depression (39 [0.4%] vs 34 [0.3%]; RR 1.14 [0.72–1.80]; p = 0.6) or any psychiatric disorder (96 [0.9%] vs 90 [0.9%]; RR 1.06 [0.79–1.41]; p = 0.7). Any adverse effects of lowering cholesterol might be expected to emerge only after some years of lower cholesterol levels, but there was no suggestion of an excess of non-vascular death even during the later years of the study (figures 2 and 4 ). For example, there were similar rates of non-vascular death in each treatment group during years 3 & 4 (RR 0.97 [0.81–1.17]; p = 0.8) and during years 5+ (RR 0.93 [0.77–1.13]; p = 0.5). Nor did lowering total cholesterol by an average of about 1.2 mmol/L for 5 years produce an excess of non-vascular mortality in any of the different types of patient studied (figure 7 ). For example, inverse associations between cholesterol concentrations and mortality have been reported from some observational studies among the very elderly [ 32 , 33 ]. But no adverse effect on non-vascular mortality was seen among the 5806 participants in HPS who were aged 70 years or older at entry (RR 0.95 [0.80–1.13]; p = 0.6) or, indeed, even among the 1263 participants who were aged 75–80 years at entry (60 [9.8%] vs 78 [12.0%]; RR 0.78 [0.56–1.09]; p = 0.1). Similarly, despite concerns from non-randomised observational studies about higher mortality in association with low cholesterol concentrations, there was no adverse effect on non-vascular mortality even among the 4072 participants with pretreatment total cholesterol measurements below 5.0 mmol/L (RR 0.99 [0.77–1.26]; p = 0.9), in whom simvastatin allocation reduced total cholesterol concentrations to an average of 3.5 mmol/L (Table 1 ). Figure 7 Effect of simvastatin allocation on non-vascular death in participants subdivided by presenting features . Symbols and conventions as in Figures 1 & 5. Effects on all-cause mortality The lack of any overall effect of cholesterol-lowering therapy on non-vascular mortality in different circumstances (figure 7 ) suggests that the proportional reduction in all-cause mortality in any particular circumstance is likely to depend chiefly on the fraction of deaths that are due to vascular causes and on the proportional reduction in vascular mortality that is produced by lowering LDL cholesterol. For example, in the present study, the 13% proportional reduction in all-cause mortality produced by lowering LDL cholesterol by an average of about 1 mmol/L with statin therapy reflects the combination of the highly significant 17% reduction in the two-thirds of deaths due to vascular causes and the lack of any significant difference in the remaining one third of deaths from non-vascular causes (table 2 ). Figure 2 illustrates this combined effect on all-cause mortality over time, and the similar proportional reductions in total mortality in different types of participant (figure 8 ) reflect the consistent beneficial effects of simvastatin allocation on vascular death, and consistent lack of effect on non-vascular deaths, across subgroups. Figure 8 Effect of simvastatin allocation on all-cause mortality in participants subdivided by presenting features . Symbols and conventions as in Figures 1 & 5. Effects on cancer incidence Overall, there was no evidence that reducing total cholesterol from an average of 5.4 mmol/L to an average of 4.2 mmol/L (table 1 ) produced any adverse effect on the incidence of first cancer (excluding non-melanoma skin) at any site following randomisation (814 [7.9%] simvastatin vs 803 [7.8%] placebo; RR 1.00 [0.91–1.11]; p = 0.9: table 3 ). Moreover, no significant excess of these cancers emerged even during the later years of the study (figure 9 ). Nor was there any significant evidence that cholesterol-lowering therapy produced adverse effects on the cancer incidence rate in any particular type of participant studied (figure 10 ). For example, by contrast with a marginally significant adverse trend in one recently reported randomised trial of statin therapy [ 34 ], simvastatin allocation was not associated with a significant excess of cancer among the 5806 participants aged 70 years or older at presentation (RR 1.02 [0.88–1.19]; p = 0.8). Furthermore, whereas non-randomised observational studies have found higher cancer rates in association with low cholesterol levels [ 1 , 2 ], no adverse effect was seen on the cancer incidence rate in this large randomised comparison among the 4072 participants with pretreatment total cholesterol measurements below 5.0 mmol/L (RR 0.87 [0.71–1.07]; p = 0.2). Table 3 Effect of simvastatin allocation on site-specific cancer incidence Cancer site Simvastatin-allocated (10,269) Placebo-allocated (10,267) Event rate ratio (& 95% CI) P-value Respiratory Lung/larynx 171 (1.7%) 157 (1.5%) 1.08 (0.87 – 1.34) Other 8 (0.1%) 10 (0.1%) 0.79 (0.32 – 2.00) Subtotal: Respiratory 179 (1.7%) 167 (1.6%) 1.06 (0.86 – 1.31) 0.6 Gastrointestinal Upper GI 74 (0.7%) 62 (0.6%) 1.18 (0.84 – 1.65) Colon/rectum 114 (1.1%) 131 (1.3%) 0.86 (0.67 – 1.11) Other 41 (0.4%) 33 (0.3%) 1.23 (0.78 – 1.94) Subtotal: Gastrointestinal 228 (2.2%) 223 (2.2%) 1.01 (0.84 – 1.22) 0.9 Connective tissue Female breast 38 (1.5%) 51 (2.0%) 0.74 (0.49 – 1.12) Melanoma 17 (0.2%) 10 (0.1%) 1.66 (0.78 – 3.54) Other 5 (0.0%) 7 (0.1%) 0.71 (0.23 – 2.20) Subtotal: Connective tissue 60 (0.6%) 68 (0.7%) 0.87 (0.62 – 1.24) 0.4 Genitourinary Renal 23 (0.2%) 22 (0.2%) 1.04 (0.58 – 1.86) Bladder 74 (0.7%) 90 (0.9%) 0.81 (0.60 – 1.11) Prostate 145 (1.9%) 145 (1.9%) 0.99 (0.79 – 1.25) Gynaecological 19 (0.7%) 18 (0.7%) 1.05 (0.55 – 2.00) Other 6 (0.1%) 6 (0.1%) 0.99 (0.32 – 3.08) Subtotal: Genitourinary 259 (2.5%) 272 (2.6%) 0.94 (0.80 – 1.12) 0.5 Haematological Leukaemia/lymphoma 42 (0.4%) 32 (0.3%) 1.30 (0.82 – 2.05) Other 23 (0.2%) 23 (0.2%) 0.99 (0.55 – 1.76) Subtotal: Haematological 64 (0.6%) 52 (0.5%) 1.22 (0.85 – 1.75) 0.3 Other & unspecified 54 (0.5%) 57 (0.6%) 0.94 (0.65 – 1.36) 0.7 ALL CANCERS* 814 (7.9%) 803 (7.8%) 1.00 (0.91 – 1.11) 0.9 * Prespecified that analyses of cancer incidence were to exclude non-melanoma skin cancer (243 [2.4%] simvastatin-allocated versus 202 [2.0%] placebo-allocated; p = 0.06). Figure 9 Effect of simvastatin allocation on incidence of first cancer by period of follow-up . Symbols and conventions as in Figure 1. Excludes non-melanoma skin cancer. Figure 10 Effect of simvastatin allocation on cancer incidence in participants subdivided by presenting features . Symbols and conventions as in Figures 1 & 5. Excludes non-melanoma skin cancer. Any adverse effects of cholesterol lowering on cancer might be expected to be restricted to cancers at particular sites: for example, respiratory, gastrointestinal and haematological cancers have been associated with low cholesterol concentrations in observational studies [ 1 , 2 ]. Substantial numbers of respiratory, gastrointestinal and genitourinary cancers were reported during HPS, so it provides a reasonably reliable (and unbiased) assessment of whether lowering cholesterol by more than 1 mmol/L for about 5 years affects the risks of such cancers. There was no suggestion of any excess in the incidence of cancers at these sites, either overall (table 3 ) or separately among participants who presented at younger or older age or with lower or higher cholesterol concentrations (figure 11 ). Even in a study of this size, however, too few cancers occurred at some particular sites (e.g. haematological cancers) for reliable assessment, but an on-going meta-analysis of individual patient data from all major statin trials should be able to provide such information [ 35 ]. Figure 11 Effect of simvastatin allocation on site-specific cancers subdivided by age and total cholesterol at study entry . Symbols and conventions as in Figures 1 & 5. Excludes non-melanoma skin cancer. Individuals with a history of non-melanoma skin cancer remained eligible for inclusion in HPS (although those with a history of other cancers did not), and non-melanoma skin cancer reported following randomisation was to be considered separately from other cancers. During the 5-year scheduled treatment period, there were more reports of non-melanoma skin cancer among the simvastatin-allocated participants (243 [2.4%] vs 202 [2.0%]: RR 1.20 [0.99–1.44]), only one of which was associated with death. This difference is not conventionally significant (p = 0.06), even before allowing for the multiple comparisons involved. Moreover, it did not appear to reflect a clear excess in either basal cell carcinomas (124 vs 99; RR 1.24 [0.96–1.61]; p = 0.1) or in squamous cell carcinomas (137 vs 122; RR 1.12 [0.87–1.42]; p = 0.4). Nor did this slight excess of non-melanoma skin cancer become more apparent with more prolonged treatment, as might be expected if it was causally related. Indeed, the excess was observed largely during years 1 & 2 (107 [1.0%] vs 76 [0.7%]), with little apparent difference during years 3 & 4 (91 [0.9%] vs 86 [0.9%]) or years 5+ (45 [0.5%] vs 40 [0.4%]). Discussion The large size of the Heart Protection Study, its prospective randomised design and the inclusion of a broad range of participants allow it to assess reliably both the efficacy and the safety of cholesterol lowering in a variety of different circumstances. The present results demonstrate that lowering LDL cholesterol by about 1 mmol/L (38 mg/dL) for 5 years with a statin reduces the rate of death from vascular causes by about one-sixth, with no apparent adverse effect on non-vascular mortality or morbidity. This proportional reduction in vascular mortality was remarkably consistent among the different types of participant studied, including women as well as men, older as well as younger individuals, and those who entered the study with below average cholesterol concentrations. Furthermore, the lack of any significant hazard was also consistent among the different types of participant, and there was no suggestion of any adverse effects emerging with more prolonged follow-up. Consistent beneficial effects of cholesterol lowering on vascular mortality and morbidity The prespecified analyses for assessing the benefits of statin allocation in different types of participant were to be of major vascular and coronary events. Because much larger numbers suffered at least one such event, analyses of those outcomes can help interpret the observed effects on the smaller numbers of deaths from vascular causes. For example, the proportional reduction in vascular mortality observed among the 5082 participating women was very similar to that among the men. But since fewer women took part and their absolute risk of vascular death was somewhat lower, this result did not reach conventional levels of statistical significance on its own (p = 0.08). Even so, the very definite and highly significant reductions in major vascular events observed among the participating women (figure 6 ) indicate that the reduction in vascular death, and hence in all-cause mortality, among the women is real [ 36 , 37 ]. Similarly, the highly significant reductions in major vascular events in the elderly and those with below average cholesterol concentrations at baseline reinforce the consistent reductions in vascular mortality of about one-sixth in these groups. Given the two-thirds compliance during HPS, actual use of 40 mg simvastatin daily in this population would lower LDL cholesterol by about 1.5 mmol/L (57 mg/dL), and this would probably reduce the vascular death rate by about one quarter. Furthermore, the continued divergence in the vascular death rate during successive years of follow-up (reinforced by the similar effect on major vascular events) suggests that more prolonged statin therapy would produce even larger absolute reductions in vascular mortality. Despite the proven benefits of statins in people with coronary heart disease, it has been suggested that statins may have adverse effects among people with overt heart failure [ 31 ]. Only small numbers of participants died from heart failure during HPS, but simvastatin allocation was associated with a marginally significant reduction in heart failure hospitalisations or deaths during the treatment period. Patients with occlusive arterial disease or diabetes who had coexistent heart failure were still eligible for the study provided they were not breathless at rest. Although the presence of heart failure at study entry was not routinely recorded, ACE inhibitors were chiefly used for heart failure or hypertension during the recruitment period (1994–7), which predated evidence from the HOPE trial of benefit in other circumstances [ 38 ]. Among participants using ACE inhibitors at baseline, many of whom were likely to have had heart failure, simvastatin allocation significantly reduced the risk of major vascular events (495 [24.9%] simvastatin vs 568 [28.5%] placebo; RR 0.84 [0.75–0.95]; p = 0.006), and there was a non-significant trend towards fewer vascular deaths (265 [13.3%] vs 285 [14.3%]; RR 0.93 [0.78–1.09]; p = 0.4). These results are not consistent with any substantial adverse effect of statin therapy on heart failure, and suggest that the beneficial effect on major vascular events is likely to outweigh any small adverse effect that might exist. Subsequent analyses of HPS based on baseline blood levels of brain natriuretic peptide (which is a sensitive and validated marker of heart failure [ 39 ]) should help further to assess the effects of statin treatment in patients with heart failure (as will on-going trials in such individuals [ 40 ]). The significant 13% (SE 4) reduction in all-cause mortality observed in HPS reflects the combined impact in this high risk population of the significant 17% (4) reduction in vascular mortality produced by lowering LDL cholesterol by 1 mmol/L and the lack of any significant effect on deaths from non-vascular causes during 5 years of treatment. Since the proportion of vascular to non-vascular deaths will differ between populations, the observed reduction in all-cause mortality is not readily generalisable to other situations, whereas the reduction in vascular mortality may well be. Direct assessment of the effect of cholesterol lowering on total mortality could also obscure potentially important differences in the effects on cause-specific mortality in particular circumstances [ 41 ]. Consequently, the separate analyses of vascular and non-vascular mortality (and morbidity) presented here provide both more sensitive and more generalisable evidence, not only of any beneficial effects of cholesterol-lowering statin treatment but also of any hazards in particular circumstances. No evidence of adverse effects of cholesterol-lowering on non-vascular mortality or morbidity HPS involved much larger numbers of non-vascular deaths, cancers and other serious non-vascular outcomes than any previous cholesterol-lowering study, as well as including large numbers of participants with relatively low cholesterol concentrations. It can, therefore, help address remaining uncertainty as to whether associations in non-randomised observational studies between lower total cholesterol concentrations (e.g. <4 mmol/L) and higher rates of particular non-vascular conditions are causal or, instead, due to confounding or reverse causation [ 3 , 4 ]. Reassuringly, among the 20,000 participants in HPS, lowering total cholesterol by an average of 1.2 mmol/L (46 mg/dL) for 5 years did not appear to have any adverse effect on non-vascular mortality or morbidity. Although there were too few deaths from some particular causes to assess the effects of cholesterol-lowering directly, the results for relevant non-fatal outcomes were of help in overcoming these limitations. For example, despite inverse associations in observational studies between cholesterol concentrations and respiratory mortality [ 1 , 2 ], the randomised evidence from HPS does not indicate any adverse effect of lowering cholesterol on either respiratory mortality or on the much larger number of hospitalisations for fatal or non-fatal respiratory illness (or, indeed, on lung function). Similarly, the apparent lack of any adverse effect in HPS for deaths from cancer, haemorrhagic stroke, liver or renal disease is reinforced by the results for the larger numbers of related non-fatal outcomes. Some non-randomised observational studies have found lower cholesterol concentrations to be associated with higher mortality in the elderly [ 32 , 33 ], and there has been uncertainty about the effectiveness of cholesterol-lowering in older people [ 37 ]. In HPS, however, allocation to cholesterol-lowering statin therapy not only reduced vascular (and total) mortality among 5806 participants aged 70 or over at presentation, but there was also no apparent increase in non-vascular mortality. More recently, a randomised trial reported a marginally significant excess with statin allocation among the 444 patients aged 70–82 at presentation who developed cancer during 3 years of treatment [ 34 ]. But that result may well represent a chance finding, since no significant excess was seen with statin allocation among the 674 HPS participants aged at least 70 at presentation who developed cancer during 5 years follow-up, nor has any such excess been reported from the other large statin trials [ 23 ]. Chance may also explain the slight (but non-significant) excess of non-melanoma skin cancers observed during HPS, since that excess was largely confined to the early years of the study, was seen with both squamous and basal cell carcinomas, and is not supported by published data from other major statin trials excluding HPS (724 [4.2%] statin vs 699 [4.1%] placebo; odds ratio 1.04; 95% CI 0.93–1.15; p = 0.5) [ 12 , 17 , 22 , 42 ]. If lowering cholesterol really did have adverse effects on non-vascular mortality or morbidity [ 43 ] then this might be more apparent either among participants who entered HPS with below average cholesterol levels (and so had their cholesterol concentration reduced to very low levels) or after more prolonged exposure to treatment during the later years of follow-up. But even among the 4072 participants whose pretreatment total cholesterol was below 5.0 mmol/L, lowering total cholesterol to an average of 3.5 mmol/L (133 mg/dL) for 5 years was not associated with any excess of non-vascular mortality or morbidity. Nor did any adverse effects begin to emerge with more prolonged treatment and follow-up during the trial. Nevertheless, cancer and other risks may take many years to become manifest, and extended follow-up for mortality and morbidity in HPS (as well as in some other statin trials [ 44 ]) will help assess any longer term effects. Efficacy and safety of large cholesterol reductions As might be expected from the approximately log-linear association in observational studies between vascular disease risk and cholesterol concentrations [ 2 , 5 ], a 1 mmol/L LDL cholesterol reduction in HPS from about 4 mmol/L to about 3 mmol/L (i.e. about 155 to about 116 mg/dL) reduced the risks of vascular death by about one sixth and of other major vascular events by about one-quarter, and so too did reducing it from about 3 mmol/L to about 2 mmol/L (i.e. about 116 to about 77 mg/dL), without any evidence of adverse effects. Recently, a randomised study of more intensive statin treatment versus a standard regimen in 4162 patients found that lowering LDL cholesterol to 1.6 mmol/L [62 mg/dL] rather than to 2.5 mmol/L [95 mg/dL] for about 18 months produced a 16% (95% CI 5–26) reduction in major vascular events [ 45 ]. These findings indicate that any thresholds below which lowering LDL cholesterol does not safely reduce vascular disease risk are at much lower concentrations (e.g. below 2 mmol/L [77 mg/dL] of LDL cholesterol or 3.5 mmol/L [135 mg/dL] of total cholesterol) than are typically seen in Western populations. Several large-scale trials that are currently assessing more intensive statin regimens [ 46 - 48 ] will provide further information as to whether even more substantial cholesterol reductions are not only effective at lowering vascular disease risk but also safe. Conclusion Based on large numbers of deaths and other relevant outcomes, the present results show that lowering LDL cholesterol by an average of 1 mmol/L produces substantial reductions in vascular (and, hence, all-cause) mortality in a wide range of individuals at increased risk of occlusive arterial disease (as well as reducing their risks of heart attacks, strokes and revascularisation procedures). These results also provide considerable reassurance that lowering total cholesterol concentrations by more than 1 mmol/L for an average of 5 years does not produce adverse effects on non-vascular mortality or cancer incidence, even among those who had their cholesterol concentrations reduced to very low levels. Indirectly, this observation provides some reassurance about the likely efficacy and safety of the more intensive cholesterol-lowering achievable with higher-dose or newer statins, and with combinations of standard statin doses and drugs acting through other pathways (such as resins or cholesterol absorption inhibitors). The present results provide further evidence of the benefits and safety of using statin therapy routinely in anyone (irrespective of their initial cholesterol concentration or other factors, such as age or gender) in whom a reduction in their vascular disease risk of about one third would be considered worthwhile. Competing interests The Clinical Trial Service Unit has a staff policy of not accepting honoraria or other payments from the pharmaceutical industry, except for the reimbursement of costs to participate in scientific meetings. Coordinating centre members of the writing committee (J Armitage, R Collins, L Bowman, S Parish, R Peto) have, therefore, only had such costs reimbursed. P Sleight has received honoraria as well as such reimbursement of costs. Authors' contributions MRC/BHF Heart Protection Study Collaborative Group Writing Committee -Jane Armitage, Rory Collins, Louise Bowman, Sarah Parish, Peter Sleight and Richard Peto. JA was involved in data collection, analysis and interpretation, and drafted the manuscript; RC conceived and designed the study and was involved in data collection, analysis and interpretation, and drafting of the manuscript; LB was involved in data collection and drafting of the manuscript; SP was involved in data collection, analysis, validation and interpretation; RP conceived and designed the study, and was involved in analysis and interpretation; PS was involved in the study design and interpretation. All authors provided comments on and approved the final manuscript. Steering Committee -R Collins (principal investigator), T Meade (chairman), P Sleight (vice-chairman), J Armitage (clinical coordinator), S Parish and R Peto (statisticians), L Youngman (laboratory director), M Buxton, D de Bono (deceased), C George, J Fuller, A Keech, A Mansfield, B Pentecost, D Simpson, C Warlow; J McNamara and L O'Toole (MRC observers). Data Monitoring Committee -R Doll (chairman), L Wilhelmsen (vice-chairman), K M Fox, C Hill, P Sandercock. Collaborators - (doctors; nurses; receptionists): Aberdeen Royal: N Benjamin, J Webster; J Jamieson; L Donald. Bassetlaw Hospital: R Blandford; L Carrington, H McMahon; D Cheetham. Royal United, Bath: J Reckless; L Brice, R Carpenter, J Christmas; C Flower. Bedford: I Cooper; S Frampton, E Pickerell; J Wells. Belfast City: M Scott; V Crowe, A Shaw; L Shannon. Birmingham City: S Jones; G Faulkner, A Lavery, H O'Leary, R Watson; C Capewell, S Hughes. Birmingham Heartlands: S Bain, A Jones; G Holmes, C Jewkes; T Bellamy, P Harrison. Queen Elizabeth, Birmingham: N Buller; J Hooks, H Jones, E Smith, P Vint, R Watson; P Crook, J Williams. Bishop Auckland General: M Bateson; P Cawley, P Gill; L Hawkeswell, K Simpson. Royal Bournemouth: M Armitage; C Cope, J Tricksey, M Wilson; S Cottrell. Princess of Wales, Bridgend: C Jones; M Llewellyn, P Smith; T Woodsford. Royal Sussex County, Brighton: R Vincent; E Joyce, N Skipper; P Peters. Bristol Royal Infirmary: M Lemon (late), D Stansbie; A Hagos Kidan, M Halestap; A Gibbons, J Meredith. Frenchay, Bristol: C Dawkins, M Papouchado; L Baker, K Boulton, C Dawe; A Lewis, J Wisby. Addenbrooke's, Cambridge: M Brown; J Emeny, W Smith, D Thurston, D Trutwein; M Cornwell, D Lloyd. Castleford & Normanton: C White; M Hudson, M Khalifa, N MacKereth, J Woolford; G Martin. St Peter's, Chertsey: M Baxter; R Chambers, S Glenn, J Kerr; G Golesworthy, A Watts. Corby Community: G Baines; J Groom, L Price; I Barlow. Leighton, Crewe: S Mallya; J Maiden, M Nash; V Lowe. Derbyshire Royal Infirmary: M Millar-Craig, A Scott; S Cozens, J Hannah, M Hinwood, S. Hopcroft, M Margetts, H Waterhouse; J Millward. Darlington Memorial: J Murphy; M Charters, B Graham; M Banks, M Boon, C Cassidy, R Nobbs, Dewsbury District: T Kemp; P Turner; S Sheldrake. Russells Hall, Dudley: M Labib; R Pearson, J Sidaway; P Davies, M Hodgkiss. Queen Margaret, Dunfermline: D MacLeod; R Stuart; J Albrock, J Fisher, F Stuart. Edinburgh Royal Infirmary: C Swainson; S Glenn, J Johnston, S Sadler; M Curren, S Feirnie, L Stenhouse. Western General, Edinburgh: R Lindley, C Warlow; A Kenny, F Waddell; M Brownlie, I Guilar. Derriford, Plymouth: A Marshall, J Went; S Clarke, A Inman, J Simmonds; B Duook, G Mortimore, A Pascoe. Glasgow Royal Infirmary: S Cobbe; C Campbell, H Young; M Keeble. James Paget, Great Yarmouth: S Absalom; L Baillie, N Bracey; L Falco, D Stone. Hartlepool General: G Tildesley; B Carr, G Longstaff, A Turner, H Wilkinson; S Wilkinson. Hillingdon: R Hillson; D Brookes, B Capper, N. Mahabir, K Price; V Badrick. Huddersfield Royal Infirmary: H Griffiths; J Fitzgerald, S Lewis; P Campbell. Kettering General: G Baines; J Cullen, G Claypole, J Lomas; A Rogers. Royal Lancaster Infirmary: A Brown; J Cheshire; J Rowley. Leeds General Infirmary: S Ball, C Prentice, A Hall; P Atha, K Caffrey, W Currie, K Drury, C Hague, S Hall, P Maguire, C Rose, R Watson; A Buxton, A Wedgwood. St James University, Leeds: S Gilbey; W Currie, K Drury, S Hall, C Rose, J Wilson; M Vaughan. Walton Centre, Liverpool: P Humphrey; J Blocksage, R McSloy, K Ost, L Owen, S Saminaden, D Watling, J Wiseman; J Davies. Ealing General, London: A Kehely, J Kooner; I Corbett, J Peters, K Price, S Trainor; M Van Goethem. Guy's & St Thomas', London: J Chambers; M Crawshaw, A Jones, J O'Sullivan, S Powell, M Reoch, J Sanders; M-F Beament, B Fangrad, Y Williams. North Middlesex, London: S Banim, T Crake; B Ford, V Glynn; S Ismail. Royal Brompton, London: N Buller, A Coats; L Aitken, E Cruddas, K Serup-Hansen; D Nosworthy, N Reilly. Whittington, London: S Coppack, J Malone-Lee; P Clifton, A Holmes; L Camplin. Luton & Dunstable: D Peterson, C Travill; S Gent, A Hunter, C Stroud; K Griffiths. Macclesfield District General: E Davies; M Mason, A Robinson; S Belfield. Maidstone: J Chambers; L Bispham, J Massey, A Mercer, J Sheppard; S Burrage. Manchester Diabetes Centre: K Cruickshank; KL Chan, V Wharfe, J Woodward; F Alexander, Y Williams. Manchester Royal Infirmary: M Walker; P Campbell, J Day, S Edwards, B Kelly, P Nicholson; S Barrett, S Gleeson. North Manchester General: M Savage, J Swan; D McSorland, Gillian Thompson, C Waywell; C O'Neill, L Wharton. Royal Victoria Infirmary, Newcastle-upon-Tyne: P Adams, R Lindley, N Cartilidge; M Mace, M Thompson; J Hulmes. Radcliffe Infirmary/John Radcliffe, Oxford: J Armitage, R Collins, P Sleight; S Beebe, M Campbell, J Godden, S Goodwin, A Lawson, H Lochhead, P Whitbread; S Knight, A Taylor, S Turner; Royal Alexandra, Paisley: I Findlay; C Campbell, J Hunter, H Young; E McNally. Whiteabbey, Newtownabbey: P Crowe; V Crowe, B Hunter, A Shaw; L Shannon. North Tyneside Health Care Centre, North Shields: R Curless, R Lindley, P McKenna, S Roberts; A Black, J Martin; M Burt. Northampton General: J O'Donnell; T Burdett, S Marsh, J Woodward; R O'Hare, C Owen. Poole General: A McLeod; M Richardson; C Reeves. Halton General, Runcorn: R Mallya; J Forshaw, J Hodson; H Lenden, G Osborn. St Helier: J Barron; A Ballard, B Docherty, M McDonnell, S Ritson, D Tyler; S Carter, C Rigney. Conquest, St Leonard's-on-Sea: R Wray; K Gaughan, J Sinclair; J Burleigh, J MacDonald. Royal Hallamshire, Sheffield: G Venables; C Doyle, M Fox, L Mundey; D Thompson, S Rowley. King's Mill Centre, Sutton-in-Ashfield: R Lloyd-Mostyn; D Bailey, I McKenzie; R Bamford. Singleton, Swansea: P Thomas; R Thomas; C Alexander, R Chohan, K Wood. Princess Royal, Telford: N Capps; D Donaldson, C Stiles, L Tonks; S Crank. Manor, Walsall: A Cunnington, P Giles; N Groves, E Walton; W Dance. Watford General: M Clements; C Feben, A Hunter, E Walker; L Atkins, R Kaiser, R Williats. Sandwell District General, West Bromwich: E Hughes; J Elson-Whitaker, S Sumara, C. Verow; G Banks, R Glover, K Hall. Worcester Royal Infirmary: A Munro, C Pycock, D Tibbutt; J Cadwell, M Greenwood; M Betts. Worthing: M Signy; E Joyce, C Wrapson; G McCourt, R Moore. Wycombe General: S Price, R Regan; M Aldersley; P Pendry. Coordinating Centre administration and computing, Clinical Trial Service Unit, University of Oxford: J Barton, C Bray, and K Jayne (administrative coordinators), V Booker, H Bojowsky, R Brooker, M Corbett, J Crowther, A Grantham, C Harwood, D Haywood, J Heineman, C Hope, C Indge, R Jones, S Jones, R Kanahan, K Kidney, M King, S Knight, H Lang, C Mardsen, C Mathews, G Meade (deceased), H Monaghan, K Murphy, A Naughton, A Owers, A Peto, S Pickworth, G Pocklington, A Radley, S Southren, K Szumczyk, R Tong, E Wincott; Clinical support and outcome adjudication: J Armitage and R Collins (study coordinators), A Keech and S MacMahon (piloting and planning), C Baigent, L Bowman, K Burbury, Z Chen, R Clarke, S Dunachie, V Frigi, M Landray, E Lau, C Sudlow, C Turnbull; Statistics and computing: S Parish and R Peto (statisticians), P Harding, M Lay, and K Wallendszus (computing coordinators), N Bruce, A Charles, A Cody, N Goodwin, R Greenlaw, B Hauer, P McCabe, A Palmer, C Peto, A Rowe, S Wilson, A Young, A Young; Coordinating Centre laboratory, Clinical Trial Service Unit, University of Oxford: L Youngman (director), K Kourellias, S Clark, and M Radley (laboratory coordinators), K Bhamra, L Buckingham, M Bradley, T Chavagnon, B Chukwarah, C Colominas, S Crowley, K Emmens, S Edwards, J Gordon, J Hill, A Lee, C Lennon, M McAteer, N Miller, S Norris, H Priestley, J Taylor, J Wintour, M Yeung; Nurse Monitors and trainers: S Beebe, M Campbell, J Fitzgerald, J Godden, A Lawson, S Lewis, H Lochhead, M McDonnell, M Nash, P Whitbread. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079845

EMBASE search strategies for identifying methodologically sound diagnostic studies for use by clinicians and researchers

Background Accurate diagnosis by clinicians is the cornerstone of decision making for recommending clinical interventions. The current best evidence from research concerning diagnostic tests changes unpredictably as science advances. Both clinicians and researchers need dependable access to published evidence concerning diagnostic accuracy. Bibliographic databases such as EMBASE provide the most widely available entrée to this literature. The objective of this study was to develop search strategies that optimize the retrieval of methodologically sound diagnostic studies from EMBASE for use by clinicians. Methods An analytic survey was conducted, comparing hand searches of 55 journals with retrievals from EMBASE for 4,843 candidate search terms and 6,574 combinations. All articles were rated using purpose and quality indicators, and clinically relevant diagnostic accuracy articles were categorized as 'pass' or 'fail' according to explicit criteria for scientific merit. Candidate search strategies were run in EMBASE, the retrievals being compared with the hand search data. The proposed search strategies were treated as "diagnostic tests" for sound studies and the manual review of the literature was treated as the "gold standard." The sensitivity, specificity, precision and accuracy of the search strategies were calculated. Results Of the 433 articles about diagnostic tests, 97 (22.4%) met basic criteria for scientific merit. Combinations of search terms reached peak sensitivities of 100% with specificity at 70.4%. Compared with best single terms, best multiple terms increased sensitivity for sound studies by 8.2% (absolute increase), but decreased specificity (absolute decrease 6%) when sensitivity was maximized. When terms were combined to maximize specificity, the single term "specificity.tw." (specificity of 98.2%) outperformed combinations of terms. Conclusion Empirically derived search strategies combining indexing terms and textwords can achieve high sensitivity and specificity for retrieving sound diagnostic studies from EMBASE. These search filters will enhance the searching efforts of clinicians.

Background Accurate diagnosis is essential for both the clinical care of patients and research about disease conditions. Clinicians increasingly use online access to evidence in the course of clinical care as well as for continuing education and research [ 1 ]. For most clinicians and researchers the current best evidence published in health care journals is usually first widely accessible through major biomedical databases such as MEDLINE and EMBASE. However, information retrieval in these databases can be problematic due to the scatter of relevant articles across a broad array of journals, the very dilute concentration of high quality, relevant studies in a very large database, and the inherent limitations of indexing in any large bibliographic database, amplified by clinicians' lack of search skills [ 2 ]. EMBASE searches, for example, take place in the milieu of over 9 million citations from over 4,600 journals with between 6,000 and 8,000 citations added weekly [ 3 ]. Researchers have developed search strategies to assist clinicians with searching, the majority of which have been developed for MEDLINE when searching for therapy and review articles [ 4 - 10 ]. More recently, search strategies have been reported for the retrieval of diagnostic studies in MEDLINE [ 11 - 14 ]. This is an important development because clinicians must be able to efficiently retrieve the increasing amount of innovation and new knowledge concerning diagnosis and the burgeoning number of validated treatments for specific conditions that are contained in these large electronic databases. Using search strategies or filters can assist clinicians with this retrieval. In addition to searching MEDLINE, clinicians may wish to search other electronic databases such as EMBASE to cover their topic of interest more comprehensively. EMBASE is complementary to MEDLINE in that EMBASE provides greater coverage of the European and non-English language publications and provides broader coverage in such areas as psychiatry and toxicology [ 3 ]. In the early 1990s, our group at McMaster University developed search filters for use by clinicians and researchers on a small subset of 10 journals and for 4 types of journal articles (therapy, diagnosis, prognosis and causation [etiology]) [ 15 , 16 ]. This research was updated and expanded using data from 161 journals indexed in MEDLINE from the publishing year 2000 [ 17 - 20 ]. These search strategies have been adapted for use in the Clinical Queries interface of MEDLINE . Clinicians can easily access and use these search strategies by going to the Clinical Queries page in PubMed. We now report the extension of this research for EMBASE, including the information retrieval properties of single terms and combinations of terms for maximizing the sensitivity and specificity of identifying methodologically sound primary (original) studies on the diagnosis of health disorders. These search strategies will assist clinicians and researchers when searching for relevant, high-quality articles in EMBASE. Methods We compared the retrieval performance of methodological search terms and phrases in EMBASE with a manual review of each article for each issue of 55 journal titles for the year 2000. Overall, research staff hand-searched 170 journal titles. These journals were chosen based on recommendations of clinicians and librarians, Science Citation Index Impact Factors provided by the Institute for Scientific Information, and the ongoing assessment of their yield of studies and reviews of scientific merit and clinical relevance for the disciplines of internal medicine, general medical practice, mental health, and general nursing practice (list of journals provided by the authors upon request). Of these 170 hand-searched journals, 135 were indexed in EMBASE. Search strategies were developed using a 55-journal subset chosen based on those journals that had the highest number of methodologically sound studies, that is, studies that clinicians should be using when making patient care decisions. This selection enriches the sample of target articles, improving the precision of estimates of search term performance and simplifying data processing, but is unlikely to bias the estimates of the sensitivity and specificity of search terms. We compiled an initial list of search terms, including index terms and textwords from clinical studies. Input was then sought from clinicians and librarians in the United States and Canada through interviews of known searchers, and requests at meetings and conferences. We compiled a list of 5,385 terms of which 4,843 were unique and 3,524 returned results (list of terms tested provided by the authors upon request). Examples of the search terms tested are 'criterion standard', 'cut point', 'sensitivity', and 'ROC curve', all as textwords; 'diagnosis', the index term, and the index term 'diagnostic test', exploded (that is, including all of this term's indexing subheadings). As part of a larger study [ 21 ], research staff performance was rigorously calibrated before reviewing the journals and inter-rater agreement for identifying the purpose of articles was 81% beyond chance (kappa statistic, 95% confidence interval (CI) 0.79 to 0.84). Inter-rater agreement for which articles met all methodological criteria was 89% (CI 78% to 99%) beyond chance [ 21 ]. Six research assistants then hand-searched all articles in each issue of the 55 journals and applied methodological criteria to determine whether the article was methodologically sound for evaluation of a diagnostic test. The methodological criteria applied for studies of diagnosis were as follows: Inclusion of a spectrum of participants; objective diagnostic ("gold") standard or current clinical standard for diagnosis; participants received both the new test and some form of the diagnostic standard; interpretation of diagnostic standard without knowledge of test result and vice versa; and analysis consistent with study design. The proposed search strategies were treated as "diagnostic tests" for sound studies and the manual review of the literature was treated as the "gold standard". We determined the sensitivity, specificity, precision and accuracy of each single term and combinations of terms in EMBASE using an automated process. Borrowing from the concepts of diagnostic test evaluation and library science, sensitivity for a given topic is defined as the proportion of high quality articles for that topic that are retrieved; specificity is the proportion of low quality articles not retrieved; precision is the proportion of retrieved articles that are of high quality; and accuracy is the proportion of all articles that are correctly classified [ 22 ]. Individual search terms with sensitivity > 25% and specificity > 75% for a given purpose category were incorporated into the development of search strategies that included 2 or more terms. All combinations of terms used the Boolean OR, for example, "predict.tw. OR specificity.tw.". The Boolean AND was not used because this strategy invariably compromised sensitivity. For the development of multiple-term search strategies to optimize either sensitivity or specificity, we tested all 2-term search strategies with sensitivity at least 75% and specificity at least 50%. For optimizing accuracy, 2-term search strategies with accuracy > 75% were considered for multiple-term development. In the development of diagnosis search filters, 6,574 search strategies were tested. In addition to developing search strategies using the Boolean approach described above, we also evaluated the potential for improving performance using logistic regression. Two approaches were taken. First, we took the top performing Boolean search strategies and ORed additional terms to these base strategies using stepwise logistic regression. The level of significance for entering and removing search terms from the model was 0.05. Adding terms to the model stopped when the increase in the area under the ROC curve was < 1%. Second, we developed search strategies from scratch with stepwise logistic regression using these same cut-off values. Both logistic regression approaches were compared with the Boolean approach to search strategy development when developing strategies for treatment articles and prognostic articles for MEDLINE. Treatment and prognosis were chosen because they represented the best and the worst cases for MEDLINE search strategy performance. For both purpose categories, the logistic regression approaches to developing search strategies did not improve performance compared with search strategies developed using the Boolean approach described above. Thus, for subsequent purpose categories, including diagnosis and databases, including EMBASE, the Boolean approach was used for search strategy development. We also tested search strategies published by other researchers for detecting diagnosis studies. Results Indexing information was downloaded from EMBASE for 27,769 articles from the 55 hand-searched journals. Of these, 433 were classified as diagnosis, of which 97 (22.4%) were methodologically sound. Search strategies were developed using all 27,769 articles. Thus, the strategies were tested for their ability to retrieve articles about high quality diagnosis studies from all other articles, including both low quality diagnosis studies and all non-diagnosis studies. Table 1 shows the best single term for high-sensitivity, high-specificity, and best balance of sensitivity and specificity. The single term, "di.fs." (Ovid syntax for diagnosis as a floating subheading) produced the best sensitivity of 91.8% while keeping specificity at 76.4%. Specificity was maximized at 98.2% using the single term "specificity.tw.", but this was achieved at the expense of sensitivity, 62.9%. The single term "diagnos:.mp." (Ovid syntax for the appearance of "diagnos:" in any one of the title, abstract or subject headings), produced the optimal balance between sensitivity (89.7%) and specificity (84.7%). Table 1 Single Term with the Best Sensitivity, Best Specificity, and Best Optimization of Sensitivity and Specificity for Detecting Studies of Diagnosis in EMBASE in 2000. Values are percentages (95% confidence intervals). Search term OVID search* Sensitivity (n = 97) Specificity (n = 27672) Precision† Accuracy (n = 27769) Best sensitivity (keeping specificity ≥ 50%) di.fs. 91.8 (86.3 to 97.2) 76.4 (75.9 to 76.9) 1.4 (1.1 to 1.6) 76.5 (76.0 to 77.0) Best specificity (keeping sensitivity ≥ 50%) specificity.tw. 62.9 (53.5 to 72.5) 98.2 (98.1 to 98.4) 11.0 (8.4 to 13.6) 98.1 (97.9 to 98.3) Best Optimization of Sensitivity & Specificity‡ diagnos:.mp. 89.7 (83.6 to 95.7) 84.7 (84.3 to 85.2) 2.0 (1.6 to 2.4) 84.8 (84.3 to 85.2) *Search strategies are reported using Ovid's search engine syntax for EMBASE. †Denominator varies by row. ‡Based on the lowest possible absolute difference between sensitivity and specificity. di = diagnosis; fs = floating subheading; tw = textword (word or phrase appears in title or abstract); : = truncation; mp = multiple posting – term appears in title, abstract, or subject heading. Sensitivity = the proportion of high quality articles for that topic that are retrieved; specificity = the proportion of low quality articles not retrieved; precision = the proportion of retrieved articles that are of high quality; accuracy = the proportion of all articles that are correctly classified. Combinations of terms with the best results for sensitivity, specificity and optimization of sensitivity and specificity are shown in Table 2 . Combinations of terms improved on single search term performance for sensitivity. The 3-term search strategy, "di.fs. OR predict:.tw. OR specificity.tw.", achieved a sensitivity of 100% with a specificity at 70.4%. The single term "specificity.tw." had the highest specificity, outperforming all 2- and 3-term combinations. A 3-term combination resulted in the optimization strategy achieving slightly above 89% for both sensitivity and specificity (Table 2 ). Table 2 Combination of Terms with the Best Sensitivity, Best Specificity, and Best Optimization of Sensitivity and Specificity for Detecting Studies of Diagnosis in EMBASE in 2000. Values are percentages (95% confidence intervals). Search Strategyn OVID search* Sensitivity (n = 97) Specificity (n = 27672) Precision† Accuracy (n = 27769) Best Sensitivity (keeping specificity ≥ 50%) di.fs. OR predict:.tw. OR specificity.tw. 100.0 (100.0 to 100.0) 70.4 (69.8 to 70.9) 1.2 (0.9 to 1.4) 70.5 (69.9 to 71.0) Small drop in sensitivity with a substantive gain in specificity diagnos:.mp. OR predict:.tw. OR specificity.tw. 96.9 (93.5 to 100.0) 78.2 (77.7 to 78.7) 1.5 (1.2 to 1.8) 78.3 (77.8 to 78.8) Best Specificity (keeping sensitivity ≥ 50%) specificity.tw. 62.9 (53.5 to 72.5) 98.2 (98.1 to 98.4) 11.0 (8.4 to 13.6) 98.1 (97.9 to 98.3) Small drop in specificity with a substantive gain in sensitivity specificity.tw. OR accurac:.tw. 73.2 (64.4 to 82.0) 97.4 (97.2 to 97.5) 8.8 (6.9 to 10.8) 97.3 (97.1 to 97.5) Best Optimization of Sensitivity & Specificity‡ sensitiv:.tw. OR diagnostic accuracy.sh. OR diagnostic.tw. 89.7 (83.6 to 95.7) 91.6 (91.3 to 91.9) 3.3 (2.9 to 4.4) 91.6 (91.3 to 91.9) *Search strategies are reported using Ovid's search engine syntax for EMBASE. †Denominator varies by row. ‡Based on the lowest possible absolute difference between sensitivity and specificity. di = diagnosis; fs = floating subheading; : = truncation; tw = textword (word or phrase appears in title or abstract); mp = multiple posting – term appears in title, abstract, or subject heading; sh = subject heading. Sensitivity = the proportion of high quality articles for that topic that are retrieved; specificity = the proportion of low quality articles not retrieved; precision = the proportion of retrieved articles that are of high quality; accuracy = the proportion of all articles that are correctly classified. Slight modifications to the above-noted most sensitive and most specific search strategies led to some attractive trade-offs in sensitivity and specificity (Table 2 ). For instance, by replacing "di.fs" with "diagnos:.mp." in the most sensitive search strategy ("diagnos:.mp. OR predict:.tw. OR specificity.tw.") specificity increased (70.4% to 78.2%) at the price of a small decrease in sensitivity (100% to 96.9%). Additionally, by ORing "accurac:.tw." to "specificity.tw.", to the most specific search strategy, sensitivity increased by 10.2% (62.9% to 73.2%) with a small decrease in specificity (98.2% to 97.4%). Our search strategies were simpler and compared well with two previously published strategies by Bachmann and colleagues for retrieving diagnostic test studies from EMBASE [ 23 ]. The most sensitive search reported by Bachmann and colleagues, an 8-term strategy, had a sensitivity of 96.9% in our database compared with 100% for our 3-term strategy (difference 3.1%, 95% CI -0.8% to 8.7%) (Table 3 ). The most specific search reported by Bachmann and colleagues, a 2-term strategy, had a specificity of 90.9% in our database, compared with 98.2% for our 1-term strategy, but our strategy was less sensitive (62.9 vs. 79.4, difference 16.5%, CI 3.8% to 28.9%). Unlike Bachmann's study, our study evaluated the methodological rigor of diagnosis studies, and thus the performance of search strategies compared here is for detecting methodologically sound diagnostic studies. Table 3 Comparison of previously published search strategies with search strategies developed our database. Values are percentages. Search Strategy OVID search* Sensitivity Specificity Precision Accuracy Bachmann's most sensitive search [22] sensitiv:.tw. OR detect:.tw. OR accura:.tw. OR specific:.tw. OR reliab:.tw. OR positive.tw. OR negative.tw. OR diagnos:.tw. 100.0 Not reported Not reported Bachmann's strategy tested in our database 96.9 72.3 1.2 72.4 Our most sensitive search di.fs. OR predict:.tw. OR specificity.tw. 100.0 70.4 1.2 70.5 Difference (our strategy – Bachmann) 95% CI for the difference 3.1 -0.8 to 8.7† -1.9 -2.7 to -1.2‡ 0 -1.9 -2.7 to -1.2‡ Bachmann's most specific search sensitiv:.tw. OR detect:.tw. 73.7 Not reported Not reported Bachmann's strategy tested in our database 79.4 90.9 3.0 90.8 Our most specific search specificity.tw. 62.9 98.2 11.0 98.1 Difference (our strategy – Bachmann) 95% CI for the difference -16.5 -28.9 to -3.8‡ 7.3 7.0 to 7.7‡ 8.0 5.6 to 11.0‡ 7.3 6.9 to 7.7‡ *Search strategies are reported using Ovid's search engine syntax for EMBASE. †Differences are not statistically significant. ‡Differences are statistically significant. : = truncation; tw = textword (word or phrase appears in title or abstract); di = diagnosis; fs = floating subheading. Sensitivity = the proportion of high quality articles for that topic that are retrieved; specificity = the proportion of low quality articles not retrieved; precision = the proportion of retrieved articles that are of high quality; accuracy = the proportion of all articles that are correctly classified. Discussion Our study documents search strategies for use by clinicians and researchers that can help discriminate relevant, high-quality studies from lower quality studies of the diagnosis of health disorders and articles that are not about diagnosis. Those interested in all sound articles on diagnosis, for example researchers conducting systematic reviews of diagnostic tests, will be best served by the most sensitive search. If systematic reviewers wish to include diagnostic test articles that fail the methodological criteria we set, they will still be well served by starting with this strategy: in addition to retrieving all sound studies, the suboptimal specificity (70.4%) of our most sensitive search strategy means the many lower quality diagnostic test studies will also be retrieved. Reviewers may then use additional means to ensure that all pertinent studies are retrieved. Those with little time on their hands who are looking for a few good articles on diagnosis, most likely clinicians, will probably be best served by the most specific strategies. Clinicians could further broaden their search by using the strategies that optimize sensitivity and specificity while minimizing the difference between the two as these strategies provide the best separation of "hits" (target citations) from "false drops" (undesired citations) but do so without regard for whether sensitivity and specificity are affected. In all cases precision was low. This is the inevitable result of a low proportion of relevant studies for a given purpose in a very large, multipurpose database. This means that clinicians and researchers will continue to need to invest their time in discarding irrelevant retrievals. While low precision in searching can be of concern, the low values here should not be over-interpreted: we did not limit the searches by clinical content terms, as would usually be the case in clinical searches. Precision might be enhanced by combining search strategies in these tables with additional methodological terms using the Boolean 'AND NOT', thereby reducing the possibility of retrieving studies of lower methodological quality; however, this may decrease the sensitivity of the searches. Precision might also be increased by combining search strategies with content specific terms (e.g., "diabetes") or journal subsets using the Boolean 'AND' thus reducing the volume of literature searched. The next phases of our project will focus on finding better search strategies through using more sophisticated strategies as outlined above. Comparing the diagnostic search strategies developed for EMBASE with those that we developed for MEDLINE [ 19 ], we found that the single term "specificity.tw." was the top performer for specificity in both databases and that this term outperformed 2- and 3-term strategies. Additionally, we found that textwords outperformed most index terms for sensitivity and specificity. The only index term that was a top performer was "di.fs." or "di.xs.", which was the case for both databases. Although there are many differences between EMBASE and MEDLINE, some basic similarities are apparent, as just described. Comparing our diagnostic search strategies developed for EMBASE with those previously published [ 23 ], our strategies had fewer terms and performed at least as well. Conclusion Selected combinations of indexing terms and textwords can achieve high sensitivity or specificity in retrieving diagnosis studies cited in EMBASE. The reported search strategies will assist both clinicians and researchers when attempting the retrieve relevant, high-quality diagnostic articles. Competing interests The author(s) declare that they have no competing interests. Authors' contributions NLW and RBH prepared grant submissions in relation to this project. Both authors drafted, commented on and approved the final manuscript. Both authors also supplied intellectual content to the collection and analysis of the data. NLW participated in the data collection and both authors were involved in data analysis and staff supervision. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079846

A qualitative study of the perceptions and experiences of Pre-Registration House Officers on teamwork and support

Background Following the implementation of a new final Year 5 curriculum in one medical school we carried out a study to explore the experience of the transition from final student year to Pre-Registration House Officer (PRHO). This study looks at the experiences of two successive cohorts of PRHOs in relation to team work, support and shared responsibility in their transition from final year students to qualified doctors. The involvement of PRHOs in teams is likely to change in the development of Foundation programmes. Methods A qualitative study with semi-structured interviews with 33 PRHOs, stratified by gender, ethnicity and maturity, from two study cohorts, qualifying in 2001 and 2002, from one medical school in the UK, in their first three months following medical graduation. Results Most PRHOs reported positive experiences for their inclusion as a full member of their first ward teams. This contributed to their increasing confidence and competence in this early period of career transition. However, a number of organisational barriers were identified, e.g. incomplete teams, shift work, which produced problems in their integration for one third of newly qualified doctors. Conclusion Recently introduced policies, intended to improve the working lives of newly qualified doctors have produced both benefits and unintended adverse impacts on PRHOs. The changes of the new PRHO Foundation programme will have further impact. Foundation doctors may need to relate to wider teams with more interaction and less protection. Such changes will need to be managed carefully to protect the PRHO at a vulnerable time.

Background There is increasing emphasis on multi-disciplinary teams in modern clinical care [ 1 - 3 ]. Medical schools have attempted to embrace this through interdisciplinary learning [ 1 , 4 - 6 ] and stressing relevant attitudinal, ethical and behavioural issues [ 7 - 9 ] in relation to teamwork. Concern has been expressed about whether such teamwork skills can be effectively practised by junior doctors during the PRHO year while they cope with multiple professional, personal, physical and emotional demands [ 10 - 12 ]. The General Medical Council document, The New Doctor [ 13 ] stated that one of the professional learning outcomes of the pre-registration year is 'to work in a team and to take collective responsibility for patient care'. Several authors [ 14 - 17 ] have recognised difficulties in delivery of these goals although most PRHOs appear to acquire the ability to work in a team by the time of qualification [ 18 , 19 ]. More recent policy changes, such as The New Deal [ 20 ] might reduce the contribution of PRHOs to teamwork and decrease job satisfaction [ 21 ]. We wanted to explore how well our undergraduate curriculum, particularly the final year with 16 weeks shadowing in a student house officer role, prepared PRHOs for their new posts. In this study issues emerged about the stability of ward teams and the full integration of PRHOs in to teams. These findings may have relevance to the development of Foundation programmes. Methods Two study cohorts of 33 PRHOs were selected by convenience (16 participants from year 2000/2001) and quota sampling (17 participants from year 2001/2002), stratified by gender, ethnicity and maturity, using the respective final year student population of the Medical School as the sampling frame (see Tables 1 & 2 ). Written, informed consent was obtained from each participant. Semi-structured 1:1 interviews (see Additional file 1 ) were carried out within the first three months of their PRHO posts. Students' accounts were evaluated against the relevant professional competencies set out in The New Doctor [ 13 ] during a comprehensive evaluation of the newly developed final year in the medical school. This paper focuses on issues related to the role of the doctor within the health service, particularly 'the ability to work in a team' and 'accept principles of collective responsibility' [ 12 ]. All interviews were tape-recorded and transcribed in full. Two of the authors and an independent researcher read through the transcripts and identified a list of emerging themes. These lists of themes were then cross referenced and amended by the readers to reach agreement for the analytical framework of content analysis [ 22 ]. Throughout the data analysis the occurrence of key findings are noted by using simple counts. Combining qualitative and quantitative data allowed the authors to unpack, confirm and emphasise some similarities or differences between the different study cohorts. This approach assists in the generalisability of the findings [ 22 ] and offers evidence more convincingly (but not in a statistical significant sense) than relying on anecdotal accounts alone. This method can also contribute to the validation and credibility of qualitative research [ 22 - 24 ]. There was one key question in reference to team work but relevant information from all parts of the interview was used in the analysis (see Appendix 1). Table 1 Socio-demographic data from 16 PRHO cohort 2000/2001 Gender 11 Females, 5 Males Age 24 years (mean) Ethnicity (self-described) 5 Indian, 1 Gujarati, 1 Irish-Indian, 1 English-Chinese, 1 Sri-Lankan, 7 Caucasian Class 13 middle class, 3 working class Family status 15 single, 1 married Country of birth 15 UK, 1 outside Europe Religion 5 Christian, 3 Hindu, 5 none, 3 not stated Entry to Medical School 11 after school, 2 after a gap year, 3 mature (3/16 obtained intercalated BA/BSc during their time at Medical School) Table 2 Socio-demographic data from 17 PRHO cohort 2001/2002 Gender 9 Females, 8 Males Age 24 years (mean) Ethnicity (self-described) 1 Black-African, 1 East Asian, 7 South Asian, 8 Caucasian Class 14 middle class, 3 working class Family status 16 single, 1 married Country of birth 14 UK, 3 outside Europe Religion 5 Christian, 3 Hindu, 1 Islam, 6 none, 2 not stated Entry to Medical School 12 after school, 1 after a gap year, 1 after A level retake, 3 mature (10/17 obtained intercalated BA/BSc during their time at Medical School) Results The 33 PRHOs worked in four acute hospital trusts and seven District General Hospitals (DGHs) in England, 16 in surgical and 17 in medical wards. They commented frequently on the importance of their clinical teams. Findings from PRHOs' accounts are reported around 3 key themes: supportive environment, educational environment and organisational changes. Overall the transition from being an 'outsider' to a professional 'insider' was a welcome change for all PRHOs, including involvement in the team. Comments on finding a role were common: 'When I was a medical students in the 5 th year I was very much aware that that I didn't have a role, I felt I was sort of very superfluous to the process... 'Now I have, a definite, a clear cut role, and I know what I should be doing, and what I shouldn't be doing, and I have a continuity I have my patients and I have a team, which I have a good relationship with'. 'In the previous years we haven't been actually part of a team. We've been assigned a team but you're not working with a team....... you're not part of that patient's everyday care'. 'It's so different being a house officer than being a medical student – you're a team, like, straightaway. The first day, that's it! That's a very big difference.' 1) Supportive environment Most PRHOs (27/33) described their clinical teams in positive terms (see Table 3 ). Even so they also identified a number of specific characteristics, which had a profound influence on their first weeks working as qualified doctors. Table 3 Descriptions by 27/33 PRHOs about their medical and surgical ward teams 'The team is fine'; 'the team is working brilliantly, we gelled really quickly'; 'definitely working well' (x2); 'working well together sometimes not so well'; 'definitely I feel part of the team' (x5); 'the team is great now, but not in the first two weeks'; 'felt part of the team quickly, very quickly'; ' feel part of the team, very much so'(x2); 'it's a really nice team'(x2); 'there is a good team spirit'; 'we work as a team' (x2); 'I really like my team, they are very supportive'; our team functions very well, everybody is friendly, chats and makes jokes'; 'there has been a welcome surprise, such as the team, it has been very supportive'; 'and I got a really nice team'; 'I work well with the team and felt accepted'; 'definitely I am part of the team, you're a sort of team player really'; 'oh yeah I get on really well with them'. Constructive feedback from senior colleagues PRHOs reporting support by senior medical staff (24/33) felt that they integrated with their clinical teams early and well, especially in emergency situations. Acute or life threatening events affected them emotionally, usually with little time for debriefing. Without senior support PRHOs often felt out of their depth. When they had dealt independently with a complex clinical situation, constructive feedback from senior staff added to their positive experiences. Conversely PRHOs who received no or little supervision or response from senior staff recorded these events as a negative impact on their confidence. The latter situations were more common in surgery, where PRHOs reported that senior staff was less accessible. 'I would have quite liked an SHO [Senior House Officer] or somebody to tell me what I should be doing in that situation, or whether I should be phoning the GP or writing or speaking to him, or who actually should be talking to her [patient] and what actually telling them [the couple] ...ahm...so I basically did it all myself.' '...getting called to see a man that had collapsed on his way back from the loo, and had no blood pressure and was basically dying. And I thought: Ahhh! But I just called the SHO and said: "Can you come and help me?" And I couldn't get access in him [patient] at all, and it was just a bit scary. When the SHO arrived we sorted him out and he was then fine after that, although he died a few days later'. Sharing responsibilities and tasks A sense of collective responsibility with other team members was positively expressed by most PRHOs (26/33). The predominant ethos was one of sharing the workload between medical and nursing colleagues in the team (see Table 4 ). Table 4 Phrases used by 26/33 PRHOs about sharing work load and responsibilities 'jointly' (x 2), 'share' (x5) 'do the jobs' (x8), 'help' (x3), 'we do the same jobs', 'split the jobs', work generically as a team', 'divide jobs up', 'catch up with all the jobs', 'we have jobs to do between the house officers and all the colleagues', 'do all the work together', 'work together closely', 'plan our jobs', 'carrying out the jobs together', 'manage jobs together on the wards', 'taking care of referrals', 'do jobs as we go along' [during the ward round], 'we finish of jobs', we sort out our jobs'. *some used more than one phrase PRHOs reported that sharing jobs was not always straightforward, particularly if individual team members did not work well together (7/33), or if teams were incomplete due to staff shortage and/or locum cover (8/33). 'I mean it's [work] so erratic and our team is so disjointed! I don't really know half the patients and I don't...I don't even know if half the jobs are getting done. I mean mine all get done but I don't know whether the other half's, the other House Officers are thinking I'm doing theirs or they're doing mine, or the SHO's doing whatever'. Interprofessional working 28/33 PRHOs reported that the relationship with nursing staff changed with qualification. Former inter-professional tensions seemed due to the perceived inconvenience that medical students caused to nurses. Prior to qualification students had relied on nurses in hospital and community for help with 'log book' skills, such as catheterisation and injections, and they appreciated their patience and enthusiasm. 'Before as a medical student you were a piece of furniture according to nurses....but now, obviously you are the first-line if they want anything doing, I think in that respect there's a lot more regard....and appreciation for what you do'. However, a number of inter-professional conflicts were identified such as adjustment to professional roles (x 15), discordant communication (x9), unclear what nurses are willing or (un)able to do (x10) and others. Relationship with PRHOs in the same team 8/33 commented positively on working with other PRHOs from their final year group in the same team, making sharing the workload and responsibility easier due to closer personal relationships. However, 2/33 identified this as impacting on their teamwork and patient care when PRHOs did not get on well together and 4/33 found competition to learn additional clinical skills. 'There always has been camaraderie with other PRHOs, but at least I get to spend time around now as well, so ahm...you know it is great, when you literally you say, will you do this, I will do that, and we meet up at the end [of the day]....that is always really nice'. 'There's a mutual understanding there (amongst the PRHOs in the team], and we...amazingly, we are ever so professional on the job. I mean on the team, we're very good friends – we go out for dinner, all sorts – but when we're on the job, it's ever so professional'. 2) Educational environment PRHOs sought out educational opportunities to achieve a sense of progression after qualification within their clinical teams. Learning consisted of performing new practical skills, as well as observation, to acquire inter-personal and professional competencies. Continuity in their relationship with patients was perceived as important as team members, rather than temporary bystanders as students. Observing delicate situations Most PRHOs (23/33) had witnessed delicate clinical or ethical situations, which required advanced communication skills between team members, patients and their families, e.g. breaking bad news, communicating with uncooperative patients. The interviewees had a variety of opportunities to observe how senior medical and nursing staff within their teams managed such situations sensitively and professionally. '....it is quite interesting, watching your seniors how they deal with difficult patients: patients who are too demanding, or patients who, you know, are rude or angry, or breaking bad news, I try to sort of sit in on all that, so I can see how they [seniors] do it....' ' ... when family gets involved, it gets awfully sad [resuscitation status]. I had a patient who died; I got to know the family relatively well – two sons – and they divulged that their mother had died, and...all sorts of complex family dynamics going on there. They were quite clearly worried about their father. And then there's only so much input the house officer can do – essentially, the seniors make the decisions...' PRHOs were acutely aware that consultants were ultimately in charge of final decisions in ethical dilemmas. 6/33 PRHOs reported that they were given tasks which they felt were inappropriate, such as asking patients for consent to undergo an operation or investigation which they had never seen themselves. Learning new clinical skills from seniors 7/33 PRHOs were able to extend their practical skills, such as lumbar punctures or insertion of chest drains during their first house jobs. However, these opportunities were reduced where ward teams were incomplete and depended on the willingness and clinical competence of senior staff in their teams. Such limitations sometimes caused frustration. The message around unstable teams was often close to: 'Our team's so disjointed at the moment. The SHO that we've got is constant. But, she does 'on-calls' for other teams as well so we don't see her that often. Our Registrars change every day because they're all locums. So on some days we don't have one and some days we do ...I don't feel....I'm not learning the things I thought I'd learn.' 'There's only one regular member of the team and that's the staff grade, and so the consultant is a locum. He's been here a couple of months, or just before we started, basically he was here. The SHO is a locum SHO, who has now finished her rotation, and I've just started so there was not much stability in the team to begin with.' 3) Organisational changes Organisational changes, such as new work patterns, and inadequate staffing levels caused frustration and anxiety affecting the PRHOs emotionally and their team work. Continuity of relationships with patients Most PRHOs (25/33) described continuity of patient care in positive terms. They welcomed the fact that they could approach patients without apologies because they had a defined role and responsibility. 11/33 PRHOs reported fragmentation in their relationships with patients which they related to shift work and/or staff shortages. '...the continuity is great, you know, walking on the ward, and sort of saying "hi" to people and you know they know you, and getting on with them and kind of trying to do your best for them.' '....there's no continuity of care: we start in the morning, we finish, and then someone else comes on – and they [consultant] don't know the person. And then the next day that patient either moved to somewhere else or they've gone home. So we're not following them [patients].' New work patterns One third of PRHOs in study cohort 2001/2002 confirmed that their hospital complied with new working hours while two thirds encountered difficulties. The 2001/2002 group had a range of concerns: continuous long working hours, despite the implementation of new working patterns (12/17), loss of emergency experience at night (4/17), confusion from non-compliance or experimentation with new working hours (12/17), and unhappiness and ridicule by senior colleagues (5/17). These caused resentment and dissatisfaction amongst the majority of the cohort (12/17) more than the 2000/2001 group (5/16). Staffing levels In addition to the new working arrangements, there was a change from 2000/01 to 2001/02 in the availability and continuity of permanent staff members, doctors and nurses. This was reported only by the second PRHO cohort. One third of this group commented that team work was disrupted and undermined by incomplete teams. 'It's just working in a big hospital, and having an incomplete team was...it was just unfortunate that that was the situation when I joined...I didn't enjoy that and it upset me because, you know, it's taken me a long time to get through Medicine and I felt that that was bad.' Discussion In a few areas such as changes in rotas and hours the interviewees commented on the views of more senior doctors. These views may have influenced their responses. In order to encourage open responses interviewees were guaranteed anonymity. Interviews were conducted mainly by one interviewer, who was not part of the medical school hierarchy or trusts and had established a good rapport with the PRHOs cohorts. The interviews in this study ranged over a number of areas. They explored the preparation of PRHOs through their undergraduate curriculum and the sources of stress in the transition to the PRHO role. PRHOs discussed their experience of the working environment and their relationships with colleagues in a number of areas and team work was addressed specifically in a direct question. The themes explored in this study were identified clearly by the three authors and the independent researcher on analysis of the transcripts. The PRHOs were enthusiastic about the way their assumption of their professional role allowed integration into multi-disciplinary working with a clear change in the working relationships with nursing colleagues. This was an area that changed from their medical school experience despite efforts to give them clearly defined patient management roles during 16 weeks of experience as student house officers in hospital. It is possible that interprofessional education and even clearer integration of student house officers may help to minimise the changes on transition in future. Clear evidence about the efficacy of interprofessional education, however, has not yet been established. The supportive and educational environments facilitated by ward teams received a welcome positive reaction from the majority of newly qualified PRHOs in both study cohorts. The importance of adequate senior supervision in the early stages of the PRHO experience has been emphasised in previous studies [ 25 ]. Opportunities to observe senior staff in clinical situations were especially useful. The New Deal [ 20 ] and the New Doctor [ 13 ] were intended to improve working conditions for PRHOs. It has been suggested that introduction of 'new deal' rotas may increase psychological morbidity and reduce job satisfaction for PRHOs [ 21 ], but that a well supervised working environment may counteract reductions in hours with high work intensity [ 26 ]. In this study difficulties with working patterns appeared to have increased between the two cohorts. Since this study was performed considerable further effort has gone in to the design of compliant and suitable rotas for doctors in training. The problems were exacerbated by instability of the working teams which meant that supervision was not always adequate at these times when it needed to be most effective. Shortage of permanent staff in the ward teams was stressful and unsettling for one third of the PRHOs. These organisational difficulties affected five key areas of work: continuity of patient care, sharing responsibilities within the team, delayed integration in to the new teams, ad hoc learning opportunities to acquire new skills and ongoing lack of support from senior staff. These areas emerged without prompting from interviewers. It may be that PRHOs early in their post and low in confidence are worried by the possibility of inadequate support which might come from the unstable teams. However, the reports suggested that it was causing real concern for the interviewees who responded in this way. It is important that these areas are addressed for the introduction of the Foundation programmes. It is possible that changes in working patterns may mean that those in F1 posts in the future will have to relate to larger teams with a broader range of skills with clear responsibilities but less opportunity for continuity of care. Instability in these larger teams could be even more of a problem. Both PRHOs and more senior doctors will have to learn such new ways of working. The loss of traditional clinical "firms" also needs to be addressed in undergraduate training. Many medical schools retain periods of apprenticeship, teaching traditionally and linking a small number of students to a small clinical team. This allows the students to relate to the staff and build their confidence. The combination and interaction of these teams and the increase in day and outpatient care means that new patterns have already been developed to optimise the students' experience in the hospital environment. Changes in the student experience need to be developed in line with Foundation programmes to be sure that junior doctors are prepared for their roles on graduation. The greatest satisfaction for PRHOs in this study came from involvement with a clear role and feeling part of the team, and this will be one of the core competencies assessed in Foundation programmes. Mechanisms of working within these teams and handover arrangements need to be clear. While the increased attention on evaluation should lead to clarity of roles with appropriate supervision, the greater emphasis on evaluation could also change the nature of the relationships. Conclusion Overall this study emphasises the enthusiasm of PRHOs for well organised structures with adequate supervision in a supportive multidisciplinary environment. Over the period studied rotas and unstable staffing patterns affected this environment significantly. Competing interests Dr. Mac Cochrane received funding from the Oak Foundation for both studies. Authors' contributions HL developed study design, carried out most interviews, conducted the analyses and wrote the paper. MC supervised study, commented on study design, carried out few interviews, assisted in the data analysis. JR supervised the study, commented on study design, data analysis and contributed in the writing of paper. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Microsoft word file (teamwork additional.doc) containing details of the semi-structured interview. Click here for file

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1079847

Measuring body composition in overweight individuals by dual energy x-ray absorptiometry

Background Dual energy x-ray absorptiometry (DXA) is widely used for body composition measurements in normal-weight and overweight/obese individuals. The limitations of bone densitometers have been frequently addressed. However, the possible errors in assessing body composition in overweight individuals due to incorrect positioning or limitations of DXA to accurately assess both bone mineral density and body composition in obese individuals have not received much attention and are the focus of this report. Discussion We discuss proper ways of measuring overweight individuals and point to some studies where that might not have been the case. It appears that currently, the most prudent approach to assess body composition of large individuals who cannot fit under the scanning area would be to estimate regional fat, namely the regions of thigh and/or abdomen. Additionally, using two-half body scans, although time consuming, may provide a relatively accurate measurement of total body fat, however, more studies using this technique are needed to validate it. Summary Researchers using bone densitometers for body composition measurements need to have an understanding of its limitations in overweight individuals and address them appropriately when interpreting their results. Studies on accuracy and precision in measurements of both bone and soft tissue composition in overweight individuals using available densitometers are needed.

Background Dual energy x-ray absorptiometry (DXA) is widely used by clinicians and researchers for evaluation of bone status and soft tissue composition. While the principles of DXA technology could be found elsewhere [ 1 - 3 ] and are not the focus of this report, we address them briefly for better understanding of the discussion to follow. The underlying principle of DXA is its ability to quantify the attenuated radiation after its passage through bone and soft tissue using either K-edge filters or pulsed power sources to the x-ray tube. Subsequently, the differential attenuation of the two energies is utilized to quantify bone, lean, and/or fat tissue. The earlier DXA series are based on pencil-beam absorptiometry, where a highly collimated x-ray beam and a detector move along the rectilinear scan path. The new series employ fan-beam absorptiometry in which data are acquired either simultaneously along the entire scan line, or as rectilinear scanning with a narrow fan-beam, both resulting in a faster scanning time [ 1 ]. The fan beam densitometers have the advantage of improved geometrical resolution, but the disadvantage of errors induced by magnification effects. Within the fan beam instruments, the true fan beam densitometers have greater accuracy and precision, shorter scan time, and wider scan field than limited-angle fan beam densitometers which have inherent overlap in acquisition, smaller number of detectors, and poorer image quality [ 3 ]. The three major commercial manufacturers of bone densitometers are GE Medical Systems Inc. (former Lunar), Madison, WI; Hologic Inc., Waltham, MA; and CooperSurgical (former Norland Medical Systems, Inc.), Trumbull, CT. Although each of these companies employs a subtly different technology, our further discussion does not address the particulars of each technology and/or manufacturer. Our focus is the positioning of the overweight patients when obtaining densitometry scans and subsequent analyses of these scans, overlooked in many studies. However, for more information, the main physical characteristics of the most commonly used manufacturer/instruments are presented in Table 1 . DXA is considered one of the most precise technologies in clinical medicine when the measurement of bone mineral density (BMD) is considered, with the typical coefficients of variation between 1–2% [ 4 ]. Nevertheless, there are some limitations in BMD assessment as well. Results of in vitro and in vivo studies indicate different manufacturers, models, software versions and modes of analysis of densitometers can lead to variations in the assessed BMD and bone mineral content (BMC) in the same individuals [ 5 , 6 ]. Laskey et al (2004) found that the GE Lunar Prodigy gave significantly higher BMD, BMC and t-scores compared to the GE Lunar MD in 10 volunteers [ 6 ]. They also found that an increase in tissue depth (as in overweight individuals) caused an increase in the measured BMC and BMD for the MD model but not the Prodigy model, even when using the appropriate and same scan modes [ 6 ]. The prudent way to overcome these flaws would be to use the same instrument and software version throughout a single longitudinal study. The accuracy of DXA instruments for measurement of soft tissue is also questioned due to various methodological limitations. Some of the limitations are addressed in a recent review [ 7 ] and are generally attributed to the hardware (fan- or pencil-beam) or software versions [ 8 ]. DXA instruments from different manufactures are shown to give considerably different soft tissue assessments of the same individual [ 9 ]. Lunar and Hologic are shown to give major differences in measurements of total body and regional body fat in HIV patients (2.4–13.4% higher values for Hologic) and in body fat distribution [ 9 ]. Additionally, individuals' hydration levels may affect calculations for soft tissue [ 7 ] whereas tissue thickness may affect beam magnification, especially if the proper scan mode is not chosen and in cases involving changes in subject's weight [ 10 ]. Also, estimates for soft tissue in regions directly adjacent to the large bony areas such as the trunk, arms and head, may result in decreasing precision. During the total body scans (to obtain body composition analysis) a larger pixel size is utilized and pixels that include smaller portions of bone may be counted as lean tissue [ 10 ]. Despite the above flaws, DXA can still be used for fairly accurate assessment of soft tissue composition or its change [ 7 ], particularly for groups and large-scale epidemiological studies, provided that its limitations are considered and adequately accounted for. However, it has to be noted that DXA technology is not approved by Food and Drug Administration for the individual assessment of body composition. Currently, the most accurate method for measuring body composition is considered to be the four-compartment (4C) model in which fat free body tissue is divided into its constituent parts, namely water, protein and mineral. The 4C model then incorporates independent measurements of mineral, total body water and body density to derive body fat. The 4C model (though not a true gold standard) is often used as a criterion method to compare the accuracy of other methods for assessing body fat. This method however, is costly and time consuming and therefore not generally used in clinical settings. DXA (a two-compartment method) does not measure body water, which limits its accuracy in body composition assessment. However, since DXA offers quick and easy body fat assessment and is considered superior to many other methods, it is often used in clinical settings. Gately et al. [ 11 ] compared various body composition methods for assessing body fat in overweight and obese children. They found air-displacement plethysmography and DXA to be the most promising methods for body fat assessment in a clinical setting [ 11 ]. A study in non-obese women found DXA to be superior to waist circumference and waist-to-hip ratio in predicting intra abdominal fat [ 12 ]. The use of DXA for assessment of body composition in overweight/obese individuals increased recently due to numerous weight reduction studies. While all of the above limitations of bone densitometers have been frequently addressed, the limitations of assessing body composition in overweight individuals due to incorrect positioning and subsequent failures to properly analyze the obtained scans have not received any attention and are the focus of this report. We discuss proper ways of measuring overweight individuals and assessing their soft tissue and point to some studies where that may not have been the case. Discussion Use of bone densitometers in weight loss studies In weight loss studies where DXA is used to evaluate lean and fat tissue, overweight/obese individuals range widely in body weight and size [ 8 , 13 - 20 ]. However, the maximum size of a DXA scanning table is limited to about 193–197 cm length and 58–65 cm width, with weight limitations from 114–159 kg depending on the manufacturer and model, Table 1 . In order to fit an overweight individual within the scanning area, rice bags and straps are used to press the limbs as close to the body as possible [ 2 ]. Despite these measures, some large individuals cannot fit within the global region of the scan area. Additionally, in some cases, the space between the scanning table and the detector is not large enough to accommodate individuals with a larger chest girth, making their measurements difficult or impossible. While some authors do address these limitations when reporting their data [ 14 ], some do not describe or vaguely describe DXA assessment [ 13 , 15 , 17 , 19 ] or are unclear regarding precision of their instruments in overweight individuals [ 15 - 18 , 20 ]. In our own preliminary studies with overweight women using a Lunar pencil-beam densitometer, the coefficients of variation (CV) for different skeletal sites ranged from 0.6–1.8% [ 21 ], but those for the soft tissue were higher reaching 8.2% for fat tissue in the arms (not published). The high %CVs (range 3.1–4.3%) for fat tissue (even in normal-weight individuals) were reported by others using pencil-beam instruments [ 22 ]. Figure 1 shows a total body scan of a 104 kg woman where portions of the arms fell out of the scan area, and therefore, could not be included in the analysis of the total body soft tissue. Furthermore, since her limbs were pressed against the sides of her body, overlap of tissue occurred in the chest, arm and hip regions, resulting in inaccurate regional soft tissue analysis (namely, trunk, legs and arms). Figure 2 presents the proper positioning and analysis of total body composition in a 59 kg woman. It is obvious that the inclusion of subjects who do not fit in the global scan area might lead to questionable accuracy of both total and regional soft tissue estimates. Total body and regional soft tissue assessment When total body soft tissue assessment is the goal, it is necessary to include all parts of the body in the scanning area. In overweight subjects, overlapping of body parts may affect the total results due to increased thickness in overlapping regions. Another source of error is the head, where tissue type cannot be distinguished. Specifically, the brain tissue cannot be measured by DXA due to the surrounding skull – it has to be assumed. Therefore, the assessment of soft tissue in this region is subject to large error and it is suggested the head be excluded from total body soft tissue analysis [ 10 ]. In regional assessment, DXA utilizes the placement of standard cut-lines to assess the arms, legs and trunk (chest, abdomen, pelvis), Figure 1 and 2 . Each regional estimate may be subject to error in overweight individuals (in normal-weight ones too) if overlapping of regions occurred. Wang et al. [ 13 ] measured total and regional body fat with DXA in women (mean ± SD weight, 96 ± 11 kg) before and after weight loss. Since the positioning of the subjects was not described, it is not known whether all subjects fit within the scan area and whether tissue overlap occurred. Similar uncertainty exists in other studies [ 18 , 19 ]. In the newest study by Sun et al. [ 20 ] researchers compared the assessment of total body fat by multi-frequency bioelectrical impedance with DXA measurements as the "gold standard". The subjects in the study ranged in weight from 45 to 157 kg, with body mass index (BMI) ranging from 17 to 55 kg/m 2 , indicating some were severely obese. However, authors did not address the positioning or fitting of the obese subjects on the scanning table, therefore it could only be speculated about the adequacy of these measurements/analyses. Researchers have found estimates of abdominal fat tissue by DXA to be similar to computed tomography (CT) and MRI-derived measurements in normal and overweight individuals of wide age range, indicating DXA can accurately estimate abdominal fat [ 24 - 26 ]. The abdominal region is not a routinely defined region by DXA software and therefore, must be manually determined (see Figure 1 ), which can differ among research sites. Park et al. [ 24 ] compared abdominal adipose tissue measured by MRI and DXA in non-obese men. They defined DXA regions of interest in two different ways (between the second-lumbar vertebra and the fourth-lumbar vertebra, or iliac crest) and found both of these regions comparable with MRI total abdominal adiposity and with MRI-derived narrow abdominal slices. Bertin et al. [ 26 ] reported DXA yielded accurate measurements of abdominal adipose tissue compared with CT in overweight/obese individuals weighing 66–134 kg. They manually defined DXA abdominal region to range from the acromion to the iliac crest, a slightly different placement than the ones described above, and compared it to a 10 mm region at the fourth-lumbar vertebrae measured by CT. It is important to note that the abdominal regions of interest could be subject to potential error if the upper limbs are positioned in too close contact with the trunk, causing the overlap of the regions. Researchers have also found estimates of different regions of leg soft tissue extracted from the total DXA scans to be reliable in elderly subjects of wide weight range [ 8 , 26 , 27 ]. Similarly, fat tissue of the thigh determined by DXA was comparable to CT derived measurements in normal and overweight individuals. Tylavsky et al. [ 8 ] compared CT derived measurements of lean and fat tissue with DXA measurements of a manually defined sub-region of the mid-thigh (one-half the distance between the knee joint and the top of the femur, see Figure 1 ). They indicated a good assessment of soft tissue change by DXA in that region. It therefore appears that with large individuals, DXA should be used for assessing body composition of defined regions such as the mid-thigh or abdominal rather than the total body. Half-body DXA scans for the assessment of soft tissue Tataranni and Ravussin [ 28 ] suggest measuring soft tissue of obese individuals by scanning only one side of the body. They found total body composition results from right and left sides only differed minimally in both overweight and normal-weight individuals. The half-body scan can be performed by placing the central line of the scanning area through the midpoint of the left or right collar bone for each half-body scan. During analysis of the half-body scans, the central line is then repositioned on each half scan, and the side of the body that was not completely included in the scan area is deleted. The authors found that small errors in estimates of soft tissue can occur from imperfect positioning of the central line by the operator or by true anatomical differences between the left and right sides of the body. To minimize these errors, they suggest fat tissue be determined by multiplying percent body fat from the half-body scan by body weight, and lean tissue be determined as the difference between body weight and estimated fat tissue. Another possibility for improving accuracy of soft tissue assessment would be measuring both halves of the body, and then adding them up. However, more research on the above methods is necessary in order to make recommendations Total body bone mineral assessment in overweight individuals Similarly to problems with soft tissue assessment, there are problems with bone mass assessment when DXA is used in overweight individuals. When an individual does not fit within the scan region, there is subsequent loss of soft and bone tissue. Additionally, some anomalies in bone mass measurement during weight loss studies using different instruments were reported earlier [ 29 ]. Tothill et al. [ 29 ], re-analyzed published results of changes in total body bone mineral during weight change. The authors found weight change leads to considerable anomalies in measuring changes in bone mineral in all three brands of DXA machines (Hologic, Lunar and Norland), with the most serious ones occurring with Hologic [ 29 ]. These inaccuracies were suspected to be due to the use of different software modes (enhanced vs. standard) and the different assumptions manufactures make regarding fat distribution [ 29 ]. Phantom studies using Lunar and Hologic fan beam scanners showed bone mass measurements were not compromised by magnification effects, however, the height of bone and changes in body weight simulated with lard did affect the accuracy of BMD and BMC measurements [ 30 ]. Tothill and Hannan [ 30 ] compared Lunar and Hologic DXA fan bean scanners for measuring total body bone and soft tissue. Phantom measurements revealed that both fan beam instruments were subject to minor magnification effects, and measurements of BMD and BMC were both dependent on the height of a bone [ 30 ]. Summary Current bone densitometers are limited to a scanning area that cannot accommodate some overweight/obese individuals. Newer fan-beam densitometers have a wider scan field [ 3 ] or can accommodate individuals up to 159 kg, Table 1 , making them a better option for body composition assessment. Unless researchers are using some of the newer densitometers (with a scan table large enough to accommodate larger body sizes) they may need to rely on estimates of regional fat, namely the thigh or abdominal region when assessing the body composition of many overweight subjects. Using one or two half-body scans may provide a relatively accurate measurement of total body fat, however, more studies using this technique are needed. The results of some published studies in overweight/obese individuals need to be interpreted with caution, since they may have included subjects who could not properly fit within the scan area. Researchers using bone densitometers for body composition measurements need to have an understanding of its limitations in overweight individuals and appropriately address the stated concerns when interpreting their results. Authors also need to provide details of their DXA instrument including the manufacturer, the software version and the analysis mode used for body composition assessment when reporting their results. Studies on accuracy and precision in measurements of both bone and soft tissue composition in overweight individuals, using available densitometers, are warranted and needed. Competing interests The author(s) declare that they have no competing interests. Authors' contributions RAB wrote article drafts and evaluated the cited literature. JZI conceptualized the idea and the design of the article, revised the manuscript and completed the final version. Both authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079848

In vivo studies of genomic packaging in the dsRNA bacteriophage Φ8

Background Φ8 is a bacteriophage containing a genome of three segments of double-stranded RNA inside a polyhedral capsid enveloped in a lipid-containing membrane. Plus strand RNA binds and is packaged by empty procapsids. Whereas Φ6, another member of the Cystoviridae, shows high stringency, serial dependence and precision in its genomic packaging in vitro and in vivo , Φ8 packaging is more flexible. Unique sequences ( pac ) near the 5' ends of plus strands are necessary and sufficient for Φ6 genomic packaging and the RNA binding sites are located on P1, the major structural protein of the procapsid. Results In this paper the boundaries of the Φ8 pac sequences have been explored by testing the in vivo packaging efficacy of transcripts containing deletions or changes in the RNA sequences. The pac sequences have been localized to the 5' untranslated regions of the viral transcripts. Major changes in the pac sequences are either tolerated or ameliorated by suppressor mutations in the RNA sequence. Changes in the genomic packaging program can be established as a result of mutations in P1, the major structural protein of the procapsid and the determinant of RNA binding specificity. Conclusion Although Φ8 is distantly related to bacteriophage Φ6, and does not show sequence similarity, it has a similar genomic packaging program. This program, however, is less stringent than that of Φ6.

Background The Cystoviridae are a family of bacteriophages having genomes consisting of three segments of double-stranded RNA (dsRNA). The RNA is contained within a polyhedral capsid which is enveloped in a lipid-containing membrane [ 1 ]. Φ6 was the first and the most thoroughly studied member of this family. The packaging of the Φ6 genome was found to progress through a program that involves the sequential packaging of the plus strands of segments S, M and L in that order [ 2 ]. This program is rather stringent for Φ6 in that segment S can be packaged alone while M requires prior packaging of S and L requires prior packaging of M. Segment L can be packaged to some extent with only prior packaging of S but this is much less efficient than the normal packaging [ 3 , 4 ]. The Cystoviridae are the only family of RNA viruses that are reliably known to package their genomic segments into preformed capsids. Although the Reoviridae have an inner core particle structure strikingly similar to that of the Cystoviridae, their mechanisms of genomic packaging have not yet been determined [ 5 ]. Packaging in the Cystoviridae is driven by an NTPase motor comprised of a hexamer of protein P4 [ 6 - 8 ]. The plus strands of Φ6 have an 18 base consensus sequence at the 5' end and at about 50 bases downstream, a sequence of about 200 nucleotides that is necessary and sufficient for packaging into procapsids. The 200 nucleotide sequence is called pac and it is unique for each segment, with very limited identity in sequence between the three segments. Minus strand synthesis begins after the completion of the plus strand packaging and plus strand synthesis begins after minus strand synthesis is completed. A model has been proposed that involves the programmed changes in the binding sites for plus strands on the surface of the procapsid as a function of the amount of RNA in the particle [ 2 ]. The pac sequences of Φ6 were defined by deletion analysis of the plus strand transcripts of plasmids carrying cDNA copies of the genomic segments. In vitro packaging was used to assess the success of packaging of transcripts harboring a series of deletions. It was found that the pac sequences ended about 50 nucleotides before the first open reading frames (orfs) of the segments [ 9 ]. The pac sequences showed considerable secondary structure, with a number of stem-loops [ 10 ]. Comparison of the pac sequences of close relatives of Φ6 suggested that the stem-loop structures were important because base changes preserved the stems in a number of cases [ 2 ]. Φ8 is the most distantly related member of the Cystoviridae relative to Φ6. It has no sequence identity or similarity to Φ6 and it has significant differences in genetic and physical structure [ 11 , 12 ]. Our aim was to determine whether Φ8 genomic packaging uses pac sequences in a manner similar to that found for Φ6. Whereas the pac sequences were defined in Φ6 by in vitro packaging studies, we have found that in vitro packaging for Φ8 is non-specific when evaluated by the uptake of radioactive RNA [ 12 ]. We have therefore used in vivo transcript acquisition to define the pac sequences of Φ8 in this report. Results The determination of the pac sequences of Φ8 The pac sequences of Φ6 were determined by deletion analysis [ 9 ]. Plus strand transcripts of cDNA plasmids were prepared with deletions near the 5' ends of particular genomic segments. These RNA molecules were incubated with procapsids and the two other wild type transcripts. Minus strand synthesis was then used as the measure of packaging efficiency, since minus strand synthesis was found to be dependent upon packaging of the three genomic segments. It was found that the pac sequences ended about 50 nucleotides before the first open reading frames (orfs). These orf positions were at nucleotides 305, 367 and 270 for segments S, M and L respectively. In the case of Φ8 the first reading frames in segments S, M and L begin at nucleotides 188, 263 and 253 respectively [ 11 ](Fig. 1 ). This would put the ends of the pac sequences at about 140, 210 and 200 for S, M and L if the same relationship to orf s is operating in Φ8 as in Φ6. We have done deletion analysis on segments S, M and L and measured the efficacy of plasmid transcript acquisition into infectious phage as a measure of packaging success. Plasmid transcript acquisition is effected by the introduction of non replicating cDNA plasmids into cells by electroporation. The transcripts of the plasmids produce live phage if they are packaged and if they contain all of the necessary genes. If a particular transcript lacks a functional pac sequence, it will not be packaged and live phage will not be produced. With normal wild type transcripts one finds thousands of infectious centers produced. We found that the pac sequences are located in the 5' untranslated region of the plus strands but that they are closer to the first orfs than found for Φ6. Whereas many of the small changes in the pac sequences of Φ6 resulted in diminutions of the order of one hundred fold in acquisition frequency [ 13 ], changes of this type in Φ8 generally led to changes of less than ten fold (Table 3 ). Segment L A deletion of nucleotides 244 to 328 in L does not interfere with packaging (Table 1 , plasmid 3316). Although this deletion involves gene 14, the gene product is not required for plaque formation. Deletions starting at nucleotide 219 or earlier compromise packaging severely or completely (Table 1 ). Deletions from 329 to 745 are also tolerated if gene Hb is complemented in trans (not shown). The downstream end of the pac sequence is consequently located in the region between nucleotides 219 and 244 and therefore between 9 and 34 bases upstream of the gene 14 orf. Segment M A deletion of 196 to 215 in M compromises segment acquisition; and deletion of the BamHI site at 215 compromises acquisition (Table 2 , plasmid 3324). Creation of a HindIII site at position 238 does not reduce acquisition (Table 2 , plasmid 3348), but deletion of this site does (Table 2 , plasmid 3356). A deletion of bases 34 to 39 also compromises viability in M (Table 2 , plasmid 3043). Therefore the downstream end of the pac sequence of M is located in the region of nucleotide 242 which places it either inside of gene 10 or close to it. Segment S In the case of segment S we find that a deletion of nucleotides from 137 to 158 has little effect on the formation of viable phage (Table 3 , plasmid 2816). The deletion of 137 to 158 is interesting in that the stem at F (Fig. 2 ) is recreated and the loop, nucleotides 160 to 166, is maintained in the deletion structure (Fig. 3 ). Base changes and deletions in the loops of B, C, D and E do not show great diminution of packaging. Larger deletions such as 75 to 158 (Table 3 , plasmid 2820) or 120 to 158 (Table 3 , plasmid 2817) completely abolished plaque formation. This would place the pac region somewhere between nucleotides 40 and 139, which would make the pac region in Φ8 S about half the size of the corresponding region in Φ6. But an examination of the consequences of base changes in the exterior of this region suggests a somewhat more complicated picture. The region between nucleotides 120 and 139 is very important; but changes in the region from 160 to 170 also are significant, suggesting that the pac region extends from about 40 to 170. Although loop F at position 160 to 166 is maintained in the deletion of 137 to 158, the sequence of the loop can be changed (Table 3 , plasmid 2939) without compromising packaging. However changing bases 159 and 160 (Table 3 , plasmid 2955) prevents packaging. This suggests that the stem or the stem-loop junction may be more important than the loop. There is a stem loop structure from 125 to 147 with the loop UAG at 135 (Fig. 2 ). Changing the UAG to CUU has little effect (Table 3 , plasmid 2914). There are two U's that are unpaired in the stem and changing them so that they pair results in no change in packaging. But other changes in the stem result in poor packaging. It appears that the stems are more sensitive than the loops in the pac region in terms of interference with packaging. The downstream end of the pac sequence is again very close to, or even within, the first orf, which in this case is gene 8 (Fig. 1 ). The consequences of replacing the pac sequence of M with that of L Base changes within the pac regions of the Φ6 genome have resulted in the isolation of suppressor mutations in protein P1, the major structural protein of the procapsid and the determinant of the RNA binding sites. However, in the case of Φ8 we have found less stringency in the pac sequences and we have not found suppressor mutations in P1. In the case of Φ6 it was found that suppressor mutations in P1 could also be isolated when conditions were established that were contrary to the packaging program [ 14 ]. Packaging of segments M and L without segment S could be established in carrier states if suppressor mutations were promoted in gene 1 [ 14 ]. We had previously found that gene 8 was indispensable in Φ8, thereby making it difficult to establish carrier states in the absence of segment S. We therefore set out to select for a condition where the M segment would have the pac sequence of L and that phage would be selected for with both the M and L segments having the same pac sequences. We therefore set out to construct a plasmid with a cDNA copy of segment M but with the pac sequence of segment L. A plasmid was prepared with the cDNA copy of segment M. It was cut with BglII and XhoI which removed the first 191 nucleotides and replaced with BglII/SmaI fragment of a plasmid with a cDNA copy of L. This places the first 371 nucleotides of L at the 5' end of segment M. This plasmid is called pLM3018. Electroporation of this plasmid along with plasmids containing normal copies of L and S into cells carrying plasmids expressing SP6 RNA polymerase resulted in only one plaque as opposed to tens of thousands with normal wild type constructs (Table 4 ). The RNA of the resulting viable phage was analyzed and it was found that the pac sequence of L remained on segment M. Gene 1 on segment L was cloned as cDNA and inserted into a copy of normal segment L. The electroporation experiment was repeated with the original plasmids or with the normal S, the M segment with the L pac sequence and the new L plasmid containing the replacement of wild type gene 1 with that of the presumed mutant gene 1. The original combination gave no plaques, while the combination with the new gene 1 gave thousands of plaques as did the new L plasmid with normal M and S (Table 4 ). The yield from the latter test was ten times that of the electroporation with the chimeric M. Sequencing of gene 1 showed that a single change at U4807C had occurred. Reisolation of the small fragment between sites at 4030 and 4850 confirmed that this mutation was responsible for the ability to package the chimeric M. The amino acid change generated in protein P1 by the mutation is V242A. In Φ6, all suppressor mutations involved in packaging have been in protein P1, which is the major structural protein of the procapsid and the source of the specific binding sites for the plus strands of the genomic segments. Discussion Plus strands of the three genomic segments of bacteriophage Φ6 are packaged by core structures composed of proteins P1, P2, P4 and P7 [ 2 ]. They bind to protein P1, the major structural protein of the core and are translocated to the interior by hexamers of the NTPase P4. We had worked out the identification of sequences necessary and sufficient for packaging in bacteriophage Φ6 by in vitro packaging experiments. A region near the 5' end designated as pac is necessary and sufficient for packaging. We have established that the pac sequences of Φ6 are located in the 5' untranslated regions of the plus strands. Whereas the in vitro packaging of Φ6 is very stringent and specific, that of Φ8 was found to be non-specific when uptake of radioactive RNA was being assayed. In vivo packaging was more specific [ 12 ]. We therefore established a system of in vivo transcript acquisition to test the sequence limits for the packaging of the Φ8 genome. In this report, we have identified the pac regions for bacteriophage Φ8, a distant relative of Φ6. All three plus strands of Φ8 have a consensus sequence of 9 nucleotides at the 5' end. Again, we have found that the pac regions occupy space in the 5' untranslated regions of the plus strands. Although the pac regions of both Φ6 and Φ8 have many stem-loop structures, there is no sequence or structural similarity between the two sets. There is also no sequence similarity between the pac sequences of the three genomic segments of Φ8. While the Φ6 pac sequences extend approximately to nucleotides 230, 305 and 270 for L, M and S respectively; those of Φ8 extend to near nucleotides 244, 242 and 170. The Φ8 pac sequences end closer to the first orfs than those of Φ6. In the cases of segments S and M the pac sequences might overlap the first orfs. Changes within the pac region of Φ8 segment S are less destructive of packaging than similar changes in pac regions of Φ6. Many changes are tolerated, and in some cases the effects of changes are suppressed by other changes in the pac sequences. In some cases it is possible to rationalize the suppression in terms of the structure shown by mfold [ 15 ]; in other cases it is not clear why the suppression is effective. In Φ6, we found little suppression due to changes in the pac sequences but we found suppressors by mutation in the sequence of P1, the major structural protein of the core. Genomic RNA that is bound to procapsids can be cross-linked to protein P1 [ 16 ]. So far, we have not found suppressors of this type in Φ8 for changes in the pac sequences. However, we did obtain a suppressor mutation for another type of packaging program transgression. We found that we could exchange the pac sequence of L with that of M. The L segment could be packaged in this case if an amino acid change was present in P1. The experiments involving manipulation of the pac sequence of segment S indicate that the Φ8 packaging mechanism has a lower level of stringency than that of Φ6 [ 13 ]. Is there a benefit of low stringency in packaging for a viral system? It appears that the answer is yes in many cases. Many bacteriophages with DNA genomes are able to package host or plasmid DNA and consequently carry out transduction of host genes [ 17 ]. Low stringency offers the possiblility of the acquisition of host RNA transcripts by RNA viruses. We have seen that Φ6 can acquire plasmid transcripts that do not have pac sequences, albeit at extremely low frequencies [ 18 ]. It also allows the acquisition of genomic segments of distantly related phages. Although Φ6 cannot acquire the genomic segments of Φ13 a related cystovirus, Φ13 can acquire the S and M segments of Φ6 [ 19 ]. Influenza virus is now believed to have specific packaging of its genomic segments [ 20 ]. However the packaging is of low enough stringency that virions can acquire genomic segments from viruses that infect very different host organisms [ 21 ]. Conclusion The sequences that are necessary for packaging transcripts of the dsRNA genome of bacteriophage Φ8 are located in the 5' untranslated regions of the three genomic segments. Genomic packaging in Φ8 is much less stringent than that found for the distantly related bacteriophage Φ6. Many changes in the pac region of S are tolerated while others are suppressed by compensating changes in the RNA sequence. Changes in the packaging program can be produced by mutations that change the amino acid sequence of P1, the major structural protein of the procapsid. Methods Bacterial strains and plasmids Pseudomonas syringae HB10Y derivative LM2489 was the host for Φ8 [ 1 , 19 ]. Escherichia coli strain JM109 ( recA1, endA1, gyrA96, thi, hsdR17, supE44, relA1, λ-, Δ(lac-proAB) ), [F', traD36, proAB, lacIq, ΔM15 ] [ 22 ] was used for the propagation of all plasmids. Epicurian Super Competent cells of E. coli (Stratagene) were used for transformation after directed mutagenesis. Plasmids utilized in this study are listed in tables 1 , 2 and 3 . All cDNA plasmids used in this study have a SP6 promoter upstream of the cDNA sequences. The SP6 promoter is preceded by a BglII site. The sequences of the Φ8 genomic segments are in GenBank with accession numbers NC_003299, NC_003300 and NC_003299 for L, M and S respectively. Reverse genetics for the modification of the viral genome The simplest means of virus construction is to electroporate host cells with SP6 promoter plasmids that contain cDNA copies of the genomic segments along with a plasmid that codes for SP6 RNA polymerase. We generally add about 200 ng of each cDNA plasmid to about 2 × 10 9 cells. Using colE1 based plasmids such as pT7T319U, that transcribe but do not replicate in pseudomonads we can produce hundreds of plaques. If the polymerase plasmid is resident in the host strain, we can produce tens of thousands of infectious centers with wild type constructions. Plasmid pLM2790 is a derivative of shuttle vector pLM254 with SP6 polymerase gene of PSR3 [ 23 ] cloned into the BamH1 site [ 24 ]. In cases where we alter the pac sequences, the number of plaques can be reduced drastically. In some cases, the resulting phage contain suppressor mutations or reversions of the directed mutations. Although the 5' sequence of the Φ8 plus strands, GAAA, is more compatible with the specificity of SP6 polymerase, it is possible to use T7 RNA polymerase as well, although with somewhat lower efficacy. If one of the genomic segments has a gene for kanamycin resistance, kan , inserted into either its non coding region or replacing one or more genes, then electroporation can result in the establishment of a carrier state in which the viral genome is replicated in cells without lysis and with the expression of the drug resistance so as to form stable drug resistant colonies [ 14 ]. Production of deletions in the pac sequences of the Φ8 segments In some cases, deletions could be made by simply ligating the products of restriction enzyme cuts at unique sites. In other cases, we used oligos that contained a Pmel site that occurs uniquely in S at 160 and a jump to a vector sequence to PCR on cDNA copies of segment S. These pieces were ligated to Pmel and EcoRI in the vector to form the deletions. To make smaller deletions or base changes we used the Quick Change protocol of Stratagene (La Jolla, CA) with oligos listed in Table 5 along with their complements, which involves PCR on a plasmid template and subsequent treatment with DpnI to eliminate the template DNA. Changes in segment L usually involved PCR with linkage to the NsiI site at position 328. Changes in segment M involved deletions at restriction sites. The placing of the pac sequence of L on segment M The BglII / XhoI fragment of plasmid pLM2669 contains the pac sequence of Φ8M as well as the SP6 promoter. This fragment was exchanged with the BglII /SmaI fragment of plasmid pLM2653 which carries the cDNA copy of Φ8L (Fig. 1 ). RTPCR dsRNA was isolated from crude cell lysates by first treating with DNase, then phenol extraction and electroelution of the RNA from agarose gels. The RNA was then denatured and annealed to primer and incubated with AMV reverse transcriptase. The products were then subjected to PCR with Pfu turbo DNA polymerase (Stratagene) [ 19 ]. The resulting DNA products were cloned into pT7T319U and sequenced at the Molecular Resources Facility of the University of Medicine and Dentistry of New Jersey. Mfolds of segment S pac sequences The secondary structures of the pac sequences were determined on the mfold web server using the mfold 3.1 program of Zuker and Turner [ 15 ]. The folds were constrained so that the maximum distance between paired bases was 50. The mfold structural predictions for the Φ6 pac sequences were previously found to be similar to those found by nuclease sensitivity [ 10 ]. The structures shown in Figures 2 and 3 are not unique in that there are several structures with similar free energy values. However, stem-loop F in Figure 2 is present in all of them. Authors' contributions Both JQ and XQ devised, carried out and analyzed the directed changes in the sequences of the viral RNA molecules. LM conceived the project and drafted the manuscript. All authors read and approved the final manuscript.

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1079849

Proposal to create subspecies of Rickettsia conorii based on multi-locus sequence typing and an emended description of Rickettsia conorii

Background Rickettsiae closely related to the Malish strain, the reference Rickettsia conorii strain, include Indian tick typhus rickettsia (ITTR), Israeli spotted fever rickettsia (ISFR), and Astrakhan fever rickettsia (AFR). Although closely related genotypically, they are distinct serotypically. Using multilocus sequence typing (MLST), we have recently found that distinct serotypes may not always represent distinct species within the Rickettsia genus. We investigated the possibility of classifying rickettsiae closely related to R. conorii as R. conorii subspecies as proposed by the ad hoc committee on reconciliation of approaches to bacterial systematics. For this, we first estimated their genotypic variability by using MLST including the sequencing of 5 genes, of 31 rickettsial isolates closely related to R. conorii strain Malish, 1 ITTR isolate, 2 isolates and 3 tick amplicons of AFR, and 2 ISFR isolates. Then, we selected a representative of each MLST genotype and used multi-spacer typing (MST) and mouse serotyping to estimate their degree of taxonomic relatedness. Results Among the 39 isolates or tick amplicons studied, four MLST genotypes were identified: i) the Malish type; ii) the ITTR type; iii) the AFR type; and iv) the ISFR type. Among these four MLST genotypes, the pairwise similarity in nucleotide sequence varied from 99.8 to 100%, 99.4 to 100%, 98.2 to 99.8%, 98.4 to 99.8%, and 99.2 to 99.9% for 16S rDNA, glt A, omp A, omp B, and sca 4 genes, respectively. Representatives of the 4 MLST types were also classified within four types using MST genotyping as well as mouse serotyping. Conclusion Although homogeneous genotypically, strains within the R. conorii species show MST genotypic, serotypic, and epidemio-clinical dissimilarities. We, therefore, propose to modify the nomenclature of the R. conorii species through the creation of subspecies. We propose the names R. conorii subsp. conorii subsp. nov. (type strain = Malish, ATCC VR-613), R. conorii subspecies indica subsp. nov. (type strain = ATCC VR-597), R. conorii subspecies caspia subsp. nov. (type strain = A-167), and R. conorii subspecies israelensis subsp. nov. (type strain = ISTT CDC1). The description of R. conorii is emended to accomodate the four subspecies.

Background Taxonomic classification of members of the order Rickettsiales was originally based on relatively few phenotypic criteria [ 1 ]. Over the past twenty years, the taxonomic classification of the Order Rickettsiales has undergone many changes. On the basis of 16S rDNA sequences, Coxiella burnetii was reclassified within the Legionellaceae [ 2 ], Eperythrozoon sp. within the Mycoplasmataceae [ 3 ], and Rochalimaea sp., Grahamella sp., and Bartonella sp. within the Bartonellaceae , closely related to Brucella sp [ 4 , 5 ]. Among the remaining members of the order Rickettsiales , the Anaplasma-Ehrlichia clade was recently reorganized into 4 genera [ 6 ]; and R. tsutsugamushi was reclassified into a new genus, Orientia [ 7 ]. Within the genus Rickettsia , since the pioneering work of Philip in 1978 [ 8 ], two rickettsial strains were considered as having different serotypes if they exhibited a specificity difference of ≥ 3 and some authors have considered this to be a useful guide to speciation [ 9 , 10 ]. However, opinions divide as to whether this holds true for rickettsial strains related to Rickettsia conorii ( R. conorii ) [ 11 ]. These include Indian tick typhus rickettsia (ITTR) [ 12 ], Israeli spotted fever rickettsia (ISFR) [ 13 - 15 ] and Astrakhan fever rickettsia (AFR) [ 16 - 19 ]. Phylogenetically, these rickettsiae constitute a homogeneous cluster supported by significant bootstrap values and are distinct from other Rickettsia species (Figure 1 ) [ 20 - 22 ]. Some authors have considered these rickettsial strains as different species and proposed the names R. sharonii and R. caspii for ISFR and AFR, respectively [ 23 , 24 ]. Other authors believe that they belong to a " R. conorii complex" [ 11 , 14 , 15 , 17 , 19 ]. These rickettsiae closely related to R. conorii have been described as the causative agents of Mediterranean spotted fever, Astrakhan fever, Israeli spotted fever, and Indian tick typhus in the Mediterranean basin and Africa, Southern Russia, Middle East, and India, respectively. These rickettsioses are transmitted to humans by Rhipicephalus ticks [ 1 ]. Furthermore, most of their clinical features overlap and are characterized by a febrile illness and a generalized maculopapular rash. However, an inoculation eschar is seldom present in Astrakhan fever and Israeli spotted fever but common in Mediterranean spotted fever (Table 1 ). Recently, we have proposed gene sequence-based criteria for the identification of Rickettsia isolates at the genus, group and species levels and proposed criteria to define species [ 9 ]. Using these criteria, we have shown that ITTR, ISFR and AFR could not be considered as distinct species. Instead, they belonged to the R. conorii species [ 9 ]. However, these rickettsiae exhibit differentiable serotypes and cause diseases with distinct clinical features in defined geographic locations. The ad hoc committee on reconciliation of approaches to bacterial systematics [ 25 ] has proposed that even if related genetically, bacterial isolates within a given species could be considered as distinct subspecies if they differed phenotypically. In this study, we undertook to examine whether phenotypic differences existed among various R. conorii isolates that would enable to classify them as subspecies. We estimated the degree of genotypic variabilities among 31 isolates of R. conorii , 1 isolate of ITTR, 2 isolates and 3 tick amplicons of AFR, and 2 isolates of ISFR using multi-locus sequence typing (MLST). We incorporated 16S rDNA and glt A genes, as well as 3 membrane-exposed protein-encoding genes omp A, omp B and sca 4 (formerly gene D) [ 26 , 27 ] in MLST. To further characterize the specificities of distinct MLST types, we incorporated a prototype isolate from each of these into a multi-spacer typing (MST) assay, which we have previously demonstrated to be more discriminant than MLST at the strain level for R. conorii [ 28 ]. Furthermore, mouse serotypes were obtained for each of these MLST types. Results MLST-gene sequencing All five genes studied were amplified from all 36 studied isolates and from 3 of 57 Rhipicephalus pumilio ticks tested. All negative controls remained negative. Four MLST types were identified. Within the first type, which we named Malish type, all R. conorii isolates exhibited identical sequences for each of the five genes tested except isolates Moroccan and M1. These latter isolates showed deletions of 24 and 156 bp, respectively, at the 5'-end of the omp A gene. The second type, the ITTR type, was made of ITTR. This exhibited unique nucleotide substitutions in omp A, omp B and sca 4 genes. Within the AFR type, all isolates and tick amplicons tested showed identical sequences of the five genes studied except the Chad isolate which showed subtle nucleotide substitutions in each of them. In this isolate, the pairwise nucleotide similarity with AFR for 16S rDNA, glt A, omp A, omp B and sca 4 genes was 99.7, 99.5, 99.5, 99.2, and 99.8 %, respectively. The fourth type, ISFR type, which included both ISFR isolates, showed the same nucleotide substitutions in each of the five genes tested. This differentiated them from other rickettsiae. As representative strains for each MLST genotype, we have chosen R. conorii isolate Malish, ITTR isolate Indian, AFR isolate A-167, and ISFR isolate ISTT CDC1. Among representatives of the four MLST genotypes, the degree of pairwise nucleotide similarity ranged from 99.8 % between AFR and ISFR to 100 % between R. conorii and ITTR for the 16S rDNA gene (Table 3 ). For glt A, such similarity ranged from 99.5 % between Malish (or ITTR) and ISFR to 100 % between R. conorii and ITTR. For the 5' end of omp A, the degree of pairwise sequence similarity ranged from 98.1 between AFR and ITTR to 99.8% between R. conorii and ITTR. This pairwise similarity ranged from 98.5 between ITTR and ISFR to 99.8% between R. conorii and ITTR for omp B. For sca 4, the pairwise sequence similarity varied from 99.2 between ITTR and ISFR to 99.9% between R. conorii and ITTR. The dendrograms obtained using the three different tree building analysis methods showed similar organization for the five rickettsiae studied. We identified two monophyletic groups with the trees so constructed. One included R. conorii and ITTR and the other, ISFR and AFR (Figure 2A ). The clustering of these rickettsiae was supported by high bootstrap values. Both AFR isolates were grouped together for each of the five genes tested. MST genotyping ITTR showed unique 232-bp dks A / xer C (type P, GenBank accession number AY836513) and 153-bp mpp A / pur C (type F, AY836517) spacer sequences. However, it also showed a 200-bp rpm E / tRNA-fMet spacer sequence identical to that of R. conorii type B (AY345092); ISFR had unique 232-bp dks A / xer C (type Q, AY836510), 153-bp mpp A / pur C (type G, AY836514), and 200-bp rpm E / tRNA-fMet (type C, AY836518) spacer sequences; AFR isolates A-167 and Chad showed unique dks A / xer C of 232- (type R, AY836511) and 167-bp (type S, AY836512), respectively. They also showed identical 153-bp mpp A / pur C (type H, AY836516) and 200-bp rpm E / tRNA-fMet (type D, AY836519) spacer sequences that were different from those of other strains. When combining the genotypes obtained from each of the three spacers into a MST genotype, each of the four rickettsiae showed a distinct MST genotype. The MST genotype so obtained was different from that of R. conorii . However, on pairwise comparisons of the four strains, both AFR strains showed similar spacer types. The phylogenetic tree inferred from the concatenated spacer sequences showed an organization similar to that obtained from MLST when Malish strain was taken as R. conorii type strain (Figure 2B ). However, the node where ISFR branched was not supported by a significant bootstrap value. Microimmunofluorescence (MIF) serotyping When injected into mice, all five rickettsiae tested provoked antibody responses that were detectable by MIF. The endpoint titers and specificity differences (SPDs) were calculated as described in the Methods section. These values are presented in Table 4 . The antibody titres obtained in homologous sera were at least one-fold higher than those observed in heterologous sera. The SPDs for heterologous sera varied between 1 and 5. Four serotypes were identified. These included R. conorii , ITTR, both AFR isolates, and ISFR. The dendrogram inferred from the matrix based on SPDs (Figure 2C ) showed that ITTR occupied a position that slightly differed from its phylogenetic organization. Instead of forming a cluster with R. conorii , it occupied an intermediate position between R. conorii and ISFR (Figures 2A and 2B ). Discussion We propose to create subspecies within the R. conorii species to accommodate both the genotypic homogeneity and the serotypic, MST genotypic, geographic and pathogenic diversities of rickettsia strains within this species. Defining a species within the Rickettsia genus has long been difficult but rickettsiologists have agreed to use mouse serotyping as one criterion [ 8 ]. This classification scheme served as a basis for the development of genotypic criteria at the species level [ 9 ]. However, confusion still exists about the taxonomic status of rickettsiae. This is especially the case for rickettsiae which are closely related to classic R. conorii isolates. Philip et al . [ 8 ], using mouse serotyping, have demonstrated that R. conorii isolates Malish, Moroccan and Kenya were closely related antigenically to ITTR and that they belonged to a single serotype. Using complement fixation, Bozeman et al . [ 29 ] were unable to distinguish ITTR from R. conorii isolates. In contrast, others, using mouse polyclonal antibodies [ 14 ], and monoclonal antibodies [ 11 ], have demonstrated substantial differences between ITTR and R. conorii isolates. ISFR and AFR could not be distinguished using PCR-RFLP [ 17 ] until omp A was used as target gene [ 30 ]. In 1993, we have demonstrated that AFR differed from both ISFR and R. conorii in terms of SDS-PAGE mobility and PFGE profiles [ 19 ]. Although they are difficult to distinguish serologically, the cross-reacting epitopes they share are located on antigenic proteins that differ in SDS-PAGE mobility and hence in molecular weight [ 19 ]. In 1995, Walker et al. , using serotyping, Western bloting, monoclonal antibody reactivity and PCR amplification of the tandem repeats within omp A, concluded that ISFR belonged to the R. conorii species [ 15 ]. In 1998, Dasch and Jackson differentiated R. conorii from ISFR using PCR-RFLP [ 23 ]. Recently, using a combination of genotypic criteria, we have demonstrated that the genotypic differences among ITTR, AFR and ISFR were too subtle to classify them as new species [ 9 ]. Instead, they belonged to the R. conorii species. In the present study, we have demonstrated, using MLST, that rickettsiae closely related to R. conorii isolate Malish are very homogeneous. Except isolates Moroccan and M1 which showed one omp A deletion of 24 and 156 bp, respectively, all studied R. conorii isolates had identical sequences for the five genes studied and thus constituted a reliable taxon. The ITTR isolate showed unique sequences of omp A, omp B and sca 4 genes. Among isolates and tick amplicons of AFR, the degree of MLST sequence similarity was of 100% except for the Chad isolate which is genotypically slightly different from AFR isolate A-167 [ 31 ]. In another study, we have found identical omp A sequences in four tick amplicons from Kosovo and AFR isolate A-167 [ 32 ]. We have also observed serologic, antigenic and genetic homogeneities among two tick and one human AFR isolates from Astrakhan [ 19 ]. Thus, except the Chad isolate which was slightly different genotypically, AFR, with three strains and ten tick amplicons studied, appeared to form a homogeneous taxon. The two isolates of ISFR tested showed 100% pairwise nucleotide similarity in all five genes studied. As we have compared only two isolates, one may argue that this may not be representative of the diversity of omp A in ISFR. However, Walker et al . found four human ISFR isolates from Israel to be highly homogeneous antigenically as well as genetically [ 15 ]. Bacellar et al. described three human isolates of ISFR from Portugal that showed 100% similarity in 16S rDNA, glt A and omp A sequence with those of ISFR isolate ISTTCDC1 [ 33 ]. Giammanco et al . identified a tick rickettsial isolate in Italy that had 100% omp A sequence similarity with ISFR isolate ISTTCDC1 [ 34 ]. Therefore, it appeared that our ISFR isolates were representatives of the same homogeneous taxon. In the first part of the study, we have identified four MLST genotypes which were subjected to further evaluation. Among representatives of these MLST genotypes, the minimal pairwise similarity in the nucleotide sequences of 16S rDNA, glt A, omp A, omp B, and sca 4 genes between the rickettsial isolates tested and R. conorii , was 99.8%, 99.4%, 98.3%, 98.5%, and 99.3%, respectively. Thus, All four isolates tested fulfilled the criteria to belong to the R. conorii species [ 9 ]. Phylogenetically, the members of the R. conorii complex have been demonstrated to form a stable group distinct from other Rickettsia species (Figure 1 ) [ 20 - 22 ]. We found that within this cluster they consistently formed two phylogenetic clusters (Figure 2A ). One of these clusters included R. conorii and ITTR, and the other, AFR isolates and ISFR. Using MST, we identified five genotypes. However, using pairwise comparison of spacer sequences, we identified only four genotypes similar to those obtained by MLST. MST-based phylogeny also demonstrated a similar pattern (Figure 2B ). Serotypically, we could identify four serotypes which, according to the definition of species proposed by Philip [ 8 ], would classify them as different species: 1) R. conorii isolate Malish; 2) ITTR; 3) AFR; and 4) ISFR. These four serotypes also showed distinct MST genotypes. We demonstrated a discrepancy between the genotypic homogeneity of these rickettsiae, which classified them within the R. conorii species [ 9 ], and their MST genotypic, serotypic, geographic, and pathogenic heterogeneity. For ITTR, an additional discrepancy was observed between its phylogenetic and serotypic classifications (Figures 2A and 2B versus 2C ). Such findings are in accordance with those of Walker et al . who had shown substantial differences between ITTR and R. conorii isolates [ 11 ]. We also agree with these authors who hypothesized that all members of the "so-called" R. conorii complex belonged to the R. conorii species [ 15 ]. Conclusion We have demonstrated that R. conorii and closely related strains exhibit a high level of conservation in nucleotide sequence despite genotypic specificities, and phenotypic specificities including serotyping, epidemiological and clinical characteristics. Therefore, according to the accepted criteria of the ad hoc committee on reconciliation of approaches to bacterial systematics which allow the creation of subspecies for genetically-close isolates which express phenotypic differences [ 25 ], we are justified in creating new subspecies for these organisms. Emendation of the description of Rickettsia conorii Rickettsia conorii (Brumpt 1932). The characteristics of this taxon are similar to those described by Weiss and Moulder for the species [ 1 ]. The species contains four subspecies. The minimal degree of pairwise nucleotide similarity of the R. conorii species is of 99.8 % for 16S rDNA, 99.4 % for glt A, 98.3 % for the 5' end of omp A, 98.6 % for omp B, and 99.3 % for sca 4, by comparison with isolate Malish. Description of Rickettsia conorii subsp. conorii subsp. nov Rickettsia conorii subsp. conorii (co.no'ri.i N. L. gen. n. conorii , of Conor, in honor of A. Conor who, in collaboration with A. Bruch, provided the first description of fièvre boutonneuse in 1910 [ 35 ]). The characteristics are the same as those of the species. Transmitted to humans through the bite of Rhipicephalus sanguineus ticks. Grows in Vero cells at 32°C in antibiotic-free Minimal Essential Medium supplemented with 2 % fetal calf serum and 2 mg/ml L-glutamine. The type strain is Malish, ATCC VR-613, which was isolated in South Africa by J.H.S. Gear in 1946. This type is the most common and representative of the isolates of R. conorii subsp. conorii subsp. nov. The nucleotide sequence of the 16S rRNA, glt A, omp A, omp B, and sca 4 genes have been deposited in the GenBank database under the accession numbers, AF541999, U59730, U43806 [ 30 ], AF123721 [ 22 ], and AF163008 [ 20 ], respectively. To be classified as R. conorii subsp. conorii subsp. nov., a rickettsial isolate should exhibit a minimal pairwise nucleotide sequence similarity of 100%, 100%, 99.9%, 99.9%, and 100% for the 16S rDNA, glt A, omp A, omp B, and sca 4 genes, respectively, with those of R. conorii isolate Malish, ATCC VR-613. R. conorii subsp. conorii subsp nov. strain Malish has also been deposited in the official collection of the WHO Collaborative Center for the Diagnosis and Study of Rickettsioses in Marseille, France. Description of Rickettsia conorii subsp. indica subsp. nov Rickettsia conorii subsp. indica (in'di.ca N. L. gen. n. indica , from India, where the Rhipicephalus sanguineus tick providing the first isolate was collected). The characteristics are the same as those of the species. Transmitted to humans through the bite of Rhipicephalus sanguineus ticks. Grows in Vero cells at 32°C in antibiotic-free Minimal Essential Medium supplemented with 2 % fetal calf serum and 2 mg/ml L-glutamine. The type strain is Indian tick typhus, ATCC VR-597, which was isolated from a Rhipicephalus sanguineus tick collected in India by C.B. Philip in 1950. The nucleotide sequence of the 16S rRNA, glt A, omp A, omp B, and sca 4 genes have been deposited in the GenBank database under the accession numbers L36107 [ 36 ], U59730 [ 37 ], U43794 [ 30 ], AF123726 [ 22 ], and AF163005 [ 20 ], respectively. To be classified as R. conorii subsp. indica subsp. nov., a rickettsial isolate should exhibit a minimal pairwise nucleotide sequence similarity of 100%, 100%, 99.9%, 99.9%, and 100% for the 16S rDNA, glt A, omp A, omp B, and sca 4 genes, respectively, with those of R. conorii subsp. indica subsp. nov., isolate Indian tick typhus, ATCC VR-597. R. conorii subsp. conorii subsp nov. isolate Indian has also been deposited in the official collections of the WHO Collaborative Center for the Diagnosis and Study of Rickettsioses in Marseille, France; of the WHO and National Reference Collection at the Gamaleya Research Institute, Moscow, Russia, and of the WHO Collaborating Reference Center for Rickettsial and Bartonella-associated Diseases, CDC, Atlanta, USA. Description of Rickettsia conorii subsp. caspia subsp. nov Rickettsia conorii subsp. caspia (cas' pi.a N. L. fem.adj. caspia , from Mare Caspium, the Latin name of the Caspian sea where the disease caused by this rickettsia is endemic). The characteristics are the same as those of the species. Transmitted to humans through the bite of Rhipicephalus sanguineus and R. pumilio ticks. Grows in Vero cells at 32°C in antibiotic-free Minimal Essential Medium supplemented with 2 % fetal calf serum and 2 mg/ml L-glutamine. The type strain is A-167, which was isolated from a Rhipicephalus pumilio tick collected in Astrakhan in 1992. The nucleotide sequence of the 16S rRNA, glt A, omp A, omp B, and sca 4 genes have been deposited in the GenBank database under the accession numbers L36100 [ 36 ], U59728 [ 37 ], U43791 [ 30 ], AF123708 [ 22 ], and AF163007 [ 20 ], respectively. The nucleotide sequence of the 16S rRNA, glt A, omp A, omp B, and sca 4 genes of AFR isolate Chad have been deposited in the GenBank database under the accession numbers AF510102 [ 31 ], AF510103 [ 31 ], AY112668 [ 31 ], AY643093, and AY643092, respectively. To be classified as R. conorii subsp. caspia subsp. nov., a rickettsial strain should exhibit a minimal pairwise nucleotide sequence similarity of 99.7%, 99.6%, 99.5%, 98.9%, and 99.6% for the 16S rDNA, glt A, omp A, omp B, and sca 4 genes, respectively, with those of R. conorii subsp. caspia subsp. nov., isolate A-167. R. conorii subsp. caspia subsp nov isolate A-167 has been deposited in the official collections of the WHO Collaborative Center for the Diagnosis and Study of Rickettsioses in Marseille, France, and of the WHO Collaborating Reference Center for Rickettsial and Bartonella-associated Diseases, CDC, Atlanta, USA. Description of Rickettsia conorii subsp. israelensis subsp. nov Rickettsia conorii subsp. israelensis (is.ra. el en' sis. N. L. gen. n. israelensis , from Israel, where the Rhipicephalus sanguineus tick providing the first isolate was collected). The characteristics are the same as those of the species. Transmitted to humans through the bite of Rhipicephalus sanguineus ticks. Grows in Vero cells at 32°C in antibiotic-free Minimal Essential Medium supplemented with 2 % fetal calf serum and 2 mg/ml L-glutamine. The type strain is ISTTCDC1, which was isolated from a Rhipicephalus sanguineus tick collected in Israel in 1974. The nucleotide sequence of the 16S rRNA, glt A, omp A, omp B, sca 4 genes have been deposited in the GenBank database under the accession numbers L36223 [ 36 ], U59727 [ 37 ], U43797 [ 30 ], AF123712 [ 22 ], and AF155058 [ 20 ], respectively. To be classified as R. conorii subsp. israelensis subsp. nov., a rickettsial isolate should exhibit a minimal pairwise nucleotide sequence similarity of 100%, 99.7%, 98.9%, 99.5%, and 99.8%, for the 16S rDNA, glt A, omp A, omp B, and sca 4 genes, respectively, with those of R. conorii subsp. israelensis subsp. nov., isolate ISTTCDC1. R. conorii subsp. israelensis subsp. Nov. isolate ISTTCDC1 has been deposited in the official collections of the WHO Collaborative Center for the Diagnosis and Study of Rickettsioses in Marseille, France, and of the WHO Collaborating Reference Center for Rickettsial and Bartonella-associated Diseases, CDC, Atlanta, USA. Methods Rickettsial strains All rickettsial strains tested and their original sources are listed in Table 2 . Rickettsial isolates for which sequences were not available were grown in Vero cell monolayers at 32°C in 150 cm 2 -cell culture flasks as previously described [ 37 ]. When Gimenez staining demonstrated the cells have been infected heavily, they were harvested using sterile glass beads and centrifuged at 12,000 g for 10 min, resuspended in fresh medium and stored at -70°C until purification of nucleic acid was carried out. All rickettsial stocks were tested and shown to be free of Mycoplasma sp using a 16S rDNA-based PCR assay as previously described [ 38 ]. Nucleic acid purification, PCR amplification and sequencing When available, we used gene sequences present in GenBank. The accession numbers for these sequences are reported at the end of the manuscript. Missing sequences were obtained in this study. Genomic DNA was extracted from 200 μl of rickettsial suspension and from 57 Rhipicephalus pumilio ticks collected in Astrakhan [ 19 ] using the QIAamp Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. For MLST, we used the previously described primers and PCR conditions to amplify 1,424-bp, 1,134-bp, 590-bp, 4,890-bp, and 3,028-bp fragments of the 16S rDNA [ 36 ], gltA [ 37 ], ompA [ 30 ], ompB [ 22 ] and sca4 [ 20 ] genes, respectively, of 29 of 36 rickettsial isolates tested for which no sequences were available in GenBank, and 57 R. pumilio ticks. In addition, we amplified and sequenced the omp B and sca 4 genes of AFR isolate Chad which had not been determined previously. Primers were purchased from Eurogentec (Seraing, Belgium). PCR reactions were carried out in a Peltier model PTC-200 thermal cycler (MJ Research, Watertown, MA). Each PCR included a negative (distilled water) and a positive control (DNA extracted from R. montanensis isolate 2–4–6 using the QIAamp Tissue Kit [Qiagen]). PCR products were visualized after electrophoresis on a 1% agarose gel. Each amplicon was purified for sequencing using the QIAquick Spin PCR Purification Kit (Qiagen) according to the manufacturer's instructions. Sequencing reactions were carried out using PCR primers and the D-rhodamine terminator cycle DNA sequencing Kit (Applied Biosystems, Foster City, California) according to the manufacturer' instructions. Sequencing reaction products were resolved by electrophoresis with an ABI Prism 3100 Automated Sequencer (Applied Bioystems). Each base position was established twice in both the forward and reverse directions. For each gene, the pairwise sequence similarity among studied isolates and tick amplicons was estimated using the multisequence alignment program CLUSTAL W [ 39 ]. Each isolate or tick amplicon exhibiting specific nucleotide substitutions within one or more of the five genes studied was considered a different MLST genotype. Then, we selected a representative isolate of each MLST genotype for further tests. When a MLST genotype incorporated more than one isolate, we selected the type strain or the first strain described in the literature as representative strain. In addition to AFR isolate A-167, we incorporated AFR isolate Chad in the phylogenetic study and the mouse immunization and MST assays because MLST sequence differences between both isolates were due to nucleotide substitutions as well as deletions. Distance matrices generated by DNADIST were determined under the assumptions of Kimura and were used to infer dendrograms by the neighbor-joining method [ 40 ] using the PHYLIP version 3.4 software package [ 41 ]. Dendrograms were also constructed by data processing with the maximum-likehood and parsimony methods (DNAML and DNAPARS, respectively, in PHYLIP). A bootstrap analysis based on 100 randomly generated trees using SEQBOOT and CONSENSE in PHYLIP was performed to estimate the node reliability of the trees obtained by three phylogenetic methods [ 42 ]. Multi-spacer typing (MST) We amplified and sequenced the dks A / xer C, mpp A / pur C, and rpm E / tRNA-fMet intergenic spacers for each of R. conorii isolate Malish, ITTR, ISFR, and AFR isolates A-167 and Chad. Primers and methods were those described previously [ 28 ]. Subsequently, spacer sequences were concatenated and used to infer the phylogenetic relationships among strains tested using the neighbor-joining method [ 40 ] as described above. Mouse immunization For each R. conorii isolate Malish, ITTR, ISFR, and AFR isolates A-167 and Chad, five BALB/c mice were inoculated intravenously (tail vein) with purified bacterial suspension (0.1 ml containing 10 4 bacteria). The concentration of rickettsial suspensions was estimated using a plaque assay as previously described [ 43 ]. On day 7, mice were boosted with a similar inoculum. On day 10, mice were anesthetized and exsanguinated by cardiac puncture [ 8 ]. Sera from each group of five mice were pooled and stored at -20°C until used for microimmunofluorescence (MIF) serotyping. MIF serotyping The MIF test was performed and the SPD was calculated using the method of Philip et al. [ 8 ]. Each of the five rickettsiae tested was compared with each other. The highest serum dilutions giving positive reactions were recorded as endpoint titer: SPD = (Aa + Bb) - (Ab + Ba), where Aa or Bb is -log2 of the endpoint titer between serum A or B and homologous antigen a or b and Ab or Ba is -log2 of the endpoint titer of serum A or B against heterologous antigen b or a. If the SPD was less than 3, the two isolates were assumed to belong to the same serotype. If the SPD was ≥ 3, the isolates were assumed to belong to different serotypes. The SPDs were used to construct a matrix, and then a dendrogram was constructed from the matrix by the unweighted pair group method with arithmetic mean (UPGMA) available in the MEGA2 software package [ 44 ]. Authors' contributions The individual parts of the work presented in the paper were conducted as follows: YZ and PEF carried out the molecular genetic studies, analyzed the sequences and drafted the manuscript. ME participated in the design of the study and helped to draft the manuscript. DR conceived the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

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1079850

Site-specific and synergistic stimulation of methylation on the bacterial chemotaxis receptor Tsr by serine and CheW

Background Specific glutamates in the methyl-accepting chemotaxis proteins (MCPs) of Escherichia coli are modified during sensory adaptation. Attractants that bind to MCPs are known to increase the rate of receptor modification, as with serine and the serine receptor (Tsr), which contributes to an increase in the steady-state (adapted) methylation level. However, MCPs form ternary complexes with two cytoplasmic signaling proteins, the kinase (CheA) and an adaptor protein (CheW), but their influences on receptor methylation are unknown. Here, the influence of CheW on the rate of Tsr methylation has been studied to identify contributions to the process of adaptation. Results Methyl group incorporation was measured in a series of membrane samples in which the Tsr molecules were engineered to have one available methyl-accepting glutamate residue (297, 304, 311 or 493). The relative rates at these sites (0.14, 0.05, 0.05 and 1, respectively) differed from those found previously for the aspartate receptor (Tar), which was in part due to sequence differences between Tar and Tsr near site four. The addition of CheW generated unexpectedly large and site-specific rate increases, equal to or larger than the increases produced by serine. The increases produced by serine and CheW (added separately) were the largest at site one, ~3 and 6-fold, respectively, and the least at site four, no change and ~2-fold, respectively. The rate increases were even larger when serine and CheW were added together, larger than the sums of the increases produced by serine and CheW added separately (except site four). This resulted in substantially larger serine-stimulated increases when CheW was present. Also, CheW enhanced methylation rates when either two or all four sites were available. Conclusion The increase in the rate of receptor methylation upon CheW binding contributes significantly to the ligand specificity and kinetics of sensory adaptation. The synergistic effect of serine and CheW binding to Tsr is attributed to distinct influences on receptor structure; changes in the conformation of the Tsr dimer induced by serine binding improve methylation efficiency, and CheW binding changes the arrangement among Tsr dimers, which increases access to methylation sites.

Background In Escherichia coli , the four methyl-accepting chemoreceptor proteins (MCPs) are distinguished by different external ligand-binding specificities for attractants and repellents [ 1 ]. The cytoplasmic domains are highly conserved by comparison [ 2 ] and bind to the signaling kinase, CheA, and the adaptor protein, CheW, which leads to the formation of a receptor/CheW/CheA ternary complex [ 3 , 4 ]. The ternary complex provides the means by which ligand binding influences kinase activity and thus cell motility; the ternary complex is in a kinase-active state without attractant bound, and kinase activity is inhibited by attractant binding [ 5 ]. Tsr is representative of the MCP family of receptors [ 1 , 2 ]; it has a remarkable α-helical structure with an end-to-end length of ~300 Å that is represented in Figure 1 [ 6 , 7 ]. It is organized as a homodimer, where each subunit consists of a four helix bundle domain located in the periplasm for ligand binding [ 8 ], two α-helical transmembrane (TM) segments, and a lengthy α-helical coiled-coil hairpin for the cytoplasmic domain [ 6 ]. The cytoplasmic domain is connected to TM2 through a linker (HAMP domain), which has been identified as a conserved element in the sequences of bacterial signaling proteins [ 9 , 10 ]. This region consists of amphipathic helices that probably adopt a compactly-folded conformation [ 11 ], but detailed structure information of this region is not yet available. The Tsr dimer has two symmetry-related serine binding sites that exhibits negative cooperativity, and binds serine with half-of-sites saturation [ 12 ]. CheW binds at nearly the opposite end of the dimer [ 13 ], with 1:1 subunit stoichiometry [ 14 ]. Four major sites of methylation have been identified in Tar and Tsr [ 15 - 17 ]. The methylation sites are clustered on the surfaces of two methylation helices found within the elongated coiled-coil hairpin structure of the cytoplasmic domain (Figure 1 ). In Tsr, three of the four major sites of methylation (residues 297, 304, and 311) are located on methylation helix I, while the fourth site (residue 493) is located on methylation helix II [ 16 ]. Methyl group incorporation has also been detected in Tsr at two other less-rapidly methylated (minor) sites; one of these sites is glutamate-502 [ 17 ], which is depicted in Figure 1 . All methylation sites are found within a recognition sequence, which has the consensus sequence of Glu- Glu -X-X-Ala-Ser/Thr, where the second Glu residue (in bold) is usually methylated [ 18 ]. Receptor methylation stabilizes the kinase-activating state of the ternary complex [ 19 - 22 ], which explains the chemical basis of adaptation as a feedback system that reversibly methylates the receptor cytoplasmic domains. For example, an increase in attractant binding rapidly inhibits the kinase, but also initiates the restoration of kinase activity on a longer time scale through an increase in the methylation level [ 23 , 24 ]. Methyl ester formation is catalyzed by the methyltransferase (CheR) and the rate of receptor methylation increases following the addition of attractant [ 25 - 27 ]. Methyl ester hydrolysis is catalyzed by the methylesterase (CheB), whose activity is stimulated significantly through transient phosphorylation by CheA [ 28 ]. To fully understand the basis of kinase regulation by ligand binding and receptor modification, the interactions between components in the excitation and adaptation branches of the signaling pathway must be determined, i.e. the interactions between proteins involved in regulating kinase activity and receptor methylation/demethylation, respectively. To the present time, this interaction has focused exclusively on the activation of CheB by transient phosphorylation, with little or no attention paid to the influence of ternary complex formation on receptor methylation. In vitro studies of attractant-stimulated increases in receptor methylation have invariably used receptor samples without either CheW or CheA present [ 18 , 29 - 31 ], the proteins that are found in receptor signaling complexes, and that are known to facilitate receptor clustering in the cell [ 32 ]. We have investigated the effect of CheW on Tsr methylation, because CheW alone can promote receptor clustering [ 32 ], and because the binding interaction with MCPs is well-defined by comparison to the entire receptor/CheW/CheA complex. The findings demonstrate that CheW significantly increases methylation at the major sites in Tsr, and that the influence of CheW is comparable to or larger than the influence of serine. Also, when CheW and serine are added together, the increase in rate is greater than the sum of the increases produced by either ligand alone. These results provide evidence that CheW plays a role in promoting receptor methylation in addition to its known role in regulating the activity of CheA within the ternary complex. Results Methylation rates at the four major sites of Tsr To examine the initial rates of methylation of the serine receptor at the four major sites individually, Tsr molecules were constructed by site-directed mutagenesis to have only one major site available. Methylation at other sites was prevented by the introduction of glutamine residues in place of glutamate. For example, Tsr with site one (E297) available for methylation, referred to as Tsr EQQQ , was used to monitor the rate of methyl group incorporation at site one. The four forms (Tsr EQQQ , Tsr QEQQ , Tsr QQEQ and Tsr QQQE ,) are collectively referred to as the single site forms of Tsr. Receptors with multiple sites available for methylation were also used, e.g. Tsr QEQE (second and fourth sites) and Tsr EEEE (all four sites), while Tsr QQQQ , which had no major sites available, was used as a control to gauge contributions from minor sites, e.g. E502 [ 17 ]. The inner membranes in which Tsr was overexpressed were isolated from plasmid-containing HCB721, which was defective in the production of CheR and CheB, and thus the receptors were maintained in the gene-encoded covalent modification pattern. HCB721 was also defective in the production of CheW (and CheA). The initial rates of methyl group incorporation for the single-site forms of Tsr are shown in Figure 2A . These experiments revealed a 7-fold larger rate of methylation for Tsr QQQE than Tsr EQQQ , and a ~20-fold larger rate than either Tsr QEQQ or Tsr QQEQ (Figure 2B and Table 1 ). Also, Tsr QQQE -containing membranes were methylated at a rate 35-fold larger than membranes containing Tsr QQQQ . This relatively low rate of methyl group incorporation into Tsr QQQQ was qualitatively consistent with previous investigations of Tsr methylation at the minor sites [ 16 , 17 ]. The rapid rate of Tsr QQQE methylation, relative to Tsr QEQQ and Tsr QQEQ , contrasted with previous investigations of E. coli and Salmonella Tar [ 18 , 27 , 29 ], which demonstrated that sites two and three were more rapidly methylated by a significant margin. A possible explanation for this difference was found by comparing the sequences of E. coli Tsr and Tar in the immediate vicinity of site four. The Tsr sequence near site four (QQNA A LV E E 493 SAAAAAAL, ref. [ 17 ]) differs from the Tar sequence at just two residues, 489 and 492 (underlined), which are serine and glutamine, respectively, at the corresponding locations in Tar. We postulated that glutamate 492 was more influential, because of its location within, and agreement with, the consensus sequence. To test this idea glutamate-492 was changed to glutamine, generating Tsr QQQE -E492Q, which matched the sequence at site four of Tar. When the methylation rates of the two forms were measured and compared, the rate of Tsr QQQE -E492Q methylation was ~35 fold lower than Tsr QQQE , which was comparable to the rate observed for the minor sites control, Tsr QQQQ . From these data it was concluded that glutamate-492 made an important contribution to Tsr methylation at site 4, and provided an explanation for the different site preferences of Tar and Tsr. The effect of serine binding on methylation rates Methylation rates of the single glutamate forms of Tsr were measured over a range of serine concentrations, shown in Figure 3 . The rates increased over a concentration range consistent with the dissociation constant for serine, ~10 μM [ 12 ], although the amount of the increase depended significantly on the site involved. In general, the more slowly methylating forms exhibited the larger relative increases, which are plotted as rates relative to the rate without serine in Figure 3B . At a saturating serine concentration (500 μM), the largest increases were observed in membranes containing TsrE QQQ or Tsr QEQQ (3 and 2-fold, respectively, using Table 1 data). A smaller, but significant, increase was observed with Tsr QQEQ -containing membranes (1.2-fold), and apart from the minor sites control membrane (Tsr QQQQ , which showed no significant effect), serine had the smallest relative effect on Tsr QQQE , the most rapidly methylated form. The relative rates plotted in Figure 3B were calculated without subtracting the Tsr QQQQ data as a background, principally because the trends in the data were not significantly affected. Qualitatively, this background subtraction increased the relative rates of the slowly methylated single site forms (Tsr EQQQ , Tsr QEQQ , Tsr QQEQ ) more than Tsr QQQE (results not shown). These data suggest that the methylation of Tsr QQQQ serves as a reliable background, an expectation that is consistent with the structural similarity of Tsr cytoplasmic fragments in different covalent states [ 6 , 33 ], yet the similarity among the C-fragment structures does not rule out the possibility that significant differences may exist in the intact receptors. Also, the use of methyl group incorporation into Tsr QQQQ as a background assumed that the rates at the individual sites could be summed to yield the overall rate, which would not take into account interactions between sites that influence methylation, such as those documented previously for Tar [ 29 ]. CheW stimulated Tsr methylation When Tsr methylation was investigated as a function of the CheW concentration, significant increases were observed at all four sites (Figure 4A ). In contrast to the increases produced by serine, the rates in the presence of CheW continued to climb up to the largest concentration tested (60 μM). These gradual increases did not mirror CheW binding to the Tsr-containing membranes used in this study, which exhibited a single-set-of-sites interaction behavior characterized by dissociation constants on the order of 10 μM (results not shown), similar to the observations of Boukhvalova et al. with Tar-containing membranes [ 14 ]. The differences between the concentration dependence of CheW binding and methylation rate increases are postulated to result from the mechanism by which CheW influences the methylation rate, which is addressed in the Discussion section. The rate increases in Figure 4 and Table 1 were CheW-specific, other unrelated proteins (60 μM BSA or 60 μM ovalbumin) had no effect on the methylation rate (data not shown), nor did 60 μM CheW-V36M (data not shown), a CheW point mutant that has been shown previously to be defective in Tar binding [ 14 ]. Like serine, CheW had the largest influence on the more slowly methylated sites. The magnitudes of these increases are more evident in Figure 4B , which plot the rates relative to the rate observed without CheW. CheW had the most substantial effect on Tsr EQQQ , Tsr QEQQ and Tsr QQEQ , which were methylated between four to six fold more rapidly at the largest CheW concentration (Figure 4B and Table 1 ). The methylation rate of Tsr QQQE increased about two-fold under these conditions. Altogether, the larger concentrations of CheW produced rate increases that were comparable to, or larger than, the increase in rate generated by serine. The large concentrations of CheW used in these experiments, relative to the amounts of Tsr, are unlikely to be found in the wildtype cell [ 34 ]. CheA is also normally present in the cell, which has been observed to bind to inner membrane preparations containing Tsr [ 35 ]. We chose to study the effect of CheW in isolation, because of the complex nature of receptor/CheW/CheA interactions. Depending on the concentration, CheW may either promote or compete with CheA binding [ 35 , 36 ]. Also, published estimates of receptor-CheW and receptor-CheA interactions at saturation are 1 and < 0.2, respectively [ 14 , 35 ], which implies that a greater fraction of Tsr molecules in the methylation samples are likely to be influenced by CheW than CheA. Nevertheless, some experiments were conducted to determine if significant rate increases could be observed with more modest CheW concentrations (~1:1 CheW:Tsr), but in the presence of CheA, because there is evidence to suggest that CheW can bind synergistically with CheA under the appropriate conditions [ 35 , 36 ]. Methylation was measured in samples containing 7 μM Tsr EQQQ , 6 μM CheW, and CheA at several concentrations. These rates are shown in Figure 5 and are expressed relative to Tsr EQQQ without either CheW or CheA. They provide evidence that CheA can augment the increases produced by CheW alone. A thirty percent increase was observed with 6 μM CheW (no CheA), while an increase of eighty percent was observed when 1.5 μM CheA was also present. No increase in the methylation rate was observed when CheA was added without CheW (data not shown). Thus, the combined effect of CheW and CheA was more pronounced than CheW alone at the same concentration, which suggests the effect of CheW acting alone at larger concentrations may reflect the situation within the ternary complex at smaller CheW concentrations. The effects of serine and CheW are synergistic Table 1 also summarizes the results of experiments that tested the effect of a combined serine and CheW stimulus. These data were used to calculate the sums of the rate increases generated by 0.5 mM serine and 60 μM CheW in separate samples (Rate (serine) + Rate (CheW) - 2 × Rate (noligand) ), which were compared to the increases that resulted when both ligands were present simultaneously (Rate (serine & CheW) - Rate (no ligand) ). Figure 6 is a histogram of these rate increases, showing both the individual rate increases summed together (striped) and the rate increases produced by the simultaneous addition of serine and CheW (black) for all four single site forms and Tsr QQQQ . Figure 6 provides clear evidence of a synergistic effect between serine and CheW at all sites except site 4. The ratios of the combined increases relative to the summed increases, (Rate (serine & CheW) - Rate (no ligand) )/(Rate (serine) + Rate (CheW) - 2 × Rate (no ligand) ) represent a single numerical index for the extent to which the combined increases were greater than the sums (Table 2 ). A value equal to 1 indicated that stimuli were additive (site 4); values significantly greater than one characterized the site as synergistic toward the two ligands (sites 1, 2 & 3). Table 3 also presents these initial rates in the form of rate increases, expressed as differences between serine-stimulated and unstimulated rates, to facilitate explicit comparisons between rate increases produced by serine when CheW was also present versus when it was not. At every site, CheW magnified the increase in the methylation rate produced by serine. The effect was most significant in Tsr EQQQ , the increase produced by the addition of serine in the presence of CheW was 4-fold larger than in the absence of CheW (an increase of 0.017 s -1 versus 0.004 s -1 ). The effect of CheW on receptors with multiple available sites Tsr QEQE and Tsr EEEE -containing membranes were used to test the effect of CheW on receptor methylation when more than one site was available. Figure 7 shows rate data collected as a function of the CheW concentration. Without CheW (or serine) the rates observed with Tsr QEQE and Tsr EEEE samples were similar to each other (~0.011 s -1 ), a result that was not unexpected, because Tsr QEQE and Tsr EEEE both possessed the rapidly-methylated fourth site. Moreover, the rates observed for these samples in the absence of CheW ( v QEQE , v EEEE ) were in approximate agreement with rates calculated from sums of single-site Tsr data, i.e. v QEQE = v QEQQ + v QQQE - v QQQQ and v EEEE = v EQQQ + v QEQQ + v QQEQ + v QQQE - 3 v QQQQ , indicating that individual rates were approximately additive. This approximate additivity suggested that CheR-mediated interactions between adjacent sites, which were postulated to occur in a previous study of Tar [ 29 ], were not significant in these samples, although the additivity observed here might mainly reflect the dominant contribution of site four. The rate increases in Figure 6 produced by the addition of 60 μM CheW displayed a trend qualitatively consistent with additivity among the sites of methylation, reflected in the fact that the rate increase with Tsr EEEE was greater than the rate increase with Tsr QEQE . A quantitative assessment based on rate increases (Δ v ) observed in the data of Figure 7 , i.e. the rate with 60 μM CheW present minus the rate without CheW, and increases calculated from the single-site data in Table 1 in the manner indicated above, generated respective measured and calculated estimates for Δ v of 0.005 and 0.012 s -1 for Tsr QEQE , and 0.016 and 0.022 s -1 for Tsr EEEE . In both situations, the observed increases fell short of those calculated with the assumption of site additivity. On one hand the greater availability of sites in Tsr EEEE and Tsr QEQE work in favor of larger methylation rates, but this might be offset by unfavorable electrostatic interactions within and between receptor dimers that interfere with methylation and become more significant with an increasing number of glutamates present. The discrepancy between the observed and calculated values of Δ v may then reflect the fact that CheW-binding does not completely overcome these interfering electrostatic effects. Yet CheW-binding does provide a more significant increase in the rate of methylation of both Tsr QEQE and Tsr EEEE ; the observed values of Δ v are larger for Tsr QEQE and Tsr EEEE than the single site forms. These larger increases are qualitatively consistent with similar CheW-induced changes in the interactions between Tsr dimers, and changes in dimer structure of all the forms of Tsr (single-site and multiple-site forms). Discussion An understanding of the features essential to receptor methylation and its role in chemotaxis has developed through a sustained effort that has ( i ) identified the sites of methylation and the methylation consensus sequence [ 15 - 18 ], ( ii ) demonstrated that increases in methylation result from attractant binding [ 26 , 27 , 29 ], ( iii ) established the role of interdimer methylation [ 30 , 37 , 38 ], and ( iv ) probed the factors influential in methylation site – CheR active site interactions [ 29 , 39 , 40 ]. These studies have been conducted almost exclusively on receptor samples without any of the cytoplasmic signaling proteins present other than CheR. However, it is now appreciated that a substantial fraction of the receptors in the cell are often located in patches at the cell poles [ 32 ], and are part of the signaling complexes that form with CheW and CheA [ 3 , 4 , 32 , 41 ]. Their influences on receptor methylation have not been systematically studied. A single experiment in the study of Tar transmethylation conducted by LeMoual, Quang and Koshland failed to find an influence of ternary complex formation on Tar methylation, possibly because insufficient amounts of CheA and CheW were present [ 37 ]. Levit and Stock [ 22 ] made the qualitative observation that the methylation of Tsr proceeded to a greater extent in the presence of CheA and CheW, which is in agreement with the results of the experiments presented here. Figure 1 maps the locations of the serine and CheW binding sites in relation to the sites of methylation on the dimer model of Tsr [ 6 ]. The serine and CheW binding sites are nearly at opposite ends of the dimer, and thus their effects are communicated in opposite directions toward the centrally-located sites of methylation. The panels in Figure 8 provide schematic representations of the methylation region from one subunit, which help to summarize the relative rates of methylation at the four sites without ligand, in the presence of either serine or CheW, and in the presence of both ligands (Figure 8A to 8D , respectively). The values next to the four sites ( not in parentheses) are relative to the rate at that site without ligand, while the rate values inside the parentheses are relative to site 4 under the same conditions. The following observations are made from these data: ( i ) the increases in the methylation rate generated by high concentrations of CheW are larger than the increases generated by serine. ( ii ) The effects of serine and CheW are synergistic at sites one, two and three. This leads to the additional conclusion that the rate increases produced by serine are significantly larger when Tsr is saturated with CheW (see also Table 3 and Figure 6 ). ( iii ) The differences in rate between Tsr QQQE and all other single site forms of Tsr lessen as serine and CheW are added, to the extent that Tsr EQQQ is methylated more rapidly when both serine and CheW are present. CheW and serine: different influences on Tsr structure? Our results demonstrate that serine and CheW binding to Tsr both increase the methylation rate, and that the effects of CheW and serine are synergistic (super additive). These behaviors are consistent with the individual and combined effects of two allosteric activators that have similar effects on receptor structure. Yet a qualitative argument, based on the known influences of serine and CheW on receptor signaling activity, suggests otherwise. To illustrate this point, we assume the simplest situation, one in which Tsr is engaged in a two state equilibrium between kinase activating and kinase inhibiting states. Based on experimental data, serine binding shifts this equilibrium toward the kinase inhibiting state; a state that is also characterized by more rapid methylation. On the other hand, CheW is expected to either shift the equilibrium toward the kinase-activating state or at least not influence it, which according to our argument is characterized by a smaller methylation rate. Clearly, the experimental results are not consistent with this picture (CheW binding increases the methylation rate), which thus suggests that the binding of CheW and serine produce different effects on the structure and arrangement of Tsr in the membrane. The distinct locations and properties of CheW and serine binding are also consistent with this idea. Serine-induced changes in Tsr structure are expected to be like aspartate-induced changes in Tar, which have been thoroughly investigated (reviewed in ref. [ 1 ]). One effect of aspartate binding on Tar is consistent with a propagated piston-like motion of TM2 relative to TM1 within the Tar dimer. Attractant binding shifts TM2 toward the cytoplasm; this motion is correlated with increases in the methylation rate and the inhibition of CheA activity (ref. [ 1 ] and references therein, [ 42 ]). Sites one, two, and three are clustered on the first methylation helix and joined to TM2 through the linker (Figure 1 ). In view of this circumstance, it is perhaps significant that the serine-stimulated rates relative to the rates without ligand ( r = v +serine / v no ligand ) display the trend r EQQQ > r QEQQ > r QQEQ ~ r QQQE (Figure 8B ), which is opposite to the trend in separation between the methylation sites and the end of TM2 in the primary structure. The trend is not evident in the increases in rate produced by CheW alone ( r EQQQ ≈ r QEQQ ≈ r QQEQ > r QQQE , Figure 8C ), but it is evident again when both serine and CheW are present (Figure 8D ), although the values of r are larger. It is as though the effect of serine binding on methylation diminishes with increasing distance from TM2, but because serine binding also serves to inhibit the kinase, it must also exert an influence at the CheW (and CheA) binding site(s). This suggests that the nature of the influences produced by serine at the CheW/CheA binding site and methylation sites are distinct, which may reflect the differences in the processes of receptor methylation and kinase regulation. Attractant-induced changes in receptor structure that influence methylation seem to be restricted to the receptor dimer binding attractant [ 43 ], while kinase regulation is a cooperative process shared among several receptor dimers [ 20 ]. In contrast to the attractant/receptor complexes, high-resolution structural information is not available yet for the CheW-receptor complex; consequently specific models for changes in receptor structure are difficult to develop. Nonetheless, the distinguishing features of serine and CheW binding provide some clues to the basis for different influences. CheW binds to MCPs at a location in the primary structure between methylation sites, i.e. between sites 1–3 and site 4, in contrast to attractants like serine, which have an identifiably more direct connection to the first three sites (Figure 1 ). As noted above, one of the changes in structure generated by serine may be mediated primarily through one of the two helices in the cytoplasmic domain. In contrast, the methylation data suggests that CheW exerts a more uniform influence on both methylation helices, because CheW (when added alone) produces significant rate increases at all four sites. The mechanism of the CheW-mediated increase As it is currently understood, receptor methylation involves two interactions between the MCPs and CheR, a tethering interaction and an active site – methylation site interaction. The tethering interaction involves specific binding of a conserved pentapeptide (NWETF) at the C-terminus of Tar and Tsr to CheR [ 44 ], and provides a mechanism to increase the CheR concentration in the vicinity of the methylation sites. The available evidence suggests that the tethering interaction is significantly stronger than active site – methylation site interactions [ 43 , 44 ], and together the two interactions allow CheR tethered at the C-terminus of either Tar or Tsr to visit the multiple sites of methylation many times through the lower affinity (and presumably more labile) active site – methylation site interactions before a methyl group transfer event takes place. The small turnover number of CheR under saturating substrate concentrations (~7 min -1 ) [ 45 ], is consistent with this scenario. Importantly, this active site – methylation site interaction can, and in some circumstances must, occur in receptor dimers that are different from the receptor dimer involved in the tethering interaction [ 20 , 37 , 38 , 43 ]. According to this line of reasoning, the increases in the methylation rates generated by CheW could result from increases in ( i ) the fraction of CheR tethered to Tsr at the C-terminus, ( ii ) the number of methylation sites accessible to tethered CheR, and/or ( iii ) an increase in the probability of methyl group transfer per active site – methylation site encounter. An increase in the recruitment of CheR (possibility i ) requires that a substantial fraction of the CheR tethering sites are obscured in the absence of CheW. This is not supported by previous binding experiments involving CheR and Tsr-containing membranes, which demonstrated that ~90% of the CheR tethering sites on Tsr are accessible in the absence of CheW [ 46 ]. Moreover, it is estimated that ~70% of CheR is tethered to Tsr in the methylation experiments, based on the measured CheR-Tsr binding affinity ( K a = 4 × 10 5 M -1 ) [ 44 , 46 ] and the concentrations of Tsr and CheR used here. Thus, the increases in rate produced by CheW cannot be accounted for by an increase in the recruitment of CheR to the membrane. Possibility ii , that tethered CheR could access a larger number of methylation sites when CheW is bound, is consistent with the observations, because as noted above, CheW promotes receptor clustering [ 32 ]. Electron microscopic images of cells and isolated membranes with overexpressed Tsr indicate that receptor molecules are packed closely together in the membrane [ 7 , 47 ]. CheW binding may either promote further clustering or a change in the arrangement of receptors that are already clustered, which serves to increase the number of methylation sites that can be accessed by tethered CheR molecules. To generate the rate increase, this larger number of sites must lead to a larger number of active site – methylation site encounters per unit time. Because of the complexity of these samples we cannot yet be specific about how this might occur, i.e. changes in the interactions between receptor dimers, between CheW and CheA, and between MCPs and CheW (and CheA) could all be involved. If binding CheW produces a change in the arrangement of Tsr dimers, either via clustering or some other mechanism, without generating a significant shift in the equilibrium between the kinase-activating and kinase-inhibiting receptor states, then the logical inconsistency presented at the beginning of the section is circumvented. In this respect, possibility iii , which postulates an increase in the probability of methyl group transfer, seems less likely, although we cannot formally exclude it. If CheW binding were to produce a change in the conformation and/or dynamics of Tsr, which is a consequence of possibility iii , it seems likely that this would shift the equilibrium between the kinase-activating and kinase-inhibiting states in a manner inconsistent with the experimental results. This line of reasoning is also predicated on the expectation that serine binding shifts the equilibrium toward the kinase-inhibiting/methylation-active state, a state in which the receptor dimer is a better substrate for methylation. Consequently, the effects of CheW and serine are multiplied, because each new site to which CheR has access as a result of CheW binding also has a better chance of being methylated due to serine binding. Overall, an influence of CheW binding that increases the number of methylation sites accessible to CheR provides a plausible explanation of the results. Implications for adaptation Adaptation is the ability of a cell to return toward its prestimulus behavior following an increase or decrease in a stimulus. For E. coli adaptation is (near) perfect; that is the return to the prestimulus swimming behavior (tumble frequency) following a persistent attractant stimulus is complete (in most circumstances). This property is robust [ 48 ], i.e. the extent of adaptation is not strongly influenced by the relative amounts of receptor and methyltransferase in the cell [ 49 ]. Yet the chemotactic ability of E. coli is tolerant to variations in the adapted state; bacteria with significantly different adapted state tumble frequencies can be similarly efficient in chemotaxis assays [ 50 ]. Moreover the kinetics of adaptation are not a robust property of the system; the time required to return to the prestimulus behavior is strongly influenced by relative abundances of receptor and methyltransferase [ 48 , 49 ]. Taken together, these observations suggest that the kinetics of adaptation is more essential than either the specific value of the adapted state tumble frequency or a requirement for perfect adaptation. The large influence that CheW can have on the methylation rate suggests that it plays an important role in determining the kinetics of adaptation in the cell. To achieve adaptation to a specific ligand, it seems necessary for changes in methylation level to occur primarily on the receptors to which the ligand binds. This requires ligand-specific increases in methylation [ 43 ], and perhaps also receptor-specific decreases in receptor demethylation. CheW appears to play an important role here, because the absolute difference in rate between serine-stimulated and unstimulated rates are larger in the presence of CheW. Also, the ligand-specific responses observed in transmethylation experiments involving Tar and Tsr [ 43 ] are not affected by the presence of CheW (F. Antommattei, unpublished observations ). Finally, we noticed that site four differs from the other major sites in two respects: it is the most rapidly methylated site (under most conditions) and it is influenced least by ligand. In contrast, sites one, two and three are methylated more slowly, but are influenced more significantly by serine, especially with CheW present. In E. coli , Tsr is the most abundant receptor, and Tsr together with Tar comprise the large majority of receptors in the cell [ 34 ]. Its abundance suggests that Tsr has a strong influence on the kinetics of adaptation and setting the tumble frequency of the adapted state. In this respect, it appears that three of the major sites are involved in serine-specific adaptation, while site four, which is influenced little by ligand, plays a role in buffering the methylation level on all the MCPs in the cell. Conclusion It has been shown that the binding of CheW to an MCP (Tsr) substantially increases the rate of receptor methylation. In addition, because the serine-stimulated increases in the methylation rates are magnified by CheW, the specificity of ligand-stimulated methylation is increased. Given the importance of methylation and demethylation kinetics to adaptation and efficient chemotaxis, these findings suggest that CheW has an important role in methylation, in addition to its known role in CheA regulation. The effect of CheW is consistent with a change in the arrangement of receptor dimers, e.g. by clustering, which increases the access of CheR to more sites via the transmethylation mechanism. Methods Plasmids and biochemical reagents The IPTG-inducible E. coli tsr expression plasmids pHSe5. tsr QEQE and pHSe5. tsr EEEE were used to generate membrane samples of Tsr in the wildtype (Q297, E304, Q311, E493) and unmodified (E297, E304, E311, E493) levels of covalent modification at the major modification sites, respectively [ 17 ]. The single site tsr expression plasmids, pAC02, pAC01, pAC03, and pAC04, were constructed from either pHSe5. tsr QEQE or pHSe5. tsr QQQQ by site-directed mutagenesis with the Quickchange™ procedure (Stratagene, Inc.) to express Tsr proteins in which only one of the major methylation sites coded for a glutamate residue (E297, E304, E311, or E493, respectively), while the three remaining sites coded for glutamine residues. These four forms of Tsr are referred to throughout this paper as Tsr EQQQ , Tsr QEQQ , Tsr QQEQ and Tsr QQQE , respectively. pAC04 was subjected to an additional round of PCR mutagenesis to change the codon corresponding to glutamate-492 to glutamine, which resulted in a plasmid (pAC05) that directed the expression of Tsr QQQE -E492Q. The identities of these tsr constructs were verified by sequencing (Davis Sequencing, Davis, CA). [ 3 H- methyl ] SAM (16.1 Ci/mmol) was obtained from Amersham Biosciences. Other chemicals were reagent grade and were obtained from either Sigma Chemical Co. or Fisher Scientific. Preparation of Tsr-containing inner membranes Inner membrane vesicles were prepared as previously described [ 3 , 12 ]. Cells expressing Tsr were grown in LB medium supplemented with 150 mg/L ampicillin at 33°C. Expression was induced with 0.5 mM IPTG in mid log phase cultures, 3 hours prior to harvest. Vesicles were prepared by osmotic shock, isolated on sucrose gradients of 30, 40, and 50%. The middle fraction, of three, was collected and analyzed on Coomassie-stained 12.5% SDS-PAGE for purity. Tsr concentrations were determined by scanning densitometry against an affinity-purified Tsr standard. Single-use aliquots of vesicles were stored at -75°C in buffer (10 mM Tris-HCl, 1 mM EDTA, 1 mM PMSF, and 5% w/v Glycerol, pH 8.0). Protein purification CheR and CheW were purified according to published procedures [ 36 , 45 ]. The purified proteins were stored -75°C in 20 mM Tris-HCl, 1 mM βME, 1 mM PMSF, pH 8.0 (CheR) and 10 mM piperizene, 100 mM NaCl, 0.5 mM EDTA, pH 6.0 (CheW). Concentrations were estimated by the Lowry assay (D C Protein Assay, Bio-Rad Laboratories) according to the manufacturer's instructions. Methylation assays In vitro methylation was measured at 25°C and was initiated by mixing Tsr-containing membranes with [ 3 H methyl] SAM and CheR in methylation buffer (100 mM sodium phosphate, 5 mM EDTA, 2 mM βME, pH 7.5), to generate assay samples with Tsr, SAM and CheR concentrations of 8, 9 and 1 μM, respectively, in a reaction volume of 100 μL. When the effects of ligands were tested, CheW and/or serine were added 30 and/or 5 minutes prior to the initiation of the reaction, respectively. At various times after initiation, 20 μL aliquots were removed and quenched with 10 μL 3X SDS-sample buffer followed by 4 minutes at 100°C. Fourteen microliters of the quenched and cooled reaction aliquots were resolved on 10% SDS-PAGE, then stained briefly with Coomassie to identify the Tsr bands, which were excised, placed in scintillation vials with 1 mL 1 M NaOH, and overlaid with 3 mL scintillation fluid. The samples were incubated overnight before measuring 3 H content by scintillation. All data presented in Results were compiled from duplicate experiments, conducted with independently prepared samples, and uncertainties were either represented by the standard errors of the mean from these duplicates or based on error propagation. List of abbreviations β-ME, β-mercaptoethanol; CheA, autophosphorylating kinase; CheB, methylesterase; CheR, methyltransferase; CheW, SH3-like adaptor protein; IPTG, isopropyl β-D-thiogalactopyranoside; LB, Luria broth; MCP, methyl-accepting chemotaxis protein; MH, methylation helix; PMSF, phenylmethylsulfonyl fluoride; SAM, s -adenosyl-L-methionine; SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis; Tar, aspartate receptor; Trg, ribose-galactose receptor; Tsr, serine receptor; TM, transmembrane segment. Authors' contributions AC carried out preparation and purification of the Tsr inner membrane samples, designed and conducted the methylation experiments and participated in drafting the manuscript. RMW conceived the study, participated in its design, and coordinated and drafted the manuscript. Both authors read and approved the final manuscript.

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1079851

Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis

Background Outbreaks with mass mortality among common carp Cyprinus carpio carpio and koi Cyprinus carpio koi have occurred worldwide since 1998. The herpes-like virus isolated from diseased fish is different from Herpesvirus cyprini and channel catfish virus and was accordingly designated koi herpesvirus (KHV). Diagnosis of KHV infection based on viral isolation and current PCR assays has a limited sensitivity and therefore new tools for the diagnosis of KHV infections are necessary. Results A robust and sensitive PCR assay based on a defined gene sequence of KHV was developed to improve the diagnosis of KHV infection. From a KHV genomic library, a hypothetical thymidine kinase gene (TK) was identified, subcloned and expressed as a recombinant protein. Preliminary characterization of the recombinant TK showed that it has a kinase activity using dTTP but not dCTP as a substrate. A PCR assay based on primers selected from the defined DNA sequence of the TK gene was developed and resulted in a 409 bp amplified fragment. The TK based PCR assay did not amplify the DNAs of other fish herpesviruses such as Herpesvirus cyprini (CHV) and the channel catfish virus (CCV). The TK based PCR assay was specific for the detection of KHV and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions. The TK based PCR was compared to previously described PCR assays and to viral culture in diseased fish and was shown to be the most sensitive method of diagnosis of KHV infection. Conclusion The TK based PCR assay developed in this work was shown to be specific for the detection of KHV. The TK based PCR assay was more sensitive for the detection of KHV than previously described PCR assays; it was as sensitive as virus isolation which is the golden standard method for KHV diagnosis and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions.

Background Common carp Cyprinus carpio carpio is the most widely cultivated fish for human consumption mainly in Asian and in European countries including Israel [ 1 ]. The subspecies Cyprinus carpio koi or koi is cultivated as an expensive pet fish especially in Japan but also worldwide [ 2 ]. Since 1998, mass mortality among common carp and koi has occurred in the U.S.A, Israel, Germany, England, Italy, Netherlands, and more recently in Indonesia and Japan [ 3 - 8 ]. These outbreaks were due to a deadly infection with a newly recognized virus, the koi herpesvirus (KHV) [ 5 ]. Intensive fish culture, koi shows, domestic and international trading in the absence of health certifications or inspections have contributed to the rapid global spread of KHV [ 9 ]. Initial characterization of the virus showed that KHV resembles members of the family Herpesviridae although the virus was clearly different from two previously described herpes-like viruses from fish, Herpesvirus cyprini (CHV) and the channel catfish virus (CCV) [ 5 ]. Further characterization, including taxonomic relationships of KHV to other viruses will be dependent on more complete genome analyses. The definitive diagnosis of KHV is based on the isolation of the virus from infected fish and is time consuming. Therefore a polymerase chain reaction (PCR) assays to specifically detect the virus in fish tissues was developed by Gilad et al . and Gray et al . [ 10 , 11 ] and currently these tests are used to complement or even replace virus isolation as a means to detect KHV infections in koi and common carp. The PCR assays based on KHV DNA sequences fortuitously chosen and with no defined function [ 10 , 11 ] are more prone to lose their diagnostic value due to random mutations or deletions than a PCR assay based on sequences of essential virulence or structural genes. We identified from a genomic KHV DNA library an open reading frame (ORF) that encodes a protein with significant homology to thymidine kinase (TK) and developed a new PCR assay based on these sequences that is shown here to be significantly more sensitive than the previously described PCR assays. Results and discussion Characterization of the KHV TK When analyzing the sequence of the clones of the genomic KHV library, we identified a hypothetical thymidine kinase gene (AJ535112). At the DNA level, a low homology (Expect = 6 e-05 ) was found only with the TK of Leishmania major Friedlin (chromosome 21 cosmid L6294) (AL354533). However, when translating the DNA sequence into deduced amino acid sequences, similarities were found between the hypothetical KHV TK and the Trypanosoma brucei thymidine kinase (AF395663), the TK of Leishmania major (AL354533), and the TKs of Poxviridae (AF163844 and U94848) (Figure 1 ). We could not find any significant similarity between KHV TK and the equivalent genes in any described fish viruses. Southern blot hybridization with the labelled TK gene showed definitively that the KHV TK gene hybridized only with the DNA of KHV and not with carp DNA (Figure 2 ). The KHV TK gene had an open reading frame of 216 amino acids encoding a polypeptide with a putative molecular mass of 23.7 kDa. The similarity to eukaryotic TK may reflect a gene acquired from the host [ 12 , 13 ]. The proper nomenclature of the KHV will be solved by a complete genome analysis and not by the analysis of a single gene like in this study or by the histopathology performed on diseased fish [ 6 ]. The KHV TK gene was expressed in vivo in experimentally infected KF-1 cells as shown by RT-PCR analysis. The mRNA of the TK gene was not detected during the first 7 hours of infection (data not shown) but was detected for at least 7 days after infection (Figure 3A ). By Southern hybridization, we proved that the fragment amplified by RT-PCR was indeed the TK KHV gene (Figure 3B ). The KHV TK gene cloned into plasmid pYB1 was over expressed in E. coli ER2566. SDS-PAGE analysis of the recombinant TK showed a unique band of about 24 kDa, in agreement with the size predicted from the DNA sequence (23.7 kDa) (data not shown). Enzyme activity was determined using tritium labeled deoxy thymidine and deoxy cytidine as substrates. [ 14 ]. Preliminary analyses show that the enzyme is specifically active using methyl- 3 H Thymidine (dThd) but had no activity on deoxy (5- 3 H)cytidine (dCyd) similar to other herpesvirus TK. Acyclovir (Samchully Pharmaceuticals South Korea) and Gancyclovir (Hoffmann La Roche Basel Switzerland) in up to 100 molar excess over dThd, did not compete at all with the substrate. TK based PCR assay TK has been shown to be essential for virulence in herpes viruses [ 15 ] and therefore is a good candidate for the development of a PCR assay. The KHV infection is an emerging viral disease for common carp and koi. The highly contagious nature of this disease will unfortunately continue to negatively impact the aquaculture industry by increasing the mortality and restrictions on product movements. Rapid diagnosis of the virus is therefore essential for the control of KHV outbreaks. Currently, KHV outbreaks are assessed by multifactorial analyses comprising the post mortem observation of gross pathology lesions, the dynamics of mortality in all sizes of fish, the water temperature and the continuing mortality despite chemotherapy. The gold standard method for the diagnosis of KHV infection is based on virus isolation using KF-1 cells followed by PCR testing of the virus isolate [ 10 ]. This protocol is time consuming and therefore PCR assays alone have been suggested as possible substitutes for diagnosis of KHV disease [ 10 , 11 , 16 , 17 ]. Based on the TK sequence a newly developed PCR assay using the primers, KHV-TKf (5'-GGGTTACCTGTAC GAG-3') and KHV-TKr (5'-CACCCAGTAGATTA TGC-3') has been developed. Under optimal conditions (see Methods) this PCR assay amplified a 409 bp fragment when KHV DNA was used as template (Figure 4 ). This assay was specific for KHV and did not amplify any fragment when using CCV, CHV or KF-1 cell line DNAs as DNA templates (data not shown). A comparison of the sensitivity of two published PCR assays to the TK PCR assay (Table 1 ) using for each assay the conditions described by the authors Gilad et al. [ 10 ] and Gray et al. [ 11 ] are shown in Figure 4 and Table 2 . Using purified KHV DNA as template, the TK PCR assay, whose product was visualized by agarose gel electrophoresis, ethidium bromide staining and UV illumination, was able to detect as little as 10 fg of KHV DNA (Figure 4 ). Preliminary unpublished data showing that the genome size of KHV is around 290 kb (R. Nahary and H. Bercovier, unpublished data) is in accordance with that estimated by Ronen et al. [ 18 ] indicate that the 10 fg detected by the TK PCR assay correspond to approximately 30 virions. The two previously described PCR assays were consistently 10 to 1000 times less sensitive (Figure 4 ) than the new TK PCR. Similar results were obtained when DNA extracted from kidneys of diseased fish were tested by the three PCR assays (data not shown). Recently, real time PCR assay [ 16 ] and LAMP assay [ 17 ] have been developed for the detection of KHV DNA. The sensitivity of these two methods was not shown to be much higher than that obtained by the TK based direct PCR assay developed in this work. In addition to assessments of the specificity and sensitivity of the PCR assays, the new assay was compared to virus isolation from koi with different histories in relation to KHV infection. Koi were examined from ponds with active disease, or that had been voluntarily exposed to a KHV challenge and that were with or without disease signs at different times after the experimental exposure. Lastly, populations of koi surviving KHV in Israel were also examined. The survivor groups were the result of a procedure of exposing fish to experimental KHV infection at different seasons of the year and different water temperatures [ 18 ] with the claim that they developed resistance to KHV challenge and were free of virus (Table 2 ). The presence of KHV virus or KHV DNA in the kidney tissues of freshly collected koi was determined with the three PCR assays and the virus isolation method. The results showed that only the TK based PCR assay paralleled the virus isolation method (7 samples positive with both techniques and 6 samples negative with both) proving its sensitivity and its usefulness (Table 2 ). The two other PCR assays were similar to each other in their performances and did not produce any false positive results but resulted in false negative results (5 false negative) when compared to TK based PCR assay or virus culture. We were able to detect KHV and its DNA in fish that were intentionally infected even two months after the experimental infection (Table 2 ). These data match with the results obtained by Gilad et al. [ 16 ] using a Taqman PCR assay to detect the presence of KHV DNA in experimentally infected koi and raise the question of potential carriers in fish exposed to KHV in order to obtain immune survivors. The kidney was selected as a representative organ for virus and DNA detection because it was shown previously that KHV is found in this organ [ 10 , 11 , 16 ]. Aliquots of three kidney samples that were positive with the three PCR assays were frozen and were shown also to be positive after 3 months storage at -70°C using the three PCR assays. However the TK based PCR assay was always able to detect viral DNA in the positive samples at a 10 to 100 higher dilution than the two other PCR assays (data not shown) confirming by this its sensitivity and robustness. These first field evaluations show the advantage of the TK based PCR for detection of KHV DNA as compared to two previously described PCR assays. The TK PCR assay is based on an essential virulence gene that has little chance to be deleted and is therefore potentially more stable/robust than the published PCR assays based on KHV sequences of no defined function [ 10 , 11 ]. These initial trials show that the three PCR assays do indeed give different sensitivities with TK-PCR clearly performing the best. It was more sensitive, and unlike the other assays was able to detect viral DNA in fish samples even two months after experimental exposure to KHV. KHV DNA was not detected among koi two years post experimental exposure if survivors were not held in an infected environment. The carrier status of the survivor population remains unclear. Negative results by PCR may or may not indicate freedom of KHV infection or a viral load lower than the detection threshold. In conclusion, in all cases in our study, the TK PCR was the only direct PCR assay that always paralleled the gold standard viral isolation method. As the PCR assay is easier and less time consuming, this newly developed TK PCR assay is an excellent candidate for a simple and rapid diagnosis of KHV infection. Conclusion The TK based PCR assay developed in this work was shown to be specific for the detection of KHV. The TK based PCR assay was as sensitive as virus isolation which is the golden standard method for KHV diagnosis, and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virion particles. It was found to be 10 to 1000 times more sensitive for the detection of KHV than previously described PCR assays. Although Real Time PCR and LAMP technologies may be as sensitive if not more, than the direct PCR assay developed in this study, they have not been yet tested in field conditions and they require, at least for the real time PCR method, more sophisticated equipment and more expensive reagents than the direct PCR. Methods Viral strains, virus isolation and DNA extraction KHV-I originating from koi in Israel [ 10 ] was propagated on the koi fin cell line (KF-1) which supports the growth of KHV as described by Hedrick et al . [ 5 ]. Cells were incubated at 20°C. The channel catfish herpesvirus (CCV) strain CA80-5 served as the unrelated herpes-like virus control and was propagated on channel catfish ovary (CCO) cell line. The CCO cell line was grown in minimum essential media (MEM) supplemented with 7.5% fetal bovine serum (FBS), 50 IU penicillin/ml, 50 μg streptomycin/ml, and 2 mM L-glutamine (MEM-7.5). The FBS concentrations of the growth medium were reduced to 2 % (MEM-2) when CCO cells were infected with CCV. Cells were incubated at 25°C following inoculation with CCV. The CHV DNA was a gift from Dr. T. Sano and Dr. H. Fukuda, Tokyo University of Fisheries [ 19 ]. Viral and cell DNAs were extracted according to Gilad et al . [ 10 ]. KHV was isolated from koi head kidney tissues of infected or diseased fish according to the method described by Hedrick et al. [ 5 ] with a slight modification. Replicate wells of a 12-well tissue culture plate containing monolayers of KF-1 cells were inoculated with 0.2 ml of a 1:10 dilution (volume /volume or weight/volume) of the original head kidney homogenate. After an adsorption period of 1 h, 2 ml of MEM-2 containing 0.015 M N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), 50 IU of penicillin/ml, 50 μg streptomycin/ml, and 2 mM L-glutamine were added to each well and the plates were incubated at 20°C. The plates were observed daily for evidence of cytopathic effects (CPE). After 7 days, if there was no CPE, the medium was used to infect new KF-1 cells and incubated as above. Three such blind passages were performed before a clinical sample was described as negative for virus isolation. Cloning of the thymidine kinase KHV genomic DNA fragments following digestion with Sau 3AI were separated on 1% agarose gels adjacent to a DNA ladder. DNA fragments in the 3–6 kb region were extracted from the gel, purified and ligated overnight at 4°C into a predigested Bam HI site of the multiple cloning site arms of the Lambda Zap Express Vector (Stratagene, La Jolla, CA, USA.). The Lambda Zap Express vector allows in vivo excision and recircularization of the cloned inserts within the lambda vector to form a phagemid containing the cloned insert. Packaging of the Lambda Zap Express vector and excision of the pBK-CMV phagemid with the inserts from the Lambda Zap-Express vector were done according to the manufacturer's instructions. E. coli XLOLR colonies containing the recombinant phagemids were isolated, grown overnight in LB -Kanamycin broth at 37°C and the plasmids were extracted using the Wizard plus SV miniprep kit (Promega, Madison WI, USA). One hundred clones with an average 3 kb insert were isolated and purified. Sixty clones have been sequenced using primers T3 (5'-AATTAACCCTCACTACTAA GGG-3') and T7 (5'-TAATACGACTCACTATAGGG-3') found on both sides of the BamH I insertion site. Inserts were completely sequenced by walking using relevant primers. The sequences of 60 pBK-CMV inserts were compared with the complete virus protein and nucleotide database from the National Center for Biotechnology Information using the BLASTX and BLASTN programs [ 20 ] and the Mac Vector software (Oxford Molecular Ltd. San Diego CA. USA). Characterization of the recombinant thymidine kinase The hypothetical TK gene was amplified by PCR from purified genomic KHV DNA using the primers: IMPTK5 (5'-GTTGTTCATATGGCTATGCTGGAACTGGTG-3') and IMPTK3 (5'-GTTGTTTGCTCTTCCGCACAGGATAGATATGTTACAAGAA CG-3'). To prove that the sequences of the amplified TK originated from KHV DNA and not from a contaminating agent or fish cellular sequences, Southern blot analyses were performed. The TK gene was labeled with digoxigenin (DIG) (Roche Applied Science, Mannheim Germany) according to the manufacturer instructions. Purified TK DNA and Carp DNA (1–2 μg) were incubated with 10 U of BsaA1 or with Pvu II that cut only once within the TK sequence for 4 h at 37°C. DNA fragments were separated by electrophoresis on 1.0% agarose gels and observed after staining with 1% ethidium bromide. DNA was transferred to positively charged nylon membranes (Roche Applied Science, Mannheim Germany) and treated as recommended by the manufacturer (Roche Applied Science, Mannheim Germany). Prehybridization was performed in 10 ml DIG easy hyb solution (Roche Applied Science, Mannheim Germany) within a roller bottle at 65°C for 30 min. Hybridization was carried out in 6 ml of DIG easy hyb containing 250 ng of DIG labeled probe at 65°C overnight. Membranes were washed twice with low stringency wash buffer (2 × SSC+0.1% SDS) at room temperature followed by two washes with high stringency wash buffer (0.1 × SSC +0.1% SDS) at 60°C. The membrane was blocked for 30 minutes with 40 ml blocking solution and then incubated with a 1:10000 dilution of anti Dig AP conjugated antibody (Roche Applied Science, Mannheim Germany). After washing, the membrane was placed on a plastic film, evenly covered with 500 μl of ready-to-use chemiluminescent alkaline phosphatase substrate (CSPD) (Roche Applied Science, Mannheim Germany), and incubated for 5 min. at room temperature. The membranes were then exposed to super RX X-ray film (Fuji photo film, Tokyo, Japan). The amplified TK gene was cloned into vector pYB1 (Impact system, New England Biolabs, Beverly MA. USA) with an intein tag that allows the purification of the recombinant protein in a non-denaturated form without any extra residues. The TK protein was expressed in E. coli ER2566 and purified according to the manufacturer's instructions (New England Biolabs). After affinity purification on a chitin column, SDS-PAGE analysis was performed to characterize the recombinant TK and the enzymatic activity of the recombinant TK was tested. Thymidine kinase activity was determined by the DE-81 filter paper assay using tritium labeled deoxy thymidine and deoxy cytidine as substrates. [ 14 ]. A typical reaction mixture contained in 100 μl of Tris-Cl (140.0 mM) MgCl 2 (2.0 mM), DTT (1.7 mM), NaF (8.0 mM), PMSF (5.0 mM), ATP (10.0 mM), KHV TK enzyme, and substrates methyl- 3 H thymidine (dThd) (25 ci/mM) or deoxy(5- 3 H)cytidine (dCyd) (22 ci/mM) (Amersham Biosciences, UK). Substrates were used at concentrations between 5–30 mM. Reactions were carried out at 37°C. At 15 seconds intervals, 20 μl samples were removed, and spotted onto the filters. The filters were washed 4 times in 5.0 mM ammonium formate followed by a 15 min wash in Ethanol 95 %. Amount of radioactivity was determined in a scintillation counter (LS 6000TA, Beckman Instruments Fullerton CA. USA). To demonstrate that the KHV TK is expressed in an experimentally infected cell line, total RNA was extracted using the SV total RNA isolation system (Promega, Madison WI. USA) from KF-1 cells infected with KHV. Uninfected KF-1 cells served as a control. RT-PCR (Access RT-PCR system, Promega, Madison WI. USA) was performed using internal primers of the TK gene (Upstream primer TK-RTf: (5'-ATGGCTATGCTGGAAC-3'); downstream primer TK-RTrl: (5'-TGGAGCGGCT GACACG-3') on experimentally infected KF-1 cells at different time points post infection. PCR products were analyzed on 1% agarose gel. The identity of the amplified products was confirmed by Southern blot hybridization with the labeled KHV TK gene as described above. PCR assays Head kidneys were removed from koi carp infected naturally or intentionally by KHV in order to obtain survivors in Israeli farms [ 18 ]. Template DNA from head kidney of koi carp was prepared using High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim Germany) according to the manufacturer's recommendations. A TK based PCR assay to detect KHV DNA was developed using specific primers, KHV-TKf (5'-GGGTTACCTGTACGAG-3') and KHV-TKr (5'-CACCCAGTAGA TTATGC-3'), derived from for the thymidine kinase gene (AJ535112) that produced a 409 bp amplicon. The TK based PCR was done using the above primers for 35 cycles of amplification under standard conditions (95°C 5 min, then 35 cycles: 95°C 30 sec, 52°C 30 sec, 72°C 1 min and finally 72°C 10 min). Two other PCR assays were performed in parallel according to their original description and are denominated Gilad PCR (400 bp product obtained with Primers (5'-GACGACGCCGGAGACCTTGTG-3') and (5'-CACAAGTTCAGTCTGTTC CTCAAC-3') [ 10 ] and Gray PCR (290 bp product obtained with primers (5'-GACACCACATCTCAAGGG-3') and (5'-GAC ACATGTTACAATGGTGGC-3') [ 11 ] (Table 1 ). The specificity of the PCR assays (discrimination of KHV from other viruses) was assessed as described by Gilad et al. [ 10 ]. A total of 50 ng of DNA obtained from purified KHV-I, CHV, CCV, or uninfected KF-1 cells were used as templates for the PCR assays. The sensitivity of the PCR assays was determined using 10 fold serial dilutions from 10 ng to 0.001 pg of KHV-I DNA and 10 fold serial dilutions of head kidneys (boiled in distilled water for 5 min) from diseased koi carp with proven virus isolation or the extracted DNA. Whole tissue or DNA extracted from negative head kidney samples from uninfected koi carp were run as controls. The resulting amplified products were analyzed by electrophoresis on 1% agarose gels after staining with ethidium bromide. Authors' contributions HB, AE and RPH conceived the study, and participated in its design and coordination and drafted the manuscript. RN, OG, SS and AZ carried out the molecular studies (DNA preparation, PCR development and calibration) and participated in the sequence alignment. YF cloned and expressed the recombinant TK and carried out the enzymatic assays. ME prepared virus cultures and performed PCR assays in diseased fish. All authors read and approved the final manuscript.

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1079852

Identification to the species level of Lactobacillus isolated in probiotic prospecting studies of human, animal or food origin by 16S-23S rRNA restriction profiling

Background The accurate identification of Lactobacillus and other co-isolated bacteria during microbial ecological studies of ecosystems such as the human or animal intestinal tracts and food products is a hard task by phenotypic methods requiring additional tests such as protein and/or lipids profiling. Results Bacteria isolated in different probiotic prospecting studies, using de Man, Rogosa and Sharpe medium (MRS), were typed at species level by PCR amplification of 16S-23S rRNA intergenic spacers using universal primers that anneal within 16S and 23S genes, followed by restriction digestion analyses of PCR products. The set of enzymes chosen differentiates most species of Lactobacillus genus and also co-isolated bacteria such as Enterococcus , Streptococcus , Weissella , Staphylococcus , and Escherichia species. The in silico predictions of restriction patterns generated by the Lactobacillus shorter spacers digested with 11 restriction enzymes with 6 bp specificities allowed us to distinguish almost all isolates at the species level but not at the subspecies one. Simultaneous theoretical digestions of the three spacers (long, medium and short) with the same set of enzymes provided more complex patterns and allowed us to distinguish the species without purifying and cloning of PCR products. Conclusion Lactobacillus isolates and several other strains of bacteria co-isolated on MRS medium from gastrointestinal ecosystem and fermented food products could be identified using DNA fingerprints generated by restriction endonucleases. The methodology based on amplified ribosomal DNA restriction analysis (ARDRA) is easier, faster and more accurate than the current methodologies based on fermentation profiles, used in most laboratories for the purpose of identification of these bacteria in different prospecting studies.

Background Probiotics are food or preparations containing live microorganisms, traditionally regarded as safe for human use. When ingested in sufficient numbers, probiotics are believed to play an important role in the control of host intestinal microbiota and maintenance of its normal state [ 2 ]. Microbes that are frequently isolated from human and animal intestines and selected as probiotics, include species of the genera Lactobacillus , Bifidobacterium , and Enterococcus . However, some other lactic acid producing bacteria that do not normally inhabit the intestinal tract are also sometimes used as probiotics. Most of these bacteria are used as starters in dairy products and include Streptococcus , Lactococcus , Leuconostoc and Pediococcus species. Yet, as different types of lactic acid producing bacteria can affect the human intestinal microenvironment in different ways, it is important to identify which microorganisms are present in a microbial ecosystem and which species are most likely to have the potential protective effects. The precise identification of these bacteria to the species level is not an easy task. As pointed out by Tannock et al. [ 10 ], the identification of Lactobacillus isolates by phenotypic methods is difficult because it requires, in several cases, determination of bacterial properties beyond those of the common fermentation tests. In general, about 17 phenotypic tests are required to identify an isolate of Lactobacillus accurately at the species level. Most commercial kits, frequently used in laboratory routine, are able to precisely identify only 30% of vaginal and intestinal isolates from humans [ 8 ]. The establishment of a simple and fast identification method is therefore required in order to be able to deal with the large numbers of Lactobacillus isolates that can be obtained during microbial ecological studies of ecosystems such as the human or animal intestinal tracts and food products. In addition to Lactobacillus , other lactic acid bacteria (LAB) and several other groups of bacteria are found in the same habitats during ecological studies of microbiota succession. Methodological procedures to handle such a high number of isolates should be brief and precise to avoid inconclusive results. Due to the growing interest in using bacteria as probiotics or starters in dairy products, the identification of these microorganisms at species level is becoming more and more required. DNA sequencing of PCR amplified conserved genes such as rRNA ones is the most used methodology for typing microorganisms. However, it requires special equipments and dealing with hundred of different isolates is expensive. The aim of this study was to develop an easy and fast procedure to accurately identify to the species level most of Lactobacillus type and reference strains and new strains isolated from different ecological studies. In these different prospecting studies, we are looking for new Lactobacillus strains to be used as 1) a vehicle to delivery chimerical transgenes to immunize broiler chicks against coccidiosis, 2) antagonizers of pathogenic bacteria during cheese and cassava starch processing to improve food safety and quality, and 3) live medicine against women vaginosis and vaginitis. Therefore, the correct typing of these new strains is critical. Analysis of the number and size of the intergenic regions between 16S and 23S genes showed that they vary among Lactobacillus and others bacteria allowing genera discrimination (unpublished results). Other groups have published methodologies based on the fingerprinting of the variable region of the 16S rRNA [ 14 ]. We choose to amplify the 16S-23S rRNA intergenic spacers because this region allowed us to discriminate a larger number of species of Lactobacillus . Typical patterns of Lactobacillus amplicons presented three bands, corresponding to the short, medium and long spacer regions, similar to those seen in Pediococcus , Carnobacterium , Leuconostoc and Weissella species [ 4 ]. These differences in size are due to the insertion of a tRNA-Ala gene within the medium spacer or tRNA-Ala and tRNA-Ile genes within the long spacer. Bifidobacteria, enterococci, streptococci and staphylococci presented patterns with a unique band or two bands with different sizes also due insertion of tRNA genes. The methodology proposed here can replace fermentation tests for characterization of a number of clinical, health food, and laboratory isolates through PCR amplification of 16S-23S rRNA intergenic spacers followed by digestion with a set of restriction enzymes with 6 bases recognition sequences. Results The in silico predictions of restriction patterns of 16S-23S rRNA short intergenic spacer of selected Lactobacillus species and of other related bacteria frequently isolated from the same sources of Lactobacillus are shown in Tables 1 and 2 , respectively. Although for most bacteria different patterns can be predicted, some species present identical patterns. In this case, however they can be differentiated by the sizes of the two amplified bands ( E. faecium amplicons are almost 100 bp larger than E. faecalis amplicons). Fig. 1 shows a typical gel of the short intergenic spacer of Lact. rhamnosus GG digested with 11 restriction enzymes, in which DNA cleavage was observed with Hind III, Dra I and Eco RV. The uncut DNA at Dra I lane may reflect some polymorphism of the short spacer nucleotide sequences in different operon units. The patterns obtained with digestion of the 16S-23S rRNA short spacer alone could distinguish most of Lactobacillus at species level but, in some cases, such as among Lact. acidophilus , Lact. helveticus and Lact. amylovorus , they could only be grouped as 'acidophilus' clad. The separation of acidophilus group species could be achieved by nucleotide sequencing or by digestion of the others two intergenic spacers in order to search for more complex restriction patterns. Fig. 2 shows a gel with simultaneous digestion of the three 16S-23S rRNA intergenic spacers of Lact. acidophilus ATCC 4356 with the same set of enzymes used to digest the short spacers. The three spacers of Lact. acidophilus were cut with Nco I, Eco RI and Hind III enzymes, but only the long spacer was additionally digested with Sfu I. Looking at sequences deposited at GenBank, we observed that the long spacer of Lact. helveticus have a site for Sfu I, but unlike Lact. acidophilus , it can also be cut with Dra I, a feature that can be used to differentiate these species However, this theoretical finding has not yet been validated through PCR-ARDRA. The simultaneous digestion of the three intergenic spacers could therefore improve the distinctiveness of the methodology proposed here, allowing the identification of very closely related species. We tested the methodology of 16S-23S rRNA amplification and restriction digestion in a set of isolates from chicken gastrointestinal tract, human sources, and food. Table 3 shows the digestion patterns of forty-three isolates and their identification at the species level. All bacteria except Lact. gasseri , Lact. johnsonii and Lact. jensenii isolates (Reg 24, 25, 26 and 32 from chicken faeces, and PM1, 2 and 3 from human vagina) could be typed based on the restriction digestion pattern of the short 16S-23S rRNA intergenic region obtained with the 11 enzymes tested. The short spacers of these three Lactobacillus species could be cleaved only with Eco RV enzyme, and to distinguish between each species it was necessary to include the digestion profile of the medium and long spacers cleaved with Hind III, Pvu II and Bgl II. Lact. gasseri spacers were not cleaved with any of these three enzymes, while Lact. johnsonii long spacer could be cleaved with Hind III and Pvu II, and Lact. jensenii long spacer could be cleaved also with Bgl II. To validate the PCR-ARDRA identification, the forty-three short amplicons from those Lactobacillus isolates were cloned and sequenced. The level of identity between aligned sequences was above 98% for all isolates. Searching nucleotide databases we found deposited sequences of long and medium spacers of some Lactobacillus species and also of most species of Carnobacterium and Weissella , two closely related genera. Table 4 exemplifies how simultaneous cleavage of the three spacers of some Weissella species can improve the methodology of PCR-ARDRA proposed here. Discussion The precise identification of microorganisms presenting economical interest is a prerequisite to select new strains among several bacteria isolates. The lactic-acid producing bacteria associated with foods or used as probiotics include species of the genera Lactobacillus , Carnobacterium , Enterococcus , Lactococcus , Leuconostoc , Oenococcus , Pediococcus , Streptococcus , Tetragenococcus , Vagococcus and Weissella . The genus Lactobacillus is heterogeneous, presenting over 60 species showing G+C content ranging from 33 to 55 % [ 9 ]. This high heterogeneity is reflected by phenotypic tests of Lactobacillus isolates from diverse ecosystems, which show intermediate sugar fermentation profiles that hamper a more precise typing at species level. Although several DNA-based procedures are now available, most of them require special techniques or laboratory devices and can also be time-consuming, which makes identification of a hundred isolates a fastidious job. Besides their use as food microorganisms, the use of LAB as bacterial systems to express heterologous proteins or as vehicles to carry immunizing antigens after genetic modification [ 7 ] is becoming a promising issue due to the nutritional benefits and almost null pathogenicity of these bacteria. The successful expression of heterologous proteins in LAB depends on the promoter compatibility between the species or strains used as vector or hosts. As pointed out by Pouwels et al. [ 6 ], the control of transcription and translation may differ greatly between two Lactobacillus species, implying that the knowledge generated for one organism may not simply be transferred to another. Genes that are efficiently expressed in one Lactobacillus species are not necessarily expressed in other species, or are expressed with different efficiency and/or with a different regulatory mechanism. Therefore, the correct typing of new isolates with probiotic properties is crucial for the development of a successful oral vaccine, such as the one we are intending to make against avian coccidiosis delivered by genetically engineered Lactobacillus . As pointed out in Table 1 , a large number of Lactobacillus species can be typed effectively on the basis of the restriction patterns of their 16S-23S intergenic regions, after a selection step using the MRS media. This method constitutes a specific and reproducible way to identify LAB bacteria at the species level. In addition, most of other bacteria genera co-isolated with Lactobacillus in these prospective studies could also be accurately identified through 16S-23S spacer PCR-ARDRA (Table 2 ). Forty-three new strains of Lactobacillus obtained from animals, human or foods could be easily identified by this methodology, without need of gel purification and cloning (Table 3 ). The differences in intensities of the bands corresponding to the three 16S-23S rRNA spacers (Fig. 2 ) is probably due the number of rRNA operons with each spacer. This can also explain the partial digestions of shorter spacers observed occasionally such as the one observe in L. rhamnosus GG digested with Dra I (Fig. 1 ). The simultaneous digestion of all PCR amplicons corresponding to the three Lactobacillus 16S-23S rRNA spacers showed that these regions are very polymorphic and suitable for distinguishing them at species level. Similar polymorphisms were identified in DNA sequences deposited at GenBank of the three 16S-23S rRNA spacers of Weissella species (Table 4 ) and shall probably occur in Leuconostoc , Carnobacterium , Pediococcus and other genera closely related to Lactobacillus . Although we have initially employed the short spacers to distinguish Lactobacillus species as proposed by Tannock et al. [ 10 ], the main improvement described here is a typing protocol that avoids gel purification and nucleotide sequencing of the amplicons. The restriction digestion of the three spacers showed that a very good discrimination of several Lactobacillus and other bacteria could be achieved by direct digestion of PCR products followed by a single gel electrophoresis to verify the restriction pattern of the amplicons. Our procedure is faster and less expensive than the method originally proposed by Tannock's group. However, gel electrophoresis was still necessary to visualize the restricted fragments. To overcome that, we choose restriction enzyme with 6 bases recognition sites, which cut less frequently and, thus, generate simple restriction digestion patterns. Because our goal is to avoid the electrophoresis step, we are currently working on a protocol that could adapt the PCR-ARDRA into a more straightforward method such as a PCR-ELISA. With this protocol, instead of using gel electrophoresis, the positive result of a digestion reaction could be detected colorimetrically, after incorporation of labelled nucleotides, performed simultaneously with the cleavage reaction. In this way, the whole procedure can be carried out in any laboratory equipped with a PCR machine. Conclusion Forty-three Lactobacillus isolated and several strains of other bacteria co-isolated in different prospecting studies to select new bacteria with probiotic features could be processed and identified using the proposed approach. The methodology, based on the small 16S-23S rRNA spacer ARDRA is accurate but needs gel electrophoresis to visualize the digested products. In order to validate the restriction pattern obtained, we also purified and sequenced the amplicons corresponding to the short spacer. The amplification and the restriction digestion of the three spacer regions allowed us to discriminate the Lactobacillus species without the need of gel excision and purification but still requires the electrophoresis step to visualize the digested products. These results represent a first step in the development of an easier and faster protocol that may allow us to detect changes in the restriction patterns using a PCR-ELISA kit. Methods Bacterial strains and growth conditions The following bacteria were used in this study to validate the theoretical restriction patterns of Tables 1 and 2 : Lactobacillus acidophilus ATCC 4356T, Lact. brevis ATCC 367, Lact. casei ATCC 393T, Lact. delbrueckii subsp. lactis ATCC 7830, Lact. fermentum ATCC 9338, Lact. plantarum ATCC 8014, Lact. plantarum NCDO 1193, Lact. reuteri DSM 20016T, Lact. rhamnosus GG ATCC 53103, Lact. sakei NCFB 3714, Bacteroides fragilis ATCC 25285T, Bifidobacterium longum Bb46 and Bif. lactis Bb12 (Christian Hansen Laboratory, Horsholm, Denmark), cheese isolates of Enterococcus faecalis , Staphylococcus aureus , Weissella paramesenteroides , Lactococcus lactis , Streptococcus thermophilus typed by nucleotide sequencing, Escherichia coli XL1-Blue (Stratagene), and Pediococcus pentosaceus ATCC 33314. We are not able to validate the theoretical restriction patterns of several species of Lactobacillus or other bacteria and for most species several sequences were available at GenBank deposited by different research groups. Oenococcus , Carnobacterium , Leuconostoc and Propionibacterium specimens were not typed in this work but theoretical digestion patterns are also presented in Table 2 . Forty-three Lactobacillus isolates from chicken intestines, human vagina or faeces, Brazilian 'coalho' cheese, and cassava starch sour fermentation from collection of the Department of Microbiology, Universidade Federal de Minas Gerais, were typed in this study by PCR-ARDRA as proposed and verified by nucleotide sequencing to confirm species identification. All lactic acid bacteria were stored at -80°C in de Man Rogosa Sharpe (MRS) broth (Difco, Detroit, MI, USA) with 30% glycerol. Before experimental use, cultures were grown anaerobically in MRS medium at 37°C and subcultured twice in MRS. E. coli was grown aerobically in Luria-Bertani medium and Bacteroides anaerobically in BHI medium. DNA extraction Chromosomal DNA was isolated from overnight cultures of Lactobacillus sp in 10 ml MRS broth. After washing the cells with 10 ml of deionised water, the pellet was resuspended in 1 ml of 5 M LiCl and incubated for 1 h under constant shaking. Cells were washed once more with 1 ml of deionised water and the pellet was resuspended in 1 ml of protoplasting buffer (25 mM sucrose, 50 mM Tris HCl pH 8.0, 10 mM EDTA, 10 mg of lysozyme ml -1 , 100 μg of RNaseA ml -1 ). After incubation for 1 h at 37°C and centrifugation at maximum speed in a microcentrifuge for 5 min, the pellet was resuspended in 500 μl of protoplasting buffer without sucrose and lysozyme, and 100 μl of 10% sodium dodecyl sulfate were added to allow cells lysis. The mixture was extracted once with phenol, with phenol-chloroform-isoamyl alcohol (25:24:1) and with chloroform-isoamyl alcohol (24:1). After isopropanol precipitation the DNA was dissolved in 100 μl of TE buffer. PCR amplification of 16S-23S rDNA intergenic spacer The 16S-23S intergenic spacer region amplification was carried out according to Tilsala-Timisjarvi and Alatossava [ 12 ] using the primer 16-1A, 5'-GAATCGCTAGTAATCG-3', corresponding to nucleotides 1361 to 1380 of the 16S rRNA gene according to Lactobacillus casei numbering, and the primer 23-1B, 5'-GGGTTCCCCCATTCGGA-3', corresponding to nucleotides 123 to 113 of the 23S rRNA of L. casei . The reaction mixture (50 μl) contained 10 pmol of each primer, 0.2 mM of each deoxyribonucleotide triphosphate, reaction buffer with 1.5 mM MgCl 2 , 5 U of Taq DNA polymerase (Phoneutria Biotecnologia & Serviços, Brazil), and of 10 ng of template DNA. The amplification program used was: 94°C for 2 min, 35 cycles of 94°C for 30 s, 55°C for 1 min, 72°C for 1 min, and 72°C for 10 min. PCR products were electrophoresed in a 1.4% agarose gel and visualized by UV transillumination after ethidium bromide staining. Restriction digestion The 16S-23S short intergenic spacer regions of the Lactobacillus bacteria were digested by a set of 11 enzymes chosen after compilation of nucleotide sequences already deposited at GenBank and theoretical restriction digestion with Webcutter 2.0 tool (Max Heiman, ). Sph I, Nco I and Nhe I enzymes cleave inside 16S gene, Ssp I, Sfu I, Dra I, Vsp I, Hinc II and Eco RI enzymes cleave inside the intergenic region, and Avr II and Hind III enzymes cleave inside 23S gene. Eco RV enzyme cleaves inside spacer region in Lact. casei group and in the 23S gene in Lact. acidophilus group. All restriction enzymes were purchased from Promega Corporation (Madison, WI, USA). Cloning of the PCR-amplified products Lactobacillus species frequently contain three 16S-23S intergenic regions (long, medium and short spacers), in which case the smallest PCR amplicon (about 500 to 600 bp) was excised from the gel and purified with the Concert™ Rapid Gel Extraction System according to the manufacturer's instructions (Invitrogen Life Technologies, Carlsbad, CA, USA). PCR products were cleaned-up using the Concert™ Rapid PCR Purification System and cloned in E. coli XL1-Blue into pCR2.1-TOPO vector using the Invitrogen TOPO TA cloning kit (Invitrogen). PCR was performed on cell lysates of ampicillin-resistant transformants using M13 specific primers to confirm the size of the inserts. Plasmids containing a unique insert of the appropriate size were purified by the GFX™ Micro Plasmid Prep kit (Amersham Biosciences, Piscataway, NJ, USA) and were subjected to DNA sequence analysis to validate PCR-ARDRA species identification. Sequencing and sequence analysis DNA sequencing of PCR amplicons was carried out at the Núcleo de Análise de Genoma e Expressão Gênica (NAGE), Universidade Federal de Minas Gerais, using a DYEnamic™ ET Dye Terminator Cycle Sequencing kit for MegaBACE™ DNA Analysis Systems (Amersham Biosciences, USA) in combination with a MegaBACE™ 1000 automated sequencing system. Both polynucleotide strands of the cloned DNA were sequenced, using M13 forward and reverse primers. The short intergenic spacer region sequences obtained by these methods were compared to sequences from type culture and other Lactobacillus strains held in GenBank DNA database using the BLAST algorithm [ 1 ] to validate the theoretical restriction patterns used for species identification by PCR-ARDRA. Authors' contributions JLSM and RMM carried out all the experiments used for the molecular biology studies: DNA extraction, PCR amplification, gel electrophoresis, restriction digestion. MFH and SMRT participated in the sequence alignment, the discussion of the results, and the manuscript draft. EN and JRN participated in the study design. ACN conceived and designed the study, and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

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1079853

Alkane-induced expression, substrate binding profile, and immunolocalization of a cytochrome P450 encoded on the nifD excision element of Anabaena 7120

Background Alkanes have been hypothesized to act as universal inducers of bacterial cytochrome P450 gene expression. We tested this hypothesis on an unusual P450 gene ( cyp110 ) found on a conserved 11 kilobase episomal DNA element of unknown function found in filamentous cyanobacteria. We also monitored the binding of potential substrates to the P450 protein and explored the distribution of P450 protein in vegetative cells and nitrogen-fixing heterocysts using immuno-electron microscopy. Results Hexadecane treatments resulted in a two-fold increase in mRNA, and a four-fold increase in P450 protein levels relative to control cultures. Hexane, octane and dodecane were toxic and induced substantial changes in membrane morphology. Long-chain saturated and unsaturated fatty acids were shown to bind the CYP110 protein using a spectroscopic spin-shift assay, but alkanes did not bind. CYP110 protein was detected in vegetative cells but not in differentiated heterocysts where nitrogen fixation occurs. Conclusion Hexadecane treatment was an effective inducer of CYP110 expression in cyanobacteria. Based on substrate binding profiles and amino acid sequence similarities it is hypothesized that CYP110 is a fatty acid ω-hydroxylase in photosynthetic cells. CYP110 was found associated with membrane fractions unlike other soluble microbial P450 proteins, and in this regard CYP110 more closely resembles eukarytotic P450s. Substrate stablization is an unlikely mechanism for alkane induction because alkanes did not bind to purified CYP110 protein.

Background Anabaena sp. strain PCC 7120 ( Anabaena 7120) is an obligate photoautotrophic cyanobacterium that reduces atmospheric nitrogen in terminally differentiated cells known as heterocysts [ 1 ]. Approximately every tenth cell along a filament differentiates into a heterocyst under nitrogen-fixing conditions. Three developmentally regulated DNA rearrangements occur within the heterocyst chromosome which excise DNA elements integrated within the nifD , fdxN and hupL genes [ 2 , 3 ]. The element interrupting the nifD gene is 11,268 base pairs in length and carries its own site-specific recombinase, the xisA gene [ 4 ]. The nifD element is found in most heterocyst forming cyanobacteria integrated within the nifD gene [ 5 , 6 ]. It is absent from all non-heterocystous strains examined to date [ 7 , 8 ]. While factors driving the evolution of the nifD element and its associated genes remain obscure, an essential role in diazotrophic growth can be ruled out since an Anabaena strain cured of the nifD element was shown to fix nitrogen under aerobic conditions in heterocysts of normal morphology [ 9 ]. The complete DNA sequence of the nifD element reveals little about the biological functions it encodes. Nine open reading frames are known (see Fig. 1 ) only one of which can be identified by sequence homology: the first reported cyanobacterial cytochrome P450 gene, cyp110 [ 10 ]. The cyp110 gene is widely conserved in the heterocyst forming cyanobacteria including Anabaena , Nostoc and Calothrix/Fremyella . The amino acid sequence of CYP110 is most similar to the mammalian P450 family 4 fatty acid omega-hydroxylases and P450 BM3 from Bacillus megatarium , another fatty acid omega hydroxylase. However, the metabolic significance of fatty acid omega hydroxylation in bacteria remains obscure. Even for the well-characterized P450 BM3 enzyme, the identity of the in vivo substrate(s) of this protein and the pathway involved are not known [ 11 ]. Studies suggest n-alkanes act as multi-purpose microbial P450 inducers and our preliminary data suggested this was also true for cyp110 [ 12 , 13 ]. Moreover, a biosensor for hexadecane detection based on changes in cyp110 mRNA from Anabeana variabilias has been described [ 14 ]. Here we report on the pattern of expression of the cyp110 mRNA and protein in Anabaena 7120, substrate binding characteristics of CYP110, the effects of alkanes on cellular morphology, and the distribution of CYP110 in vegetative cells versus heterocysts using electron microscopy. Results Hexadecane-dependent induction of cyp110 transcripts in nitrogen-fixing and ammonia-supplemented cultures We first tested the effects of hexane, octane, dodecane and hexadecane on the growth of Anabaena 7120 cultures. All alkane additions in this study were carried out at 0.2% (v/v), the recommended concentration for microbial P450 induction [ 12 ]. In preliminary experiments, we determined that Anabaena 7120 exhibited rapid chlorosis with 0.2% hexane and octane. These treatments yielded only highly degraded RNA and were not further analyzed (data not shown). RNA samples from control, dodecane and hexadecane treated cultures were size fractionated and hybridized with cyp110 . A psbA1 specific probe was used to assess RNA quality and for normalization. The cyp110 probe identified a range of transcript sizes with a maximum of 9 kb seen in mRNA from both nitrogen-fixing and ammonia supplemented Anabaena 7120 cultures Fig. 2(a) and 2(c) . The size of the transcript identified by the cyp110 probe and the genetic organization of the nifD -element suggest that all of the open reading frames to the right of xisA on the nifD element are coordinately transcribed on a polycistronic mRNA. In support of this idea, a probe internal to the ORF6 coding region (Fig. 1 ) identified the same range of transcript sizes (data not shown). Since the xisA mRNA has not been detected with Northern blots [ 9 ], it is likely that the 9 kb transcript begins downstream of the xisA gene and terminates 3' to the orf designated adx in Fig. 1 . The psbA1 gene, which encodes the D1 reaction center protein, was used as a load control and hybridization standard. As seen in Fig. 2(b) and 2(d) , the psbA1 probe identifies a discrete 1.25 kb transcript in control RNA as previously shown [ 15 ]. Relative amounts of mRNAs were estimated by densitometry of the scanned autoradiogram. Exposure times for detection of the cyp110 transcript were 12 times longer than for the highly expressed psbA1 mRNA. The ratio of the cyp110/psbA1 signal intensities was 2.3 fold higher in hexadecane-treated cultures in the nitrogen-fixing culture (panels A&B) while a 2.2-fold increase was observed in the ammonium-supplemented culture (panels C&D). The dodecane treatment had little effect on cyp110 mRNA levels in either nitrogen-fixing or ammonium supplemented cultures. Similar results were obtained in 2 separate experiments with RNA isolated from independent cultures. The doubling of steady-state cyp110 mRNA levels in response to hexadecane treatment was independent of nitrogen status of the culture, consistent with previous data showing the entire nifD element is not required for nitrogen fixation in heterocysts [ 9 ]. CYP110 protein is only detected in hexadecane-treated cultures and localized in the membrane fraction The hexadecane effect on cyp110 gene expression was further examined using CYP110-specific antibodies to probe for the presence of P450 protein in nitrogen-fixing Anabaena 7120 cultures. As shown in Fig. 3A , the 0.2% hexadecane treatment resulted in the appearance of an immuno-reactive protein band of 50 kDa that co-migrated with a purified CYP110 protein sample. The P450 protein band intensity increased at least 2 fold within 2 hours of treatment, and at least 4 fold 24 hours after treatment with hexadecane when compared to the background, zero-time expression shown in lane 3 (Fig. 3C ). A western blot with protein samples from an untreated control culture failed to detect any immuno-reactive P450 protein (Fig. 3B ). These results were replicated with 2 independent cultures. Heterologous expression of the CYP110 protein in E. coli produced a substantial amount of membrane associated P450 protein [ 16 ]. This is a clear distinction between CYP110 and its closest sequence homolog, P450 BM3 , which has been extensively studied in part because it is a soluble P450 model protein [ 11 ]. To determine if CYP110 is membrane associated in Anabaena 7120, protein from membrane and soluble fractions separated by centrifugation were subjected to Western blot analysis. As shown in Fig. 3D , the membrane fraction appeared to contain all of the immuno-reactive P450 protein in the Anabaena 7120. This stands in contrast to P450 BM3 and most other microbial P450 proteins, and in this regard, CYP110 more closely resembles the eukaryotic P450 proteins. Spectral characterization of CYP110 protein and substrate binding assay Absorption spectra studies were recorded for CYP110 protein expressed and purified from E. coli carrying a pUC-based expression vector [ 16 ]. As shown in Fig. 4A , the CYP110 protein has a typical cytochrome P450 absorbance profile in the visible region for the oxidized, reduced and carbon monoxide bound states. CYP110 also shows the characteristic peak at 450 nm in the CO difference spectrum (Fig. 4B ). We monitored the visible spectrum while titrating a CYP110 solution with tetradecanoic acid. As shown in Fig. 4C , this resulted in a blue shift of the peak absorbance in the oxidized spectrum to 388 nm, similar to previously described P450 substrate-dependent Type I spectral shifts. This spectral shift was the same in either buffer A or buffer A + 0.2% deoxycholate. When buffer A was supplemented with 0.2% Triton X-100, no spectral shift was observed (data not shown). The titration yields the binding constant (Ks) for each substrate producing a spectral shift. A typical difference spectrum generated from the titration and the resulting double reciprocal plot are shown in Fig. 4D . The calculated binding constants for the interaction of CYP110 with potential substrate molecules of different hydrocarbon chain length are listed in Table 1 . For the saturated fatty acids tested, CYP110 displays the highest affinity for tetradecanoic acid with a K s of 0.091 mM. The unsaturated fatty acids also bound well to CYP110. Overall, CYP110 had the highest affinity for alpha-linolenic acid (Ks 0.062 mM) and arachidonic acid (Ks = 0.051 mM), long chain fatty acids with 3 and 4 double bonds, respectively. For comparison, P450 BM3 has a K s of 0.002 mM for arachidonic acid [ 17 ]. The Ks values towards arachidonic acid for the mammalian cytochrome P450s from family 4 are in the same range [ 18 ]. Fatty amines and fatty amides are known to interact with P450 ω-hydroxylases via Type II spectral shifts [ 19 , 20 ], and this was also true for CYP110. The minimal chain length for this interaction was 5 since n-butylamine did not cause a spectral shift while n-pentylamine did bind. There was a clear trend for tighter binding with longer hydrocarbon chain length, as the CYP110-dodecylamine interaction had the lowest tested Ks value (0.012 mM). CYP110 bound fatty amines 10 fold better than P450 non , which has a binding constant of 1.3 mM towards octylamine [ 21 ]. Twelve-aminododecanoic acid causes a Type I spectral shift instead of a Type II suggesting an altered mode of binding. Lu et al . [ 20 ] showed that terminal amino fatty acid derivatives interact weakly with the heme iron of cytochromes P450. The binding of CYP110 towards other fatty acid derivatives was also examined. Table 1 displays a list of compounds that displayed no spectral shift, including fatty alcohols or saturated n-alkanes. No spectral shift was observed with 11- or 12-hydroxydodecanoic acid or with methyl laurate. The unsaturated fatty acid 11-dodecenoic acid was also tested with no shift observed. Two other amines that did not display spectral shifts were the polyamine, putrescine, and cetyltrimethylammonium bromide (CTAB). Sodium dodecyl sulfate (SDS) does bind CYP110 and causes a Type I spectral shift. Although no binding constant was determined, it was observed that the maximal shift to 388 nm occurred below 0.5 mM and reverted back to 416 nm above this concentration, a phenomenon likely to be linked to the critical micelle concentration for SDS (data not shown). Alkane effects on morphology and localization of CYP110 Given the toxicity of hexane to Anabaena 7120, transmission electron microscopy was used to document changes in cell structure associated with the alkane treatments shown to induce the CYP110 expression. Vegetative cells from control, dodecane and hexadecane treated cultures are shown in Fig. 5a,5b and 5c respectively. Many dark spherical inclusions are seen in the dodecane-treated cells (Fig. 5b ). The dodecane treatment also distorts and thickens the cellular and photosynthetic membranes, creating a ruffled effect in both outer and inner membranes. Hexadecane treatment did not have the same dramatic effect on membrane morphology, however the close stacking of photosynthetic membranes seen in the control micrograph (Fig. 5a ) is lost with hexadecane treatment (Fig. 4c ). Gold-conjugated anti-CYP110 antibodies were used to determine the relative protein expression levels in vegetative cells with and without alkane treatment and to assess vegetative cell versus heterocysts expression patterns Anabaena 7120. Immunoelectron micrographs showed a greater number of anti-CYP110 polyclonal gold conjugates in dodecane and hexadecane vegetative cells (Fig. 5e and 5f ) than in control samples (Fig. 5d ). Expression levels were quantified by counting gold particles in multiple micrographs as shown in Table 2 . Immunogold particles most often clustered around dark-staining material in Anabaena 7120 vegetative cells grown in the presence of 0.2% (v/v) dodecane or hexadecane (Fig. 5e,f ). We found fewer immunogold particles/cluster in dodecane-treated samples (avg. = 22) than hexadecane treated samples (avg. = 50). Heterocysts showed significantly fewer gold particles than vegetative cells, and the number of gold particles in heterocysts did not increase with hexadecane treatment (Fig. 5g , Table 2 ). Nonspecific immuno-staining was estimated by incubating sections with preimmune rabbit IgG or with polyclonal anti-CYP110 antibodies that had been pre-adsorbed with purified CYP110 protein produced in E. coli . Very low background was seen in both the preimmune rabbit IgG primary incubation (Fig. 5h ), and sections treated with preabsorbed anti-CYP110 antibodies (Fig. 5i ). No clusters of immunogold conjugates were seen in any of the control sections. These results clearly show the majority of CYP110 protein is localized in vegetative cells and corroborate Northern and Western data showing elevated CYP110 expression in hexadecane-treated cultures. Discussion Most well characterized microbial P450s enzymes were isolated from heterotrophic organisms and are involved in the oxidative metabolism of xenobiotic compounds for energy and growth. Examples include camphor-degrading P450 CAM [ 22 ] and two herbicide inducible P450 genes in Streptomyces griseolus [ 23 ]. There is no evidence to date for cytochrome P450-dependent oxidative metabolism in the cyanobacteria. A P450-dependent N- demethylation activity is known in unicellular green algae that activates the pro-herbicide metflurazon into norflurazon [ 24 ]. Two genera of marine cyanobacteria Microcoleus and Phormidium were isolated from oil spills along the Arabian sea and reported to contribute to oil degradation [ 25 ]. While the first step in alkane degradation in many microbial systems is a P450-dependent hydroxylation [ 11 , 26 , 27 ], there are no data on the mode of alkane metabolism nor confirmation of hydrocarbon oxidation by axenic isolates of these cyanobacterial strains. Recent evidence does show that mixtures of these cyanobacteria and organotrophic bacteria of the genera Rhodococcus , Arthrobacter , Pseudomonas , and Bacillus are more effective than either group alone towards alkane oxidation in inorganic growth media [ 28 ]. The available evidence suggests that CYP110 does not participate in the degradation of alkanes. CYP110 has low overall sequence similarity to other known alkane hydroxylase enzymes like the CYP52 family [ 29 ]. Alkanes are quite toxic to Anabaena 7120, and purified CYP110 protein expressed in E. coli binds fatty acids but not alkanes using a spin-shift assay. The membrane localization and spin-shift binding data suggest that long-chain polyunsaturated fatty acids are the most likely endogenous substrates for CYP110. The endogenous function of CYP110 in Anabaena 7120 remains a mystery. Sequence comparisons indicate the protein is related to the fatty acid omega hydroxylase proteins. One possibility is that ω-hydroxylated fatty acids are further oxidized to dicarboxylic acids which then undergo β-oxidation from both ends yielding acetate, as demonstrated in eukaryotic peroxisomes [ 30 ]. Cytochrome P450 proteins in diazotrophic bacteria have been suggested to play an ancillary role in nitrogen fixation. Many years ago, Appleby proposed that a cytochrome P450 enzyme functions as an alternative high-affinity oxidase in an efficient, leghemoglobin-facilitated pathway of oxidative phosphorylation in bacteroids of Bradyrhizobium japonicum [ 31 ]. The idea was based on spectroscopic evidence for the presence of a P450 protein in B. japonicum bacteroids but not in aerobically grown cells and the blocking of the high-affinity oxidase system by the P450 inhibitor, N-phenylimidazole. More recently both bacteroids and anaerobically grown B. japonicum cells were shown to contain two immunologically distinct cytochromes P450, which were cloned and designated cyp112 and cyp114 [ 32 ]. Neither protein is expressed in aerobically grown B. japonicum cells, consistent with the Appleby hypothesis. Transposon mutagenesis of the cyp112 locus was found to block expression of both Cyp112 and Cyp114. Nevertheless, the mutant strain produced effective nodules on soybeans. These results show neither protein is essential for symbiotic function under the conditions of plant growth used in these experiments. Subsequent analysis of the B. japonicum P450 cluster identified a third P450 gene and other open reading frames suggesting an operon involved in terpenoid biosynthesis [ 33 ]. Like the Anabaena 7120 strain missing the nifD element, a quantitative phenotype related to nitrogen fixation under specific culture conditions cannot be excluded from the available evidence. B. japonicum and Anabaena 7120 are not the only diazotrophic bacteria harboring cytochrome P450 genes near clusters of nitrogen fixation-related genes. The large Sym plasmid from Rhizobium sp. NGR234 confers the ability to associate symbiotically with plants. It contains two cytochrome P450 genes whose function(s) are not known [ 34 ]. Conclusion Alkanes have been hypothesized to act as universal inducers of microbial P450 genes [ 12 ]. Consistent with this hypothesis, we observed a small but reproducible 2-fold enhancement of cyp110 transcription induced by 0.2% hexadecane. We did not test lower concentrations of hexane or dodecane to determine if non-toxic concentrations would have P450-inducing activity in Anabaena 7120. We cannot rule out the possibility that the lower solubility of longer chain alkanes in aqueous solutions may have been more important for the activity of hexadecane as an inducer than chain-length per se . The CYP110 protein displayed the highest affinity towards long-chain unsaturated fatty acids and was localized primarily in photosynthetic vegetative cells of Anabaena 7120. Unlike most prokaryotic P450 proteins, CYP110 was found to be associated with membrane fractions consistent with the solubility characteristics of its preferred substrates. Methods Cyanobacterial growth For RNA and protein isolation, cultures of Anabaena 7120 (4 L) were grown in Chu #10 media with continuous light, stirred at 30°C and bubbled with a 1% CO 2 /air mixture illuminated at approximately 100 μE s -1 m -1 . Nitrogen supplemented cultures were grown with 2.5 mM NH 4 (SO 4 ) 2 to suppress heterocyst differentiation. Treatments were carried out after cultures reached A 700 = 0.7. Control and test cultures (0.2 % dodecane or hexadecane [v/v] for 2 hours) were maintained in the same growth conditions during the treatment interval. RNA methods An amended RNA isolation procedure [ 37 ] was used for total RNA isolation. Briefly, cells were cooled by adding an equal volume of ice followed by transferring the cell paste into a mortar and freezing with liquid nitrogen. Cells were ground to a fine powder and placed in 10 mL RNA lysis buffer-pH 5.2 (0.1 M Tris, 0.1 M LiCl, 5 mM EDTA, 0.1 M NaCl, 0.1 M sodium acetate, 1 % SDS, 0.2% β-mercaptoethanol). After vortexing (2 min), 5 mL hot (60 C) NTE saturated-phenol, pH 6.0, was added, followed by 3 min of vortexing. Samples were placed at 60°C for 10 min, vortexed and 7 mL chloroform:isoamyl alcohol (24:1) added and the aqueous phase was subjected to repeated phenol/chloroform extractions until no visible material remained at the interface. Total RNA was precipitated overnight in 2 M LiCl at 4°C. RNA pellets were washed with 2 M LiCl and ethanol precipitated. After a 70% ethanol wash, the RNA was brought up in diethyl pyrocarbonate treated water. Samples of total RNA (15 μg) were separated by agarose formaldehyde denaturing gel electrophoresis. The gel was equilibrated twice in 10× SSC for 15 min and RNA was transferred onto a nylon membrane (Boehringer Mannheim) by capillary transfer. Probes were labeled using the DIG-High Prime Mix (Boehringer Mannheim) following the manufactures' recommendations. The cyp110 probe was derived from the 2 kbp HindIII pAn 207.4 DNA fragment (Fig. 1 ) containing most of the cyp110 gene sequence [ 10 ]. The psbA probe used is a 2 kbp pAn 625 Eco R1 fragment [ 15 ] containing the coding region for the D1 reaction center protein in photosystem II. RNA blots were first probed with cyp110 , stripped, and reprobed with the psbA probe. The hybridized probes were detected using the CDP- Star (Boehringer Mannheim) chemiluminescent substrate, followed by autoradiography. Prehybridizations, hybridizations, and washes were carried out as described by the manufacturer. Signal intensities from northern blots were quantified using Intelligent Quantification software (Genomic Solutions). P450 protein analysis Proteins for Western blotting were isolated from N 2 -fixing Anabaena 7120 cultures treated with 0.2% hexadecane. Cultures (2 L) were grown to an A 700 = 1.0 – 1.2 prior to hexadecane addition. Fractions of 250 mL were collected at 0, 2, 12, 24, 36, 48 and 60 h after treatment. Cells were harvested by centrifugation and resuspended in 5 mL of protein buffer: 20 mM MOPS (pH 7.4), 10% glycerol, 1 mM DTT, 20 mM KCl, 1 mM PMSF, 1 mM apoprotin and 1 mM pepstatin. Samples were lysed by three passages through a French pressure cell. Soluble and membrane fractions were separated by centrifugation at 100,000 × g for 1.5 h. Pelleted membranes were resuspended in protein buffer supplemented with 0.2% deoxycholate by gentle shaking overnight at 4°C. The CYP110 protein was expressed from a pUC8 expression vector and purified as described elsewhere [ 16 ]. Antiserum to this protein was raised in a New Zealand white rabbit using subcutaneous injections of 100 μg purified CYP110 protein in Freund's complete adjuvant. Two identical booster injections were made at 3 and 6 weeks after the first. Immune serum was collect at 6 and 12 weeks after the initial injection. Protein samples (50 μg) were separated on 12% SDS-PAGE [ 38 ] and blotted using standard methods [ 39 ]. Antiserum raised against P450 protein was used at a 1:100 dilution and detected with a peroxidase-linked anti-rabbit secondary antibody (Amersham) and the ECL Western Blot system (Amersham) using X-ray film. Oxidized, reduced and carbon monoxide reduced spectra The purified soluble fraction of CYP110 was diluted 1:10 in buffer A (20 mM MOPS pH 7.4, 20% Glycerol, 2 mM DTT, 20 mM KCl, and 2 mM PMSF). The oxidized spectra was measured with a Hewlett Packard Diode Array Spectrophotometer. Five milligrams of sodium dithionite was added, and the reduced spectra measured. CO was then bubbled into the reduced sample at a rate of 1 bubble per second for 30 seconds. The oxidized spectra was subtracted from the CO reduced spectra to yield the peak at 450 nm. The concentration of cytochrome P450 was determined by using the extinction coefficient, 91 mM -1 cm -1 , as determined for rat liver P450 [ 40 ]. Substrate binding studies Spectrophotometric titration of CYP110 with fatty acids, fatty amines, fatty amides, fatty alcohols, and n-alkanes with varying chain lengths (C 5 -C 20 ) was preformed in buffer A with and without 0.2 % deoxycholate. Purified soluble CYP110 was diluted to a concentration of 3 μM, and the baseline spectrum was measured. Substrates were then titrated into the P450 from a 100 mM stock solution in ethanol. The n-alkanes were solubilized via the method of Scheller et al . [ 29 ] by sonication of the stock solution for 10 minutes on ice to enhance solubility. The substrates were titrated to concentrations varying from 5 μM to 2 mM depending upon the compound being tested. Fatty acids, amides, alcohols, esters and n-alkanes were titrated from 50 μM to 2 mM while fatty amines were titrated from 1 μM to 700 μM. Immunocytochemical electron microscopy Nitrogen-fixing Anabaena 7120 batch cultures were grown at ambient CO 2 levels the presence or absence of 0.2% dodecane or hexadecane (v/v) under continuous light with stirring at 30 C for 30 days. Immediately upon harvesting, cells were fixed in 4% paraformaldehyde, 0.6% glutaraldehyde, 0.033 M phosphate, 0.1 M sucrose for 2 hours at 4°C. Samples were then washed three times in 7% sucrose, 0.033 M phosphate buffer. Following an ascending ethanol dehydration series (50%, 70%, 95%), the samples were infiltrated in LR white acrylic plastic (Electron Microscopy Sciences). Cells were then embedded in gelatin capsules for 24 hours at 50°C. Solidified LR-white samples were sectioned with a diamond knife and placed on carbon coated, nitrocellulose-covered, nickel grids. Sections were blocked with 1% BSA/10% goat serum in TBST (0.1% Tween, 16.5 mM Tris (pH 7.5), 137 mM NaCl, 2.7 mM KCl). The sections were incubated (2 h at room temperature) with 35 μL of the following antibodies: A. Anti-CYP110 polyclonal sera used in western blot analyses (1:400 dilution in BSA-TBST), B. preimmune rabbit IgG (1:400 dilution in BSA-TBST), or C. Immune serum preabsorbed with 2.5 mg purified CYP110 protein [ 16 ]. Following the primary antibody treatment sections were washed at least four times in BSA-TBST and incubated for one hour (room temperature) in a drop of secondary antibody goat anti-rabbit IgG (Sigma). The sections were then viewed on a Hitachi H 7000 transmission electron microscope at 80 kV. Conventional electron microscopy Following growth conditions described for immunocytochemical studies, approximately 10 7 Anabaena 7120 cells were pelleted and fixed by placing in 1 mL 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at room temperatures for 1 hour. Cells were washed twice with cacodylate buffer (15 min/wash) and embedded with 1% agar. Agar embedded samples were cut into small cubes (~1 mm) and postfixed in 1% osmium tetroxide 0.1 M cacodylate (pH 7.4) for two hours at room temperature. Samples were rinsed with deionized water and dehydrated in an ascending ethanol series. Cells were infiltrated with 50% Spurr's media (Electron Microscopy Sciences): 50% absolute ethanol, followed by at least four more infiltrations with fresh 100% Spurr's media. To allow polymerization of the Spurr's media, samples were incubated overnight at 70 C. The samples were sectioned using a diamond knife and picked up on copper grids for viewing as above. Authors' contributions ST conducted the Northern and immuno-localization experiments, analyzed and organized the data and composed initial drafts for portions of the manuscript. CRF conducted the protein purification, western and substrate-binding experiments, analyzed and organized the data and composed initial drafts for portions of the manuscript. PJL conceived the project, provided overall guidance, and wrote the final version of the manuscript. All three authors read and approved the manuscript.

D:\keerthana\PMC001xxxxxx\PMC1079853.xml

1079854

A ring-like nucleoid is not necessary for radioresistance in the Deinococcaceae

Background Transmission electron microscopy images of Deinococcus radiodurans R1 suggest that the nucleoid of this species exists as a "ring-like" body, and have led to speculation that this structure contributes to the radioresistance of the species. Since extreme radioresistance is characteristic of six other species of Deinococcus , we have attempted to correlate nucleoid morphology and radioresistance by determining whether the genomic DNA of each of these species exhibit similar structures. Results The nucleoid morphologies of seven recognized species of Deinococcus , the radioresistant bacterium Rubrobacter radiotolerans , and the more radiosensitive deinococcal relative Thermus aquaticus were evaluated using epifluorescence and deconvolution techniques. Although the nucleoids of Deinococcus murrayi, Deinococcus proteolyticus, Deinococcus radiophilus , and Deinococcus grandis have structures similar to D. radiodurans , the majority of nucleoids found in Deinococcus radiopugnans and Deinococcus geothermalis lack any specific organization. The nucleoid of R. radiotolerans consists of multiple highly condensed spheres of DNA scattered throughout the cell. The genomic DNA of Thermus aquaticus is uniformly distributed throughout the cell. Conclusion There is no obvious relationship between the shape of a species' nucleoid and extreme radioresistance. However, the genomes of all extremely radioresistance species examined are highly condensed relative to more radiosensitive species. Whether DNA in this tightly packed configuration contributes to the radioresistance of these bacteria remains unknown, but this common structural feature appears to limit diffusion of fragments generated post-irradiation even in cells incapable of repairing strand breaks.

Background The Deinococcaceae are a small family within the domain Bacteria that are distinguished by their ability to tolerate DNA double strand breaks [ 1 , 2 ]. There are eleven validly described species and seven of these tolerate what for most microorganisms is a sterilizing dose of ionizing radiation, exhibiting detectable survival after exposure to 10 kGy (1,000,000 Rad) γ radiation [ 3 ]. The ionizing radiation resistance of the four remaining species, Deinococcus indicus [ 4 ], Deinococcus frigens , Deinococcus saxicola , and Deinococcus marmoris [ 5 ] has not been reported. Ionizing radiation generates an array of DNA damage in the target cell, including many types of base damage, single, and double strand breaks [ 6 ]. Of these types of damage, DNA double strand breaks (DSBs) are considered the greatest threat to cell viability, and an excessive number of DSBs are expected to be lethal. E. coli , for example, cannot survive introduction of greater than one or two DSBs into its genome [ 7 ]. In contrast, D. radiodurans survives doses of ionizing radiation that generate greater than 100 DSBs per genome without mutation or loss of viability [ 8 - 10 ]. It is the ability to endure and accurately repair these lesions that set the deinococci apart from other species. The reasons for the deinococci's radioresistance are poorly understood. Genetic and biochemical evidence obtained from D. radiodurans , the best studied of these species, argues that recovery requires RecA-mediated homologous recombination [ 9 , 11 , 12 ]. D. radiodurans is multi-genomic, and depending on the growth phase, cells contain from 4 to 10 copies of their genome depending on growth conditions [ 13 , 14 ]. It is believed that this increased DNA content is protective in that it serves as a reservoir of genetic information that can be used during recombinational repair. In addition, it has been suggested that there may be a pre-existing alignment of homologous sequences between copies of the genome and that this alignment accounts for the remarkable speed and fidelity of DNA double strand break repair in this species [ 15 ]. However, the existence of such an alignment has not been established. Levin-Zaidman et al. [ 16 ] have reported that the genome of D. radiodurans assumes a tightly packed ring-like structure that may represent an alternative mechanism for protecting D. radiodurans from DSBs. These authors suggest this structure contributes to D. radiodurans radioresistance by preventing fragments formed by DSBs from diffusing apart during repair. Having the capacity to maintain the linear continuity of its genome in the face of the extensive fragmentation resulting from high dose ionizing radiation would provide obvious advantages to D. radiodurans . Assuming that DNA repair proteins can function within the proposed structure, gene order is preserved and the gaps generated by damage and subsequent DNA degradation could be bridged by homologous recombination with redundant genetic information available in the genome copies. Such an arrangement does not require a pre-existing alignment between sequences, but if homologous genetic information is aligned prior to irradiation, the time needed to effect repairs should be reduced. In an attempt to gain insight into whether the structures reported by Levin-Zaidman et al contribute to ionizing radiation resistance, we have combined epifluorescence and deconvolution microscopy to describe the structure of the nucleoid of the seven species of the Deinococcaceae known to be ionizing radiation resistant. We assume that if ring-like structures are required for extreme radioresistance, all deinococci will contain a similarly organized nucleoid. In addition, we have examined the nucleoid structure of a related but less radioresistant species, Thermus aquaticus , and a phylogenetically distinct, but more radioresistant [ 17 ] species Rubrobacter radiotolerans seeking to correlate variations in nucleoid morphology with radioresistance. We find little evidence to support the assertion that a specific nucleoid structure is required for bacterial ionizing radiation resistance, but note that the genomic DNA of the most radioresistant species examined is more condensed relative to more radiosensitive species, suggesting that this generic feature is passively facilitating repair processes. Results The nucleoid of Deinococcus radiodurans R1 Sequential digital images were collected at 100 nm increments from stationary phase cultures of D. radiodurans R1. Cells were stained with the membrane dye FM4-64 and the DNA specific dye DAPI (4', 6-diamidino-2-phenylindole), and the images obtained using each dye were merged to form the sequence of images depicted in Fig. 1 . In agreement with previous descriptions of this species' nucleoid, the genomic DNA of R1 exhibits a clear organized pattern [ 16 , 18 , 19 ], distinct from that observed in E. coli (Fig. 2 ) where the DAPI-stained DNA appears uniformly distributed throughout each cell. The DNA of R1 has a distinctive ring-like structure in each cell of this tetrad, suggesting that the genome of this species is spooled around a sphere or cylinder that excludes the dye. Given the size and organization of this structure we assume that the proposed core is primarily proteinaceous, but don't exclude the possibility that other macromolecules, including DNA that is inaccessible to the dye, may be present in the core. Based on the images in Fig. 1 , we believe that the DAPI-stained DNA exists as an equatorial band (or possibly a tight spiral) over the surface of this core. In each of the cells the band is tilted relative to the focal plane. For example, in the lower half of the tetrad, the bands are tilted slightly away from the viewer; toward the upper left in the cell on the left and toward the upper right in the cell on the right. Consistent with this interpretation, we find that the dye does not form a closed ring in every optical section and that the patterns vary from cell to cell. In many cells the DNA first appears in a crescent shape in one area of the cell. This crescent changes in sequential sections (Fig. 1 ), ultimately disappearing and giving way to a crescent shaped structure that mirrors where the dye first appeared. This pattern of changes is most simply explained by assuming that the DNA is on the surface of a larger structure capable of tilting about a central axis. When tilted, the ring of dye will cross the focal plane at an angle and produce a falcate shape. The nucleoid of most, but not all, species of Deinococcus is similar to that of D. radiodurans R1 D. radiophilus and D. proteolyticus have a nucleoid reminiscent of that described for D. radiodurans (Fig. 1 ). In cross section, the DAPI accumulates in a halo surrounding a circular core that excludes the dye. However, the organization of the DNA in these species differs subtly relative to D. radiodurans ; the DNA appears to completely surround the structure at the core of the nucleoid. This point is best illustrated in Fig. 3 , which depicts 20 sequential sections (moving from upper left to the lower right panel) through the DAPI-stained nucleoids in a pair of D. proteolyticus cells. (The stained membrane is not included in this image.) The first image corresponds to a focal plane at the top of the nucleoid and with each successive image; it becomes clear that the DNA forms a shell that follows the contour of an internal sphere that has not been stained. The nucleoid morphology of D. radiophilus was found to be identical to that of D. proteolyticus (data not shown). The nucleoid of rod-shaped D. grandis is distinctive, but shares similarity to that of D. radiodurans . The DNA in most cells is highly condensed, localized to a specific area of the cell (Fig 4 ). The sequential sections obtained from D. grandis suggest that the DNA surrounds an unstained core. A number of cells (~7%) exhibit additional condensed spots of DAPI stain, as illustrated in the cell on the right in Fig 4 . The significance of these "spots" is not known at present. In D. murrayi , the ring of DAPI-stained DNA is remarkably uniform (Fig. 5 ), varying little between cells. As with D. radiodurans , the center of the ring is not stained, but the number of sections containing DNA is more variable, ranging between 20 and 30. This suggests that the nucleoid of D. murrayi may be more cylindrical than that of D. radiodurans with the DNA forming a sleeve around the core. Consistent with this interpretation, most of the cells display a ring of DAPI in every optical section. In contrast to the other species discussed thus far, only 2% of D. radiopugnans and 10% of D. geothermalis cells exhibit a structurally well-defined nucleoid (Figs. 6 and 7 ). In the majority of these cells, the DNA is condensed in that it is not uniformly spread throughout the cell as was observed with E. coli (Fig 2 ), but it does not seem to be associated with an intracellular structure. Instead the DNA appears to be distributed chaotically through the cytosol. The absence of a consistent pattern in DAPI staining within most of the cells suggests that the DNA in these species does not adopt a fixed shape. The nucleoid of Thermus aquaticus The genera Thermus and Deinococcus are part of the same phylum within the domain Bacteria [ 3 ]. Because of this relationship, we sought 1) to establish if Thermus species also exhibit increased tolerance to ionizing radiation, and 2) to determine if the nucleoid of this genus bore any resemblance to that of the deinococci. We assessed the ionizing radiation resistance of Thermus aquaticus YT-1, the type species for its genus, and compared it to D. radiodurans R1. As indicated in Fig. 8 , YT-1 does not display extraordinary resistance relative to R1, but this strain is better able to survive γ radiation than the E. coli AB1157 control. After 1 kGy exposure, approximately 20% of the YT-1 population remained viable, whereas only 0.2% of the AB1157 culture survived. Images of YT-1 (Fig. 9 ) failed to show evidence of sub-cellular organization or condensation similar to that observed in the deinococcal species. The pattern obtained with DAPI most closely resembles that observed in E. coli (Fig. 2 ). The nucleoid of Rubrobacter radiotolerans Rubrobacter radiotolerans is an extremely ionizing radiation resistant bacterium that is, based on 16S rDNA-based phylogeny, unrelated to the Deinococcaceae [ 17 ]. This species is a member of a lineage within the Actinobacteria. The nucleoid of this species is quite different from that observed in the deinococci (Fig. 10 ). The DNA appears to form highly compact structures without a well-defined shape. Most cells contain two or three of these structures, and it is unclear whether each dye spot represents a unit length of genomic DNA. Based on differences in size and intensity, the spots do not appear to contain equivalent amounts of DNA, but these differences may be spurious. We suspect that the cell envelope of R. radiotolerans is less permeable to DAPI than that of the other species examined in this study. Attempts to stain the DNA of the closely related R. xylanophilus [ 17 ] were unsuccessful; the dye being unable to penetrate the cell. Differing quantities of dye entering individual R. radiotolerans cells may account for the differences in dye intensity observed. The effect of ionizing radiation on nucleoid morphology As has been shown previously, when D. radiodurans cells are exposed to high dose γ radiation they exhibit substantial DNA damage [ 10 ]. The distinctive pattern formed on a pulsed field gel by the NotI digested genome (data not shown) is initially obliterated by the DNA double strand breaks introduced during a 5 kGy exposure [ 10 , 20 ], but after three hours recovery, the pattern is restored indicating that the majority of the breaks have been repaired. To establish whether the introduction of large numbers of DNA double-strand breaks affect the distribution of DNA within the irradiated cell, we examined nucleoid structure in exponential phase D. radiodurans two hours post-irradiation, comparing the distribution of DAPI stain to that of E. coli AB1157 treated identically (Fig. 11B ). The differences in the images obtained are striking. While we are able to detect DAPI-stained material in some E. coli cells, the diffuse distribution, apparent in Fig. 2 , is no longer evident; only infrequent spots of DAPI remain. Furthermore, images of E. coli cells captured three hours post-irradiation of 5 kGy show a complete loss of the DAPI-stained material (data not shown). One reason for this may be that the E. coli genome is being progressively degraded following irradiation [ 21 ]. In contrast, the nucleoids of D. radiodurans and D. radiopugnans appear intact and retain the ability to be stained (Fig. 11A and 11D ). These images provide support for the hypothesis of Levin-Zaidman et al [ 16 , 19 ], which proposes that fragments generated by the double strand breaks may be in some manner held in place and protected from extensive degradation by cellular nucleases. The notion that the release of fragments from the damaged genome of D. radiodurans is limited is also apparent in our analysis of the effects of ionizing radiation on the nucleoid of the recA strain, rec30. The rec30 strain, which lacks the ability to carry out homologous recombination [ 8 , 9 ], is incapable of reassembling its genome following irradiation. However, like R1, the nucleoid of rec30 remains a coherent structure (Fig. 11C ) two hours post-irradiation, providing further evidence that fragments generated do not diffuse throughout the cytosol despite the presence of massive numbers of un-repaired DNA double strand breaks. Discussion Levin-Zaidman et al [ 16 ] have reported that genomic DNA of stationary phase cells of D. radiodurans is ordered as a tightly packed toroid, and it has been argued that this organization is in part responsible for D. radiodurans resistance to ionizing radiation [ 16 , 19 ]. These authors assume that the dense packaging characteristic of toroids restricts the diffusion of DNA fragments generated when cells are exposed to ionizing radiation, preventing a loss of genetic information that is needed for effective recovery from the insult. However, these authors also describe logarithmic phase cultures of D. radiodurans R1, and indicate that the nucleoids of these cells do not always exhibit a toroidal ring-like morphology even though they remain extremely radioresistant [ 2 ]. This observation seems to argue against a requirement for involvement of a specific nucleoid structure in ionizing radiation resistance. In addition, Daly et al [ 18 ] have demonstrated that growth in different media alters the organization of the nucleoid of D. radiodurans , and that the change does not correspond to changes in radioresistance. This group reports that ring-like nucleoids predominate in cultures growing in defined minimal medium, but that these cultures are more sensitive to ionizing radiation than cultures with fewer ring-like nucleoids growing in a rich medium. We initiated this study in an attempt to correlate nucleoid structure with radioresistance, reasoning that if the inferences of Levin-Zaidman et al [ 16 ] are correct, it will be reflected in morphological differences between radioresistant and radiosensitive species. We have examined species known to be ionizing radiation resistant as well as strains that are more sensitive to γ irradiation, and in agreement with Daly et al [ 18 ] find no compelling evidence that specific structures contribute to radioresistance. We base this conclusion on our failure to identify a distinctive repetitive pattern in the DAPI-stained DNA associated with the majority of D. geothermalis and D. radiopugnans cells in stationary phase culture. Cultures of D. geothermalis [ 22 ] and D. radiopugnans [ 23 ] are as radioresistant as D. radiodurans , but their genomes are more fluid, arguing that a nucleoid need not maintain a well-defined shape to sustain ionizing radiation resistance. Despite this conclusion, we note that among the extremely radioresistant species examined DAPI-stained DNA is condensed relative to what is observed in E. coli and T. aquaticus . The DAPI is localized within the more radioresistant cells, and not spread throughout the cytosol. This aggregation of DNA indicates that a basic tenet of the model of Levin-Zaidman et al [ 16 ] may be valid: specifically that the deinococci utilize mechanisms for protecting the fragments generated by strand breaks including a passive process that limits the diffusion of these fragments. This concept is most clearly demonstrated by the behavior of rec30 strain subsequent to irradiation. Despite the fact that this strain is incapable of restituting the majority of the DSBs generated, the rec30 nucleoid retains its shape. However, it must be noted that under certain conditions E. coli cells can undergo changes in nucleoid morphology similar to those reported for the deinococci. E. coli nucleoids become condensed spheres with cores of unknown composition when protein synthesis is inhibited [ 24 ], and when the mukB locus is disrupted the resulting mutant maintains a ring-like nucleoid [ 25 ]. Clearly, since E. coli strains are much more sensitive to ionizing radiation relative to D. radiodurans , the presence of a condensed nucleoid alone is not a sufficient explanation for radioresistance. Instead, we assume the combination of a condensed nucleoid structure and unique protein-dependent DNA repair mechanisms is responsible for the radioresistant phenotype displayed by the deinococci. The physical basis for the condensed nucleoid we observe is unknown. We suspect that this level of spatial organization is in large part mediated by proteins that either link copies of the genome together (assuming that a species contains more than one genome copy), or coordinates genome folding in a manner that generates the shapes we have described. For those species, such as D. radiodurans or D. murrayi , in which the nucleoid forms an obvious structure, we envision a protein lattice that acts as a scaffold that the genome is organized around. We predict these proteins are functionally analogous to the SMC (structural maintenance of chromosomes) proteins described in many eukaryotic and prokaryotic species [ 26 - 28 ]. It seems unlikely that genomic DNA is as condensed as the DNA-Dps assemblies described in some stationary phase bacteria [ 29 , 30 ]. These structures are tightly packed, almost crystalline, and it is difficult to envision how an actively metabolizing cell could function under this circumstance; the DNA needs to remain accessible to proteins that catalyze essential housekeeping functions. We also predict that the ionic composition of the cytosol of the deinococci examined favors the formation of the structures we observe. The Deinococcaceae exhibit unusually high intracellular levels of Mn +2 [ 18 ] and it is possible that accumulating this metal creates an environment that facilitates DNA condensation within this species in vivo through direct or indirect mechanisms. In vitro, the condensation of DNA can be achieved by adding a condensing agent, such as a multivalent cation, to an aqueous solution of DNA. The strong electrostatic interaction of the DNA and these cations neutralizes the repulsion of phosphate groups in the DNA backbone, and it has been shown that approximately 90% of the DNA charge must be neutralized for condensation to occur [ 31 ]. Intracellular Mn +2 may also indirectly facilitate nucleoid condensation by, for example, augmenting the function of deinococcal DNA-binding proteins. On the other hand, high Mn +2 content may have no affect on nucleoid morphology since, as described above, E. coli can have a condensed nucleoid even though this species has a much lower level of intracellular Mn +2 [ 18 ]. Years of experimental evidence irrefutably argue that DNA repair is essential for D. radiodurans recovery from high dose exposure to ionizing radiation, but this fact does not necessarily lead to the conclusion that efficient DNA repair is sufficient for extreme radioresistance. Although there has been a great deal of speculation concerning subtle differences in the properties of DNA repair proteins isolated from D. radiodurans , there has yet to be a convincing demonstration that this species' DNA repair proteins are "better" relative to homologues found in more radiosensitive species. Given this, it remains a formal possibility that D. radiodurans DNA repair proteins function within a molecular environment that enhances their effectiveness. In other words, it seems likely that there are features of deinococcal physiology that augment DNA repair processes, allowing the conventional complement of DNA repair proteins to more efficiently deal with DNA damage. We believe that the images presented here suggest that the genomic DNA of the deinococci is more condensed relative to other species. We suggest that this aggregation is protective, and that it may significantly contribute to the radioresistance of these species by confining the fragments generated subsequent to irradiation and preserving the linear continuity of the damaged genome. Conclusion This work resulted in two key observations. First, all evidence obtained is consistent with the notion that the genomes of radioresistant species are more condensed than radiosensitive species. Second, irradiation does not seem to disturb the pattern of condensation observed in the deinococci, even when we examined that pattern in a recA defective strain incapable of repairing DNA double strand breaks. Therefore, in agreement with Levin-Zaidmen et al [ 16 ], we assume that the deinococci have the capacity to passively prevent the diffusion of DNA fragments post-irradiation, and that this ability may contribute to the radioresistance of these species. Materials and methods Growth conditions Deinococcus radiodurans R1 (ATCC 13939), Deinococcus radiophilus (ATCC 27603), Deinococcus proteolyticus (ATCC 35074), Deinococcus radiopugnans (ATCC 19172), and Deinococcus grandis (ATCC 43672) were grown in TGY broth at 30°C as described elsewhere [ 3 ]. Deinococcus geothermalis (DSM 11300) and Deinococcus murrayi (DSM 11303) were grown in TGY broth at 50°C [ 17 ]. Rubrobacter radiotolerans (ATCC 51242) was grown at 30°C in a medium consisting of 1% tryptone, 0.5% yeast extract, 0.5% malt extract, 0.5% casamino acids, 0.2% meat extract, 0.2% glucose, 0.005% Tween 80, and 0.1% magnesium sulfate. Thermus aquaticus YT-1 (ATCC 25104) was grown at 70°C in Castenholz TYE medium (ATCC medium 723). E. coli AB1157 cultures were grown at 37°C in LB medium (1% tryptone, 0.5% yeast extract, and 1% NaCl). Unless otherwise indicated, all cultures were grown to stationary phase prior to microscopic examination. Cultures were harvested with the following densities as measured by OD 600 : D. radiodurans , 1.2–1.8; D. radiophilus 1.8–2.2; D. proteolyticus 2.0; D. radiopugnans 1.3–2.0; D. grandis 1.7; D. geothermalis 1.8; D. murrayi 1.3; R. radiotolerans 1.9; T. aquaticus 1.1. These values correspond to cultures with densities between 3 × 10 8 and 1.3 × 10 9 CFU/ml. Microscopy Cultured cells were stained with N-(3-triethylammoniumpropyl)-4-(6-(4(diethylamino) phenyl)hexatrienyl)pyridinium dibromide, (FM 4–64), and 4',6-Diamidino-2-phenylindole dihydrochloride, (DAPI), for the visualization of the lipid membrane and DNA, respectively. Ratios of the amounts of stationary phase culture cells to fluorescent dyes were a 3:1:1 ratio of culture cells to 0.0625 μg/μL of FM 4–64 solution to 3 μg/mL DAPI solution. The cells were then mounted on slides coated with 0.5% agarose. Figures 1 , 3 , 4 , 5 , 6 , 7 , and 10 were created by capturing two-dimensional images at a series of points along the z-axis that were set at 100 nm apart. The N2.1 filter cube from Leica Microsystems was used for FM 4–64 detection, which includes a 580 nm longpass dichroic mirror, a 515–560 nm bandpass excitation filter, and a 590 nm long pass emission filter. The A4 filter cube from Leica Microsystems was used for DAPI detection, which includes a 400 nm longpass dichroic mirror, a 360-40 nm bandpass excitation filter, and a 470-40 nm bandpass emission filter. The optical series was deconvolved with Slidebook 4.0 from Intelligent Imaging Innovations, Inc., (Denver, CO), using constrained iterative deconvolution and was then imported into Adobe Photoshop 7.0 as separate two-dimensional images. The two-dimensional images were then arranged in order as depicted. The projection image in Fig. 11A was achieved with the Slidebook 4.0 software by compiling the stack of two-dimensional images into a single two-dimensional image. All images were captured using a Leica DMRXA2 microscope and a Sensicam QE camera from The Cooke Corporation, (Auburn Hills, MI). The brightness and contrast of all images were enhanced using Adobe Photoshop 7.0. Ionizing radiation exposure Cultures (grown in the appropriate medium) of D. radiodurans (OD 600 0.12–0.19), D. radiopugnans (OD 600 1.5–1.8), rec30 (OD 600 1.4) and E. coli (OD 600 0.23) were irradiated to a dose of 5000 Grays using a Model 484R 60 Co irradiator (J. L. Sheppard & Associates, San Fernando, CA). Controls were kept at room temperature during irradiation exposure of experimental samples. All samples were incubated at the appropriate conditions immediately after irradiation. All of the cells were harvested after 2 hours of incubation and prepared for microscopy as described above. Survival curves Cultures were irradiated using the irradiator described above, and survival established by serial dilution of irradiated cultures and plating on appropriate growth medium. Three independent trials were conducted for each species examined with three replicates per trial. Sigma Plot software was used to create the survival curve. Authors' contributions JRB and JMZ conceived and designed the experiments. JMZ performed all of the experimental studies. JRB and JMZ wrote the paper.

D:\keerthana\PMC001xxxxxx\PMC1079854.xml

1079855

Genome-scale reconstruction of the metabolic network in Staphylococcus aureus N315: an initial draft to the two-dimensional annotation

Background Several strains of bacteria have sequenced and annotated genomes, which have been used in conjunction with biochemical and physiological data to reconstruct genome-scale metabolic networks. Such reconstruction amounts to a two-dimensional annotation of the genome. These networks have been analyzed with a constraint-based formalism and a variety of biologically meaningful results have emerged. Staphylococcus aureus is a pathogenic bacterium that has evolved resistance to many antibiotics, representing a significant health care concern. We present the first manually curated elementally and charge balanced genome-scale reconstruction and model of S. aureus ' metabolic networks and compute some of its properties. Results We reconstructed a genome-scale metabolic network of S. aureus strain N315. This reconstruction, termed i SB619, consists of 619 genes that catalyze 640 metabolic reactions. For 91% of the reactions, open reading frames are explicitly linked to proteins and to the reaction. All but three of the metabolic reactions are both charge and elementally balanced. The reaction list is the most complete to date for this pathogen. When the capabilities of the reconstructed network were analyzed in the context of maximal growth, we formed hypotheses regarding growth requirements, the efficiency of growth on different carbon sources, and potential drug targets. These hypotheses can be tested experimentally and the data gathered can be used to improve subsequent versions of the reconstruction. Conclusion iSB619 represents comprehensive biochemically and genetically structured information about the metabolism of S. aureus to date. The reconstructed metabolic network can be used to predict cellular phenotypes and thus advance our understanding of a troublesome pathogen.

Background Staphylococcus aureus is a pathogenic gram-positive bacterium that causes a variety of disease conditions, some life-threatening, both in hospital settings and in the community at large. Moreover, various strains of this organism have evolved resistance to some of the most clinically useful antibiotics, including methicillin and vancomycin[ 1 ]. Although the mechanisms of antibiotic resistance and infection have been elucidated, there is little published information regarding the basic and systemic biochemical function of S. aureus , especially under carefully controlled environmental conditions in chemically-defined media. While some research has been performed towards this goal[ 2 ], its scope and extent does not compare to the research undertaken for better-studied model organisms. In fact, the annotated genome sequence of a strain of S. aureus contains much more readily-available specific information regarding the organism's metabolism than does a compilation of literature data[ 3 ]. The annotated genome of a microorganism, in conjunction with biochemical and physiological data, can be used to reconstruct the metabolic network of that organism[ 4 , 5 ]. Such reconstructed networks consist of a set of chemical reactions that together comprise the known metabolic transformations that take place in a particular organism. These networks are at the genome-scale when all or most of the genes with known metabolic function are included in the network reconstruction. These network reconstructions convey the interactions between cellular components identified from the sequence annotation, and thus reconstructions can be thought of as two-dimensional annotation of the genome[ 6 ]. Genome-scale reconstructions (GENREs) represent a biochemically and genetically structured database that can be queried and interrogated using in silico analytical methods[ 7 ]. With the imposition of appropriate constraints on the reactions in the GENRE, including their exact stoichiometry and reversibility, a genome-scale model (GEM) is formulated. GEMs reflect allowable network states, or phenotypes of a cell, by defining a range of permissible solutions consistent with its mathematical representation[ 5 ]. This range of allowable states can be searched for the 'best' growth rates using linear programming methods, and the results from such computations are close to experimental observations [ 8 - 11 ]. GEMs using the constraint-based modeling formalism have been constructed for a number of microorganisms, including Escherichia coli [ 12 ], Saccharomyces cerevisiae [ 13 , 14 ], Methylobacterium extorquens [ 15 ], Mannheimia succiniciproducens [ 16 ], Helicobacter pylori [ 17 ] and Haemophilus influenzae [ 18 ], and reconstructed networks for human cells are beginning to appear[ 19 ]. GEMs are amenable to a wide variety of analysis techniques, yielding a number of interesting results, as recently reviewed[ 7 ]. Importantly, GENREs are two-dimensional annotations, are portable, and can be used for computations by different laboratories. In particular, the GENREs for E. coli and S. cerevisiae have been analyzed by groups around the world (see for a partial list). These analyses have led to several publications of general interest that focus on such diverse topics as the causes of enzyme dispensability[ 20 ], the reconfiguration of metabolism following the loss of a gene or enzyme function[ 21 ], and the distribution of metabolic fluxes in microorganisms[ 22 ]. GEMs do not only give valuable computational results, but they also provide a wealth of hypotheses that can be experimentally tested[ 8 , 23 - 26 ]. The generation of easily testable hypothesis by biological models permits model validation and improvement through iterative model building[ 23 ]. When the predictions made by a model do not agree with experimental observations, the knowledge that went into the model construction is clearly not complete regarding the area of disagreement. Importantly, an in silico model allows us to identify areas where our understanding of an organism is inadequate and where additional experimentation is needed[ 27 ]. The work reported herein describes the first manually curated genome-scale elementally and charge balanced metabolic reconstruction and model for the important pathogen S. aureus , termed i SB619 following a previously described naming convention[ 25 ], representing the first draft of its two-dimensional annotation[ 6 ]. This GENRE allows for the formulation of hypothesis ranging from relative growth capabilities on different media to the outcome of potential gene deletion experiments. Importantly, due to the curation and refinement necessary to form a functional GEM for S. aureus , the work reported contains the most comprehensive metabolic reaction list available for this significant pathogen that is consistent with known phenotypic functions. Results and discussion Basic network properties We have formulated a GENRE for S. aureus strain N315 consisting of 619 genes, 537 proteins, 640 reactions, and 571 metabolites. The entire reaction list of this GENRE is included in the supplemental material [see Additional file 5 and Additional file 2 ] and is also available at . A set of metabolic maps graphically representing the GENRE is also available at the same web address. This GENRE was built without the benefit of an earlier manually curated reconstruction and model and should be considered a first-draft to the two-dimensional annotation of the S. aureus genome. Unlike other initial GENREs[ 17 , 18 , 28 ], the S. aureus GENRE is nearly completely elementally and charge balanced. All but three reactions produce no net change in terms of chemical elements and charge. Of these three reactions, one (1,4-dihydroxy-2-naphthoate octaprenyltransferase, abbreviated DHNAOT) is never used because it is a dead-end (see below), one (phosphatidic acid synthase, abbreviated PASYN_SA) is a weighted combination of various fatty acids to form an average phosphatidate molecule for this organism, and one (2,3-diketo-5-methylthio-1-phosphopentane degradation reaction, abbreviated DKMPPD2) participates in methionine and spermidine metabolism. Most of the reactions (91%) in the GENRE are associated with one or more genes with only 9% (59) of the reactions included without a known gene. The reactions that do not have a gene association are principally transport reactions, allowing metabolites to cross the cell membrane, and reactions that involve the formation of lipids and other cell wall components [see Additional file 6 ]. The inclusion of these reactions, as well as the reactions which we were able to associate with a gene despite their absence in the genome annotation (detailed in Materials and Methods), is a legacy-data based enhancement of genome-annotation based knowledge on the metabolism of S. aureus . We provide the most comprehensive reaction list to date, including reactions that were added based on systemic analysis [see Additional file 9 ] – the GEM cannot produce biomass without them. All reactions that are associated with genes are also associated with proteins, and they are represented by what have been termed gene-protein-reaction (GPR) associations[ 25 ], which are available as Boolean statements connecting genes to reactions in the supplementary material [see Additional file 8 ] and in graphical form at . Basic properties of the reconstructed network are summarized in Table 1 . Particularly because this is a first-pass reconstruction, the network has a significant number of dead-end metabolites, as other GENREs do[ 14 , 17 , 18 , 25 ]. These dead-ends are compounds that are either only produced or only consumed by reactions in the network. Three hypotheses exist regarding the presence of a dead-end metabolite in a reconstructed network: (1) other enzymes required to produce or consume the metabolite may be missing in the reconstruction, (2) the reaction that causes the dead-end may have been misidentified based on a homology search and may not actually occur in the organism, or (3) the dead-end may exist in organism. Because the accumulation or depletion of any compound cannot occur in a steady-state, any reaction in the network involving any of these compounds cannot be used in a computed network state. In total, 108 reactions present in the reconstruction involve dead-end metabolites. All of these reactions have an associated gene and are included because of genetic evidence that they are present in S. aureus . Subsequent additions to the model will likely close some of these gaps. These reactions are further detailed in the supplemental material [see Additional file 3 ]. A reaction representing biomass formation, consisting of 58 metabolites required for cellular growth, has been defined and is detailed comprehensively in the supplemental material [see Additional file 1 ]. Key components of this reaction include amino acids, nucleotides, lipids, and cell wall constituents. Because data describing the biomass composition of S. aureus could not be located in the literature, data from Bacillus subtilis was substituted where necessary[ 29 ]; quantitative data specific to S. aureus accounts for only a small fraction of the biomass function. Although a comprehensive biomass function has been published for E. coli and used to analyze the networks of other initial reconstructions[ 17 , 18 ], this was not appropriate for S. aureus because of the differences between gram-positive and gram-negative bacteria. The relative quantities of each required metabolite were included in the biomass function when information existed, but many of the trace compounds were included in small ratios that are not quantitatively accurate. It has been shown previously that the calculated biomass production is relatively insensitive to the exact ratios used in the biomass function[ 30 ]. The biomass function is a key element of the linear programming (LP) formulation used for hypothesis generation because it allows for the computational prediction of growth. Minimal media and growth requirements Linear programming using the assumptions of flux-balance analysis (FBA) allows for the computation of feasible steady-state fluxes through a reaction network that maximize a particular objective and satisfy various constraints, including stoichiometry, thermodynamics, and enzyme capacity[ 7 , 31 - 33 ]. Specifically, we used FBA to determine fluxes leading to optimal growth subject to constraints on the usage of each reaction. This principle allowed us to systematically predict a minimal media composition capable of supporting growth of S. aureus . A literature search revealed experimental growth requirements[ 3 , 34 , 35 ] for S. aureus and they are compared with the computational predictions (Table 2 ). These requirements are for growth with oxygen, nitrate, or nitrite as a terminal electron acceptor. Computationally, we predict that S. aureus can grow with a variety of carbon sources, and Table 2 presents a glucose minimal medium because the available experimental data assumes that glucose is the carbon source. This table should be considered as a prediction of the growth requirements of S. aureus derived from its GENRE. Although some of the components in the medium seem obvious, like phosphate and a carbon source, they still serve as validation for the GEM. If we were to computationally predict that growth was possible in the absence of a carbon source, it would quickly become apparent that something was amiss with the GEM. An agreement between the computationally-predicted and the experimentally-determined requirements indicates areas where simple model predictions are consistent with existing experimental data. The primary difference between the computationally predicted growth requirements and those from experimental data is the amino acid requirement. Kuroda et. al[ 3 ] report that the six amino acids listed in Table 2 are specifically required for strain N315 to grow and speculate that, since the strain has pathways for the synthesis of all amino acids, regulation might require the presence of these amino acids. i SB619 does not account for regulatory effects and as such predicts media requirements as if regulatory processes allow any gene to be expressed at the needed levels. The transcriptional regulatory network reduces the functionality of a constraint-based metabolic model by limiting which reactions are active at a given time[ 36 ]. If enzymes required for the synthesis of a given amino acid are encoded in the genome, but are not sufficiently transcribed or translated due to regulatory processes, the cell will require that amino acid as a component of the medium even though a purely metabolic model indicates otherwise. We experimentally predict growth if any one of the nine amino acids or derivatives listed in the table is provided. Each of these compounds can either provide nitrogen through a deamination reaction or be directly converted into another compound that can provide nitrogen. S. aureus is said to require an organic source of nitrogen provided by amino acids[ 37 ], so the requirement of at least one amino acid is not surprising. It should be noted that the data from Kuroda et al[ 3 ] listing six essential amino acids is "unpublished data" and thus this information should be viewed with some skepticism. Furthermore, S. aureus has shown the ability to grow without amino acids previously thought to be required[ 37 ]; thus the amino acid requirement appears to be flexible. The differences in computationally-predicted and experimentally-determined essential amino acids highlight an area that has been historically under considered. To explore the discrepancy between experimental results and computational predictions, we elected to study in silico the effect of adding each amino acid individually to the predicted minimal media listed (with arginine present at all times). The individual results are shown in Figure 1 . We found that, on average, providing one of amino acids noted as essential from experimental data led to more biomass production than providing one amino acid not listed as essential. Using a Wilcoxon rank sum test with Matlab (The MathWorks, Inc., Natick, MA), these results are only 5.6% likely to be the result of random chance. The relative biomass production that we predict should be taken as a hypothesis that can be tested experimentally. It is not unreasonable that a pathogen would have developed regulation that leads it to require the uptake of amino acids that significantly aid its growth, especially when they are readily available in its typical environment. In essence, we predict that S. aureus can grow more efficiently by uptaking certain amino acids rather than synthesizing them, even though its genome encodes that functionality. Although one might intuitively think that this would be the case for all amino acids, the results in Figure 1 indicate that the synthesis of some amino acids is not predicted to substantially inhibit growth under the conditions studied (cys, his, met, phe, trp, tyr). Deletion Study In order to determine the effects of the deletion of a reaction from the network, as would occur in a gene knock-out experiment, FBA is used with the additional constraint that the flux through a particular reaction is zero. This allows for the rapid prediction of the results of gene deletions and also reaction deletions, as occur when a selective enzyme inhibitor is used. We calculated the effects of all single reaction and gene deletions both on minimal medium and on a rich media (consisting of all amino acids, nucleotides, and protoheme). We found that, on rich media, 130 reaction deletions and 88 gene deletions are computationally predicted to be lethal. On minimal media, 230 reaction deletions and 168 gene deletions are predicted to be lethal. These predictions are detailed in the supplemental material [see Additional file 4 and Additional file 7 ]. There are fewer lethal gene deletions than reaction deletions because some essential reactions are not associated with genes, some essential reactions are associated with isozymes, and some genes catalyze multiple reactions. When analyzed in the context of GPR associations, we found that 20 (9%) of the reactions that are essential on minimal media are associated with isozymes. This calculation indicates that gene dispensability is explained by the presence of isozymes less often than in S. cerevisiae (14.6%-27.8%) [ 20 ]. We note that the distinction between isozymes that independently catalyze a reaction and multiple gene products required simultaneously for a reaction is not always clear from the data sources used in this reconstruction. We took the results of the reaction deletion study on rich media and searched PubMed for chemical inhibitors of each of those reactions (Table 3 ). Most of the inhibitors listed do not have any published information regarding their effectiveness in S. aureus to the best of our knowledge. These computational predictions can be experimentally tested with targeted gene deletions or the inhibitors listed. Due to the diversity of biological systems in which the inhibitors were initially discovered, gene deletions would be expected to agree with our predictions more than the use of chemical inhibitors. There are a variety of reasons why any one of these inhibitors listed may have no utility whatsoever as a drug. For example, there may be no way for the inhibitor to actually enter a cell. Nevertheless, when considered as an initial guess at potential drugs, Table 3 represents hypotheses formed by a systems-level approach that has not been applied to an organism as clinically troublesome as S. aureus before. Additionally, we considered comparing the results of our gene deletion study with the results of an existing experimental approach to determine gene essentiality. Unfortunately, although we are aware of two large-scale studies on gene essentiality in S. aureus [ 38 , 39 ], there does not exist a comprehensive, publicly-available resource listing all essential genes in this organism. There is a comprehensive gene essentiality study for the related organism B. subtilis [ 40 ], but this is a different organism. Should comprehensive gene essentiality data become available for S. aureus , a comparison between experimental data and the predictions detailed herein can easily serve as validation for this model and identify problem areas. The absence of a comprehensive, publicly-available data set regarding gene essentiality in this organism is in itself powerful motivation to undertake the reconstruction detailed in this paper, as the reconstruction can rapidly predict essential metabolic genes that can later be screened experimentally as potential drug targets. Growth Phenotypes We computed the sensitivity of growth rate to oxygen uptake on a variety of different carbon sources (Figure 2 ). The carbon source uptake rate is restricted to the same molar maximum for all calculations. As expected, biomass production increases with oxygen uptake up to the point where there is no longer an oxygen limitation. In addition, the carbon sources which contain more carbon atoms also generally allow greater biomass production. For example, trehalose, which contains twice as much carbon as glucose, allows close to twice as much biomass production as an equivalent molar amount of glucose. The normalization used, explained in the figure caption, allows a quick analysis of the efficiency of the network in utilizing different carbon sources. Importantly, these predictions can be tested experimentally as a means of validating and improving the current GEM. Conclusion The work reported is the first genome-scale metabolic reconstruction for the pathogenic bacterium S. aureus and represents the first draft to its two-dimensional genome annotation. The GENRE with the GPR associations represents a chemically and genetically structured database derived from the underlying data. When the properties of this GENRE are analyzed using FBA, the model computationally predicts phenotypic states. Based on the surprising paucity of physiological growth data available for this organism, especially under carefully defined conditions, the predictions made by the GEM are best viewed as hypotheses. These hypotheses can be experimentally tested, with similar results serving as validation for the GEM and dissimilar results describing failure modes of the GEM. These failure modes can be more interesting than correct predictions because they provide direction for improvement of the GEM and point to areas in metabolism and regulation that need further investigation. The analysis of failure modes and subsequent improvement of the GEM constitute a cycle of iterative model building[ 23 , 27 ], with the potential to significantly improve a GEM. Second generation models of E. coli and S. cerevisiae have made substantial improvements over initial genome-scale reconstructions. These enhancements include greater coverage of metabolism, explicit GPR associations, more detailed enzyme localization, and better enforcement of elemental and charge balancing. We expect that future enhancements to i SB619 will improve the accuracy of the reaction network and the GPR associations. The scope of hypotheses that can be devised with a constraint-based metabolic model ranges from the relatively mundane, such as prediction of relative growth on different carbon sources, to the potentially groundbreaking, such as the determination of novel prospective drug targets. With an organism as harmful as S. aureus , any advancement of knowledge is welcome. Methods Reconstruction of the metabolic network A genome annotation for S. aureus strain N315 was downloaded from the Comprehensive Microbial Resource (CMR) at The Institute for Genomic Research (TIGR) website[ 41 ] and used to form a gene index. Each key metabolic pathway present in map form on the Kyoto Encyclopedia of Genes and Genomes (KEGG) website[ 42 ] was then examined to extract reactions that genomic data suggest occur in this strain. These reactions were matched to proteins and genes based on the information provided by both TIGR and KEGG. After this pathway-by-pathway approach, the predicted functionality of each gene in the genome was examined manually, both in the TIGR annotation and on the KEGG website to find additional metabolic reactions that are not present in any KEGG map. Inconsistencies between TIGR and KEGG were handled on a case-by-case basis to determine what functionality should be assigned to a given gene. For example, in a case where one source (either TIGR or KEGG) indicated that the gene was a "conserved hypothetical protein" but the other source listed a specific metabolic function, the gene was generally given the specific function. If both sources gave conflicting functions, a reaction was included in the model if it was present in related organisms but was only associated with a gene that had conflicting annotations if another suitable gene without conflicting information could not be found. After assembling the network based on genomic data, missing functions were noted based on physiological data regarding this organism, as well as B. subtilis and E. coli . Two books proved particularly helpful for this process[ 37 , 43 ]. Likely reactions were added to the model based on pathways present in related organisms. For example, some cell-wall components known to be in S. aureus could not be produced without the addition of reactions in various pathways present in B. subtilis . Membrane-bound transporters were added whenever evidence existed that a metabolite could enter and/or exit the cell. For example, if data indicated that a given carbon source could be used, a transporter was added to the reconstruction. Potential genes for some of these reactions were located by best-hit BLAST analysis against E. coli and B. subtilis . Each reaction without a gene association was checked against the genome annotations for E. coli and B. subtilis to determine if a gene exists in either organism for a given function. A homology search was used with any gene located in E. coli or B. subtilis against the entire S. aureus N315 genome. A reaction was putatively associated with a gene based on the E value provided by BLAST and the annotation information for that gene from both KEGG and TIGR. If a S. aureus gene did not have any functional information included in either annotation, a BLAST E value of 0.05 was considered sufficient to make an association. If functional information was present and differed from the specific reaction under consideration, a better E value would be necessary; the precise E value required was a judgment call and depended on the specificity of the annotation information. An association based on a relatively large E value should not be considered as a claim that a gene product catalyzes a reaction, but a suggestion as to a candidate gene [see Additional file 9 ]. Transporters were not associated with genes unless an annotation indicated with some degree of specificity that the gene had the given function. Whenever possible, the reactions in the network were balanced elementally and with respect to charge, where compound charge and molecular formula were based on a cellular pH of 7.2. The end result can be visualized as a stoichiometric matrix S with each column representing a reaction and each row a metabolite, with each element representing the stoichiometric coefficient. This reconstruction process, including accounting for GPR associations, and the subsequent analysis described below were done using the software program SimPheny™ (Genomatica Inc., San Diego, CA). Biomass composition Because no thorough biomass composition has been published for S. aureus , the relative production of metabolites required for growth was taken to be similar to that published for the related gram-positive organism B. subtilis [ 29 ]. The fatty acid composition of the lipids required for growth was, however, based on data specific to S. aureus [ 44 ]. Small amounts of a number of minor biomass constituents were added to the biomass function in equal amounts to account for their necessity in cellular growth. Further details are provided in the supplemental material [see Additional file 1 ]. Computation of phenotypic states and deletion study With the reconstructed metabolic network and biomass function defined, flux-balance analysis (FBA) [ 31 ] was used to find optimal growth phenotypes. Briefly, linear programming was used to find a complete set of metabolic fluxes ( v ) that are consistent with all constraints, namely steady-state network operation (eq. 1 below) and reaction rate limitations (eq. 2 below), and which maximize the production of biomass components in the defined ratio. This corresponds to the following linear programming problem: max Z = v growth Subject to S • v = 0     (1) α i < v i < β i (2) where S is the stoichiometric matrix described above, and α i and β i define the minimum and maximum allowable fluxes through each reaction v i . The flux range was set arbitrarily high for all internal reactions so that no internal reaction restricted the network, with the exception of reactions known to be irreversible, which have a minimum flux of zero. The inputs to the system were restricted where necessary (for example, limiting the amount of glucose available to the cell). The function v growth is a special reaction taking as substrates all biomass metabolites, ATP and water, and producing ADP, protons, and phosphate (as a result of the non-growth associated ATP maintenance requirement). SimPheny™ (Genomatica Inc, San Diego) was used for all FBA calculations. The value of Z computed with the above procedure can either be zero or greater than zero depending on the inputs and outputs that are allowed, corresponding to the nutrients provided in the media. A zero value is a computational prediction of no growth; this commonly occurs when an essential nutrient like a carbon source is not provided. Any value greater than zero corresponds to cellular growth. For the deletion study, each reaction was individually constrained to have zero flux and maximal biomass production was computed. Reactions were considered essential if no biomass could be produced without their usage. Gene deletions were computed in a similar manner, but all reactions requiring the presence of a given gene were simultaneously restricted to zero flux prior to computing maximal biomass production. Minimal media The minimal media was determined computationally with the systematic testing of distinct inputs. In short, different combinations of molecules were allowed to enter the reaction network until the minimal group that allowed biomass production, or non-zero Z, was found. Importantly, the minimal media computed here does not discriminate between extremely slow, inefficient growth and rapid growth; it is only concerned that some amount of biomass production is calculated. List of abbreviations CMR: Comprehensive Microbial Resource EC: Enzyme Commission FBA: flux-balance analysis GEM: genome-scale model GENRE: genome-scale reconstruction GPR: gene-protein-reaction KEGG: Kyoto Encyclopedia of Genes and Genomes LP: linear programming TIGR: The Institute for Genomic Research Authors' contributions SAB carried out all aspects of the work and drafted the manuscript. BOP conceived of the study, participated in its design and coordination, and helped draft the manuscript. All authors read and approved the final manuscript. Supplementary Material Additional File 1 Cellular biomass demand table This is a detailed, quantitative listing of the macromolecules required for cellular growth. Click here for file Additional File 2 Compound abbreviations This is a listing of the compound abbreviations used in the reconstruction and the corresponding formal compound names. Click here for file Additional File 3 Dead-end reactions This is a listing of the reactions that the current version of the model will never use because they involve a dead-end metabolite. Click here for file Additional File 4 Lethal reaction deletions on minimal media This is a listing of all of the reactions that are predicted to be essential for growth on minimal media and their corresponding gene associations. Click here for file Additional File 5 Network reaction list This is a comprehensive list of all of the reactions that are in the model. Click here for file Additional File 6 Reactions without any gene association This is a list of the reactions that are included in the model without any gene association and rationale for their inclusion. Click here for file Additional File 7 Lethal reaction deletions on rich media This is a listing of all of the reactions that are predicted to be essential for growth on rich media and their corresponding gene associations. Click here for file Additional File 8 Boolean gene-reaction associations This is a listing of reaction abbreviations along with the genes that are required for those reactions, in a Boolean form. Click here for file Additional File 9 Reactions that were added based on systemic evidence This is a list of the reactions that were added to the reconstruction based on systemic analysis (they do not appear in an obvious fashion in the genome annotation), and the gene(s) with which they are associated in cases where a gene could be located based on homology searches. Click here for file

D:\keerthana\PMC001xxxxxx\PMC1079855.xml

1079856

askMEDLINE: a free-text, natural language query tool for MEDLINE/PubMed

Background Plain language search tools for MEDLINE/PubMed are few. We wanted to develop a search tool that would allow anyone using a free-text, natural language query and without knowing specialized vocabularies that an expert searcher might use, to find relevant citations in MEDLINE/PubMed. This tool would translate a question into an efficient search. Results The accuracy and relevance of retrieved citations were compared to references cited in BMJ POEMs and CATs (critically appraised topics) questions from the University of Michigan Department of Pediatrics. askMEDLINE correctly matched the cited references 75.8% in POEMs and 89.2 % in CATs questions on first pass. When articles that were deemed to be relevant to the clinical questions were included, the overall efficiency in retrieving journal articles was 96.8% (POEMs) and 96.3% (CATs.) Conclusion ask MEDLINE might be a useful search tool for clinicians, researchers, and other information seekers interested in finding current evidence in MEDLINE/PubMed. The text-only format could be convenient for users with wireless handheld devices and those with low-bandwidth connections in remote locations.

Background ask MEDLINE evolved from the PICO [ 1 ] (Patient, Intervention, Comparison, Outcome) search interface, a method of searching MEDLINE/PubMed that encourages the creation of a well-formulated search. [ 2 ] Starting from a clinical situation, a clinician is guided through the search process by thinking along PICO elements. PICO search was developed with the busy clinician in mind, interested in practising evidence-based medicine, but unfamiliar with controlled vocabularies that could make the search more efficient. In an attempt to automate the entry of search terms into PICO elements from a clinical question, we discovered that the user could simply enter a clinical question, then let the search engine retrieve relevant journal articles. The step to allow the user to inspect the correctness of PICO elements was omitted, however, a link is provided to the PICO interface instead, so the user can manually enter search terms if the search results are deemed unsatisfactory. Although PICO helps in finding recent evidence from MEDLINE/PubMed, some users might still find searching for answers to clinical questions challenging. For some, it might be because it is time consuming to incorporate into their busy practice, difficult to learn, or perhaps, they may feel that it is not clinical enough and may not answer their question. They may find it more convenient to go directly to other subscription-only resources. Although easier to search and filtered for quality, these resources are not without disadvantages. They cover fewer journals than MEDLINE (Medical Literature Analysis and Retrieval System Online), and there is an even greater time lag between the publication of an article and its appearance in these databases than there is with MEDLINE. Other clinicians may just access resources that evidence-based practitioners might consider less evidence-based, (i.e. lesser validity) such as consulting a colleague, or reading a textbook, or perhaps, they may forego searching altogether. ask MEDLINE is intended for the clinician, researcher, or the general public who want to simply ask a question and to skip the challenge of learning how to format it in manner that will make the searching MEDLINE/PubMed efficient. It is a tool that allows the user to search MEDLINE/PubMed using free-text, natural language query, just like one would in a clinical setting, or in a conversation. A user enters a clinical question on a Web browser, and then lets the tool retrieve relevant articles in MEDLINE/PubMed. Links are provided to journal abstracts, full-text articles and related items. Moreover, ask MEDLINE is formatted for easy viewing on a wireless handheld device so it can be used while mobile, but will work equally well on a desktop computer. We report our experience in developing ask MEDLINE, and an evaluation study on its potential to retrieve references, using published, evidence-based resources. Implementation ask MEDLINE uses a multi-round search strategy. In the first round, the parser ignores punctuation marks and deletes words found on a "stop-word" list. The stop-word list includes PubMed stop words, and other words that we found by experience, to be detrimental to the search. The parser, a PHP script, then sends the modified query to PubMed Entrez' E-Utilities. The Extensible Markup Language (XML) file returned by E-Utilities indicates the category of each term in the query. Terms marked as "All Fields" denote that they are neither Medical Subject Headings (MeSH) terms nor MeSH Subheadings. These terms are checked to determine if they are found in a "MeSH Backup vocabulary." The backup vocabulary includes words other than MeSH terms, such as MeSH descriptors, that are classified as "other eligible entries". If an "All Fields" word is in the backup vocabulary, it remains in the query; if it is not, it is deleted. The remaining terms are sent back to PubMed, again through E-Utilities. Human and English language limits are always applied. If the journal retrieval count after the first round is between 1 and 50,000, the first 20 results are displayed in the user's browser and the search process terminates. Further searches are dependent on the user. The search may proceed to Round 2 under two conditions: 1) If no journals are found in the first round, a result that could signify that the search was too narrow (i.e., too many terms are searched, too many filters), the "All Fields" words are deleted from the query, even though they are found in the backup vocabulary. Only MeSH Terms and Subheadings remain (Round 2A.) 2) If the first round retrieval count is larger than 50,000 articles (an indication that the search was too broad) the "All Fields" words removed during the first round (words not found in the backup vocabulary) are put back into the query (Round 2B.) Round 2B searches contain all the MeSH terms (or MeSH Subheadings) and "All Fields" words in the original question. The updated query from either 2A or 2B is once again sent to Entrez E-Utilities. Retrieved journal articles are sent to the user. Similarly, if the count returned from second round is in the range of 1 to 50000, the search process terminates. If the second round count is still equal to 0 (denoting that the search is still too narrow) another list of "No-Go Terms", terms that when removed could result in a successful search is checked. Common MeSH abbreviations, acronyms and words like, "method," "affect," and "lead" are examples of terms on the list. New terms are continuously added to this list as they are encountered. The third round modified query is once again sent to E-Utilities and the retrieved journal articles are sent to the user. A result of 1 to 50000 citations terminates the process and displays the first 20 articles. If ask MEDLINE retrieves only one to four journal articles, a search is automatically done for related articles of the top two articles. All the articles (one to four previous) and the first 25 related articles of the first two are retrieved. As in any of the previous steps, the first 20 are displayed in the browser. In all the search retrieval pages, a link is provided for the user to manually intervene and modify the search process through the PICO interface. Links to related articles, full-text articles and abstracts are shown. Since November 2002, the British Medical Journal (BMJ) has published a POEM (Patient-Oriented Evidence that Matters) in every issue. [ 3 ] POEMs are provided to BMJ by InfoRetriever ask MEDLINE was evaluated by comparing its accuracy to retrieve an article cited as a reference in a POEM ("gold standard".) [ 3 ] Every POEM has a question with a cited reference that is relevant to the question. We entered every POEM question into ask MEDLINE, and for comparison, in Entrez, the integrated, text-based search and retrieval tool for PubMed. New critically appraised topics (CATs) from the University of Michigan, Department of Pediatrics Evidence-Based Pediatrics Web site were also used. [ 4 ] Unlike BMJ POEMs, some questions in CATs had more than one cited reference. The initial search result was examined to determine if the reference cited in a POEM or CAT was among those retrieved. Subsequent steps were taken if the reference article cited was not: 1) If the initial search retrieved journal citations, but not the specific journals cited in a POEM or CAT, the titles and abstracts were scanned to find out if they were relevant (deemed to answer the question.) If they were, related articles were retrieved, and again evaluated to determine if they matched the cited reference. 2) If no journal articles were retrieved, the question was rephrased, then searched again. Retrievals were again examined for the cited articles and relevancy to the clinical question. Overall efficiency was determined by the accuracy in retrieving a cited article and relevance of citations retrieved for citations that did not match cited references. Results Development of the search tool A simple, handheld-friendly search interface was created where users can enter free-text, natural language searches (Figure 1 .) The results are also provided in text-only mode, optimised for use by the mobile health care personnel using handheld devices. Links to abstracts, related citations in MEDLINE and full-text articles are provided. Full-text links to journal publishers are not text-based, and may require journal subscription or fees for viewing. Figure 1 ask MEDLINE is formatted for handheld devices, but will work just as well for desktop computers. The search form and retrieval pages are in text only. Evaluation of search retrievals Clinical questions in 95 POEMs and 28 CATs were searched. After first pass, ask MEDLINE found 62% of the cited articles in POEMs, while Entrez retrieved close to 14% (Table 1 .) When related articles were searched, 11.6% more were found by ask MEDLINE (8.4% in Entrez.) When three questions were rephrased, askMEDLINE, but none in Entrez retrieved two of the specific cited references, although relevant references were found to one of the questions. For 20 questions, ask MEDLINE did not find the specific cited reference, but it found journal citations that were deemed relevant and would be useful in answering the question. Entrez obtained citations for 16 (16.8%) questions that were considered relevant. Table 1 POEMs Evaluation Study. A comparison of the accuracy and efficiency of ask MEDLINE and Entrez PubMed in retrieving an exact match to cited references in POEMs in BMJ. Search Step ask MEDLINE exact match retrieved/total questions (% total) Entrez PubMed exact match retrieved/total questions (% total) A. Match at first pass 59/95 (62.1) 13/95 (13.7) B. Match after a related citation search 11/95 (11.6) 8/95 (8.4) C. Match after question rephrase 2/95 (2.1) 0/95 (0) Total exact match after A, B, and C 75.8% 22.1 No match after A, B, and C, but relevant articles retrieved 20/95 (21) 16/95 (16.8) Overall efficiency 96.8% 38.9% Citations retrieved, but not matched or relevant 3/95 (3.1) 6/95 (6.3) No citations retrieved 0/95 (0) 52/95 (54.7) Overall retrieval failure 3.1% 61% Overall, ask MEDLINE retrieved 72/95 exact matches of cited references (gold standard) in POEMs, an accuracy of 75.8%, while Entrez' accuracy was 22% (21/95.) If citations that are not the same as those cited in POEMs, but are relevant and considered satisfactory for answering the clinical question are included, ask MEDLINE's total efficiency is 96.8%. Entrez' total efficiency for finding specific and relevant citations for BMJ POEMs is 38.9% (21 specific and 16 relevant citations found.) Although citations were retrieved for all POEM questions by ask MEDLINE, three searches did not find exact matches or relevant articles (3.1%), while Entrez' results were not relevant (6.3%) for six questions. No citations were found for 52 (54% of total) questions by Entrez. University of Michigan's CATs yielded a similar total efficiency as POEMs, 96.3%, while it was 14.3% for Entrez (Table 2 .) First pass retrieval was 64.2% (Entrez 3.6%) and citation retrievals for related citations was 10.7% (Entrez 3.6%.) Four of six questions rephrased added 14.3% to the total efficiency of ask MEDLINE, while it added 7.1% to Entrez. Almost 7% of the searches retrieved relevant citations to rephrased or related articles, but none in Entrez. For CATs' questions, ask MEDLINE found 89.2% of cited references, but 14.3% for Entrez. In 21/28 questions, Entrez did not provide a specific or relevant citation, but it was only for one question with ask MEDLINE. Table 2 CATs Evaluation Study. The accuracy and efficiency of ask MEDLINE and Entrez in retrieving an exact match to cited references in CATs' questions from the University of Michigan, Department of Pediatrics Evidence-Based Pediatrics Web site. Search Step ask MEDLINE exact match retrieved/total questions (% total) Entrez PubMed exact match retrieved/total questions (% total) A. Match at first pass 18/28 (64.2) 1/28 (3.6) B. Match after a related citation search 3/28 (10.7) 1/28 (3.6) C. Match after question rephrase 4/28 (14.3) 2/28 (7.1) Total exact match after A, B, and C 89.2% 14.3% No match after A, B, and C, but relevant articles retrieved 2/28 (7.1) 0/28 (0) Overall efficiency 96.3% 14.3% Citations retrieved, but not matched or relevant 1/28 (3.6) 3/28 (10.7) No citations retrieved 0/28 (0) 21/28 (75) Overall retrieval failure 3.6% 85.7% Discussion ask MEDLINE is part of a project to develop easy-to-use resources at the point of care that has the functionality of an expert searcher. [ 5 ] Special consideration was given to healthcare personnel who use handheld devices in wireless environments, hence the emphasis on text rather than images. The results of the evaluation using POEMs in BMJ and CATs from the University of Michigan seem to indicate that it may a useful addition. MEDLINE now contains more than 13 million citations from over 4,000 journals, with approximately 40,000 added monthly. The Internet also holds millions of Web pages that archive medically related information. The task of the user then is to find the information one needs, sift through the good, bad and dangerous, and after considering other factors (patient information, laboratory tests, personal experience) apply them to the management of a patient. This is the practice of evidence-based medicine. Providing decision support for evidence-based practice at the point of care is the goal of ask MEDLINE. Several special vocabularies were considered and tested for ask MEDLINE- - Unified Medical Language System (UMLS), International Classification of Diseases: 9th revision (ICD9), but MeSH was selected because it is the controlled vocabulary used for indexing articles for MEDLINE/PubMed. MeSH terminology presents a reliable means of retrieving information that may use different terminologies for the same concept. The multi-round algorithm was developed through testing. The difference in accuracy rates between the POEMs (75.8%) and CATs (89.2%) is most likely explained by the greater number of cited references in CATs. POEMs only had one, but CATs had one or more per question. We considered a single match to a reference is a positive count. The overall efficiency of finding a citation match in POEMs and CATs was consistent, around 96%. We are unable to account for the discordance with between POEMs (38.9%) and CATs (14.3%) questions. Some POEMs' questions did not return exact matches although retrievals may have been relevant, but when the questions were reformulated, positive matches were obtained. One example was the questions, "Is low dose aspirin safe and effective for the prevention of thrombotic complications in patients with polycythaemia vera?" which returned 51 relevant citations but no exact match. When it was rephrased to, "Does low dose aspirin prevent thrombotic complications in polycythaemia vera?" the second citation matched the cited POEMs reference. The question, "Is a prolonged period of antithrombotic pretreatment effective for reducing adverse outcomes in patients with unstable coronary syndromes?" returned close to 40,000 citations, but when rephrased to, "Is prolonged antithrombotic treatment indicated in unstable coronary syndromes before intervention?" the first citation was an exact match. There were questions that did not match despite attempts to rephrase them. One, "Is a one day treatment of Helicobacter pylori as effective as a seven day regimen in patients with dyspepsia?" The citations retrieved were all relevant, but none was the exact match because there were many treatment regimens variations. Some questions were quite easily modified to obtain exact matches, like, "Does azithromycin given to patients with acute coronary syndromes prevent recurrent ischaemia?". But by simply deleting the "s" in syndromes, the first of 25 relevant citations was a match. Other examples are shown in Table 3 . Table 3 Examples of rephrased questions. Minor modifications of some questions increased retrievals of exact matches of cited references. a POEMs, b CATs Original question Result before rephrasing question (Citation match/total retrieved) Rephrased question Result after rephrasing question (Citation match/total retrieved) a What is the risk that any given mole will become a melanoma? 0/31 What is the risk of any given mole transforming into a melanoma? 1/50 a Are ear temperatures reliable? 0/511 Is measurement of ear temperature reliable? 6/23 b In children with an acute febrile illness, what is the efficacy of single-medication therapy with acetaminophen or ibuprofen compared with combination therapy combining the two medications in reducing fever while avoiding adverse effects? 0/0 In children with fever, is acetaminophen or ibuprofen alone better than combined, while avoiding adverse effects? 8/25 Currently, there are not many free-text, natural query search engines that are specifically developed for MEDLINE/PubMed. Requiring clinicians to learn and master search methods for efficient searching is unrealistic. [ 6 ] It is even more important for the general public, unfamiliar with medical terminology, who are seeking medical information from MEDLINE/PubMed in increasing numbers, but who frequently do not use effective strategies. [ 7 ] Providing a natural language search tool that mimics a clinician's practice habits at the point of care, but with the functionality of an expert medical librarian might be beneficial. ask MEDLINE also uses MeSH and omits variation that other search engines have that may impact search results as some have found. [ 8 ] Google is a very powerful search engine that searches the entire World Wide Web, including biomedical Web sites. There are ongoing discussions in biomedical lists on the merits of Google and PubMed. They search different databases and comparisons between the two are not valid. ask MEDLINE is a more modest tool and certainly not comparable to either of the two. Conclusion Evaluation of ask MEDLINE based on two collections of evidence-based resources suggests that it may be a useful resource for clinicians, researchers and the general users interested in finding relevant medical information. Its text-only format will make it convenient for users of wireless handheld devices to use it in the clinical setting where wireless Internet access is available. It will also be helpful for those in with slow connections to the Internet especially those in remote locations in developing countries. Efforts to improve its functionality and clinical usefulness for evidence-based medicine are continuing. Availability and requirements Project name: Development of evidence-based medicine tools Project home page: Operating system: platform independent Programming language: PHP Other requirements: Apache, MySQL, PHP License: Free, anyone may use the service Any restrictions to use by non-academics: None, anyone may use the service Competing interests The author(s) declare that they have no competing interests Authors' contributions PF conceived of the study and design, participated in the development of the search tool, carried out the evaluation studies and drafted the manuscript. FL participated in the design of the study and wrote the search script. MA participated in the study design, evaluation study and gave final approval of the version to be published. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

D:\keerthana\PMC001xxxxxx\PMC1079856.xml

1079857

Personalized online information search and visualization

Background The rapid growth of online publications such as the Medline and other sources raises the questions how to get the relevant information efficiently. It is important, for a bench scientist, e.g., to monitor related publications constantly. It is also important, for a clinician, e.g., to access the patient records anywhere and anytime. Although time-consuming, this kind of searching procedure is usually similar and simple. Likely, it involves a search engine and a visualization interface. Different words or combination reflects different research topics. The objective of this study is to automate this tedious procedure by recording those words/terms in a database and online sources, and use the information for an automated search and retrieval. The retrieved information will be available anytime and anywhere through a secure web server. Results We developed such a database that stored searching terms, journals and et al., and implement a piece of software for searching the medical subject heading-indexed sources such as the Medline and other online sources automatically. The returned information were stored locally, as is, on a server and visible through a Web-based interface. The search was performed daily or otherwise scheduled and the users logon to the website anytime without typing any words. The system has potentials to retrieve similarly from non-medical subject heading-indexed literature or a privileged information source such as a clinical information system. The issues such as security, presentation and visualization of the retrieved information were thus addressed. One of the presentation issues such as wireless access was also experimented. A user survey showed that the personalized online searches saved time and increased and relevancy. Handheld devices could also be used to access the stored information but less satisfactory. Conclusion The Web-searching software or similar system has potential to be an efficient tool for both bench scientists and clinicians for their daily information needs.

Background The rapid growth of publications available in the Medline and other sources raises the question how to search efficiently while maintaining acceptable relevancy. Without the assistance from domain experts such as medical professionals or librarians, retrieving relevant information from the Internet remains a difficult task. For bench scientists, it is important to monitor articles in their fields constantly on a weekly if not daily basis. For a clinician, it is important to access the patient records anywhere and anytime. These searching procedures often involve relatively simple and similar procedures. For example, they go to certain websites such as the Medline, type in certain words or terms, and then search accordingly using the search engine of that site. Different words or combination reflect different research topics. The returned results were viewed and discarded. The similar search may be repeated next time by typing the same words/terms again. The objective of this study is to automate this tedious procedure by recording and parsing theses words and websites into a database and to search automatically using software. The person who records the words could be a librarian or a person familiar with medical subject heading (MeSH). The advantage of using MeSH terms is to increase both recalls (sensitivity) and relevancy (specificity) since Medline is indexed through the MeSH. The software could be software agent that automatically performs certain procedure such as searching the Web using words from the database. The advantage of using software is to save time, especially when the search is conducted during night time. It is usually faster due to less congested network traffic. The returned search results were stored locally on a server and could be updated regularly. We have developed such a system and implemented in our Cancer Center website. We emphasized the security issues due to the potential of the system in retrieving information from privileged sources such as a patient record system. A Web-based user satisfaction survey showed that the personalized information search increased both efficiency and relevancy. We concluded that the Web-searching agent or similar system has potential to be an efficient tool for both research scientists and clinicians to get desired information automatically from various sources. Implementation System architecture and the agent software The system was created as a Web-based search, storage, and presentation system with both wired and wireless components. We include the wireless component due to ever increasing popularity of personal digital assistant (PDA) such as a pocket PC, Palm pilot, or other handheld devices in personal information access. The wired component was based on the Ethernet that links the users and the web server. The wireless component included several wireless LANs (WLAN) with Access Point (AP) was centrally controlled by an Access Control Server (ACS, Cisco Company) for Authentication, Authorization and Accounting (AAA). All wireless clients had Extensible Authentication Protocol (EAP)-enabled as described before [ 1 ]. The scheduling software agent called Schedule Wizards was installed and programmed to use the stored user preference such as words, websites, and journals in the database to search the various information sources automatically. Database, coding, schedules, automatic search, storage and visualization The database was developed based on Microsoft (MS) Access. User-defined scripts were inserted for using the agent software. Web-based administrative interface for information presentation, and preference updates were created by using MS FrontPage. Hyper Text Markup Language (HTML), active server pages (ASP) with that links web interface with the database server through open database connectivity (ODBC), VBscript, and Structured Query Language (SQL) computer languages were applied for preference collection and storage. The Boolean expressions "AND", "OR" and "NOT" were applied to filter the articles, focus the search, and increase relevancy. The returned search results were stored locally, as is in a html file, in a MS Internet Information Server (IIS) and updated daily. Each user preference has option to have 4 or more topics that each includes four MeSH terms, a website, a update schedule, and as many journals, which are coded using their International Standard Serial Number (ISSN). The four terms are linked by two "AND" Boolean expressions and one "NOT" Boolean expression. The users log onto the website to view the updated search results without typing any words. The system has potentials to retrieve similarly from non-MeSH-indexed literature or a privileged information source such as a clinical information system where user account information are needed for authentication. The security is thus addressed by applying access control strategy coupled with user account management. Online survey and data analysis An online survey form was created and the survey conducted during the testing period. The questions had been designed to collect user information, to survey the user satisfaction and to determine the efficiency and effectiveness of the system. Although not the focus of the study, questions related to recall (sensitivity) and precision (specificity) were also included. Statistical analysis using paired t-test assuming equal variance was conducted to compare time spent before and after using the system. The evaluation of the relevancy is based on the user-defined criteria. Theoretically, both the relevancy and recall using MeSH may be higher than the search based on unmodified words, since parsing from regular words into MeSH terms could increase recall and precision from the MeSH-indexed Medline. Results System and database development The system adopted a client and server architecture to centralize search, storage and management as shown in Figure 1 . The software agent runs on the server using the data for the database to search the Medline. The Active Director (AD) of a Windows 2000 (W2K) Server is used as a Network Access Server (NAS) that negotiates with ACS for the AAA services. The database includes various tables to record information about user preference (words/MeSH terms, website, search schedule, et al). All the information can be updated over the Web using an online preference form. The overall search contains multiple steps as shown in the Figure 2 . The system administrator discusses with end users for setting up user accounts and recording their preferences. A librarian or any person familiar with MeSH can do the same. A Web-based form is available for the user to view current preferences and update the preference over the Web. The recorded words are then matched to MeSH terms. The modified preference is then parsed into the code that the software agent will apply to perform the search accordingly and automatically. The search is conducted automatically according to the user preference mostly during the night. This is to take advantage of the less-congested network traffic during the night time. Figure 1 Web-based, wireless information access and security management. The AP 350 was configured to use ACS for authentication of the EAP-enabled wireless devices over the WLAN. The IIS offers presentation interfaces for the stored information. A W2K Server running AD was used to mimic a NAS to communicate with ACS through RADIUS protocol. The NAS has enabled RAS for the ACS. Figure 2 The automatic search procedure by the Medline Agent. The automatic search includes the recording, parsing and storage of user preferences (steps 1–3), code-generation and search by the software (steps 4–5), storage (step 6) and presentation (steps 7–9) or the retrieved information. User preferences were stored in the database and will later be parsed into the executable code. The software performed search automatically according to the designated schedule and preference such as website, terms and sources. The retrieved information was stored within the IIS and presented over the Web. The Web interfaces for visualization The system was implemented in a testing Web server and Web access interface designed for individual user. Figure 3 showed one of the typical Web interface for interested topics. Clicking on the related linkage left column leads to the PubMed search page, as shown in the right column. Theoretically, the user could have as many topics but we limited it to around four for the testing purposes. The users do not have to type in any words since those words have been recorded, optimized through MeSH website , and used in generating the updated linkages to the publications. Figure 3 The Web-interface for clients/users. The automatic search was conducted as scheduled based on the user preferences. Individual users logon to their own interface to see publications from the Medline (upper section in left panel) and from the other sources (lower section of the left panel). The right panel show the sources when linkages on the left panel are clicked. The listed publications reflects the updated search results. Clicking on the linkage on the right panel leads to the abstracts just like you search the Medline regularly. The system was also implemented in the Cancer Center Microarray Shared Facility web site to retrieve and update regularly the Microarray-related publications, as show in the Figure 4 . The disease sites such as Breast and Colorectal et al were selected since the related clinical trials are currently conducted in the Center. Investigators view the updated publications without typing a word. The searching terms has been mapped through MeSH tool in Entrez PubMed website in order to increase the sensitivity and specificity. Figure 4 The Web-interface for microarray-related publications. The automatic search was conducted as scheduled based on the predetermined MeSH terms. The left panel includes the microarray-related technology (upper section) and clinical applications (lower section), which are different sites studied. The right column shows the interface of Entrez PubMed. The listed publications reflects the updated search results. Clicking on the linkage in the right panel leads to the abstracts just like you search the Medline regularly. Coding of personal agent to use the database The Schedule Wizard was the scheduling software we used to manage the scheduled procedures such as daily backup and searching job using Web browsers such as MS Internet Explorer (IE). The software was programmed to turn on at nighttimes to search and retrieve relevant information from the Medline. Various search engines over the Web were used to search the user predetermined topics. The returned search results were stored as a HTML file and saved locally on a server within a MS IIS. The scheduled search and retrieval were performed according the user-predefined schedule on a daily, weekly or monthly base. The visualization interfaces were linked to those stored results. Part of a sample code for a single search (one topic) is listed below: START "C:\PROGRAMS\Internet Explorer\IEXPLORE.EXE" "C:\PROGRAMS\Internet Explorer" WAIT 3 KEYS [Ctrl-TAB] [ENTER] IFTIME 300 WAIT 3 KEYS [Ctrl-A] KEYS "0028-4793" OR "0098-7484" OR "1532-0464" OR "0007-1447" OR "0026-1270" OR "1475-3898" OR "0003-4819" AND Radiology Information Systems AND Computer Storage Devices AND Computer Communication Networks [ENTER] WAIT 3 KEYS [Alt-f] [a] KEYS C:\Inetpub\wwwroot\Agent\All in one\RISprefered.htm [ENTER] WAIT 1 KEYS [TAB] [ENTER] WAIT 3 END KEYS [Alt-f] [c] The above example is to search and retrieve information regarding "radiology information system", "storage devices" and "networking". The preferred journals are indicated as ISSN codes. The line 1 is to start MS IE followed by 3 second for the browser to be fully loaded. Line 3 is to direct the agent to the PubMed Entrez searching page with the default searching filed. Line a) to line k) conducts the search and downloads the searching results in a folder called "All in one" and named "RISprefered.htm" within the IIS. The "IF" statement reinforces the rule that the search will be terminated with "END" if time lasts more than 300 seconds. This is to ensure timely closure of searching window in order not to interfere with the next scheduled searching task. Many search procedures could be concatenated forming a single search task, which could be executed all at once. The stored search results are immediately available for viewing through the browser after an authorized user logs on to the system and is directed to the stored HTML file in IIS. Effectiveness and efficiency of the Medline search by user satisfaction survey The main goal of our system was to save time for bench scientists and clinicians. We tested the feasibility of an automated search process based on user preferences. Although not the focus of the study, the question related to recall and precision were asked in the survey. Similar to the sensitivity and specificity, recall refers to the proportion of relevant documents in the collection retrieved by a query [ 2 - 5 ], and precision the proportion of the relevant documents returned by the query [ 2 ]. From the online survey, the system was rated as useful and easy to use, as shown in the Table 1 . Most users did not know MeSH concept well and intend or recommend using the MeSH and the system. The time spent to retrieve similar amount of information decreased from 2.2 hours to 1 hour. Almost 70% information retrieved was considered relevant. Among the relevant information, one third was considered new to them. All these suggest that the system or similar products may potentially be useful tools to improve search efficiency and relevancy as well. The keys may hinge on individualization and specification. User information also suggests that search process is time-consuming process and may prompt more use of MeSH terms in the future. Table 1 The system saves time and improves online search efficiency (n = 35) Questions Criteria Scale Mean ± SD The system useful 1 for not at all and 9 very useful 7.5 ± 1.4 easy to use 1 for very difficult to 9 very easy 6.6 ± 2.3 will use it regularly 1 for not and 9 certainly will 6.5 ± 2.2 recommend using it 1 for not and 9 highly 7.5 ± 2.0 Efficiency time prior to using it 0.5, 1.0, 1.5, ...4.0 hrs/wk 2.2 ± 1.3 time after using it 0.5, 1.0, 1.5, ...4.0 hrs/wk 1.0 ± 1.2* Relevancy relevant articles 10, 20, ..., 90% 69.3 ± 19.4 new among the relevant 10, 20, ..., 90% 33.6 ± 15.7 Users know MeSH before 1 for not at all and 9 for expert 3.2 ± 2.0 new to computer 1 for new and 9 for expert. 6.3 ± 2.4 reasons to try it too busy to search 74% too time consuming 43% too many irrelevant reports 17% frustrated when searching 11% *p < 0.001 using paired t-test assuming equal variances as compared with hours spent weekly prior to using system. System stability, wireless access and security The stability and reliability of the system were tested with tasks scheduled 5-minutes apart. The average time for one search and retrieval task, which includes many searching topics concatenated, was 5–10 minutes. It will serve more than 8,000 users if a dedicated computer is used to conduct search on a monthly basis. Among all scheduled tasks, most (99%) were successfully accomplished during 5 months of testing period, except those during power outages and network downtime (data not shown). The wireless LAN managed wireless clients from a centralized ACS as reported before [ 1 ] and shown in the Figure 1 . We chose to include the wireless component due to the increasing popularity of the technology. The security concern has to be addressed first. It has been reported that through open-air clear text transmission of Wired Equivalent Privacy (WEP) keys and Media Access Control (MAC) addresses increased vulnerability [ 6 ]. A W2K Server running AD and Domain Name System (DNS) were thus implemented and used to enhance the security. AD and DNS mimic a Network Access Server that was needed for the wireless clients to communicate with ACS through Remote Access Dial-in User Service (RADIUS) and EAP-based protocol. No apparent weakness has been reported yet. The NAS has enabled a Remote Access Service (RAS) for the ACS. The ACS takes advantage of Windows security management features as applied in AP management. The centralized control of all wireless access points and clients enhanced the secure transmission of the stored information over the WLAN. Discussion Standardized procedure and automated online search Our study is in an attempt to standardize and to automate a time-consuming but relatively simple and similar searching procedure, not trying to do traditionally considered Information Retrieval (IR) [ 7 - 10 ] text mining [ 11 ] or Automatic knowledge extraction [ 12 ]. To reduce the time spent on the Medline search, different approaches such as stored procedures, filtering and librarian assistances have been applied. Locally-stored procedures, however, is less accessible than stored information, since the procedures have to be stored locally and executed again. Web-based search system may provide user-friendlier presentation and accessibility. Software agent is a robot software program that carries out set of operations on behalf of a user with some degree of independence or autonomy. Functionally, an software agent can be as simple as an autonomous software programs that assists the user's daily routine such as reading electronic mail and maintaining a calendar. The Multi-Agent Retrieval Vagabond on Information Networks (MARVIN) has been developed for search from clinical systems [ 13 ]. The system is not, however, tailored to individual need. It is possible to automatically search, retrieve, and present information by the joined efforts of both end users and system administrators and others [ 14 ]. The personalized automatic searching and presentation system will help bench scientist and clinicians in their daily information acquisition. Individualized search may enhance both relevancy and efficiency Individualized online retrieval from the MeSH-indexed Medline or other sources such as a local medical information system was expected by many clinicians to improve their decision-making [ 15 ]. Many commercial products intend to personalize the user preference. The low specificity of a profession-based rather than individual-based filtering system limits their capability to maintain precision for a research scientist or other medical professional, e.g., an oncologist of B cell lymphomas, let along when personal criteria of relevancy may change over time [ 16 ]. The real challenge is how to extract individual needs into controlled vocabulary such as MeSH in order to achieve highest possible relevancy and efficiency [ 14 ]. Understanding the user's need and knowledge of the searching area may result in higher quality search from, e.g., MeSH-index sources such as Medline. Mobile devices and security management for privileged information The widely used mobile devices such as Pocket PC or Palm Pilots are getting relatively inexpensive and may be applied in daily information access [ 17 ]. The use of wireless access to broadband services may mean that even full motion video applications could be supported over long distance [ 18 ]. We foresee a wider use of wireless devices for daily information needs. The security concerns for accessing retrieved information through both wired and wireless network still prevent the healthcare organization from deploying them. One of the approaches to minimize the vulnerability is to control the remote and/or wireless clients' access through RADIUS and EAP-enabled AP management [ 1 ]. Another approach is to use digital certificate and secure web servers [ 19 , 20 ]. In addition, personal-identifiable data from patient should be guarded with highest possible security measure [ 21 ], especially because of the Health Care Insurance Portability and Accountability Act (HIPAA). Combined efforts of technical, organizational and behavioral approaches are needed to guard the stored information and at the same time to make authorized access easier. The limitation of the system and future directions There are several limitations related to the study. First, individualization of the searching strategy involved the interaction between system administrator and end users for their preferences thus limit the automatic nature of the system. Further study to automate the procedure based on the online preference modification is needed to expand the user pool. Promotion of MeSH awareness is another way to increase its use and enhance recall and relevancy for MeSH-indexed sources. Secondly, WLAN provided access to the stored information within the campus. Real time Internet access including Virtual Private Network (VPN) and digital certificates should be further tested although our preliminary results indicated the possibilities (data not shown). Thirdly, most current users were recruited among users within Medical Informatics field including teachers and students or research labs in Biology. Expansion into other fields will further demonstrate the usefulness of the system. Lastly, pocket PC does not seem to be a good choice for viewing images due to its small screen and low speed wireless connection. Other handheld devices with bigger screen and higher visibility such as tablet should be tested for files like images and electrocardiogram. Conclusion The primary goal of this study is to test the feasibility of automated search from various sources, especially from online literature sources such as the MeSH-indexed Medline and access the stored information through a Web interface. The survey analysis based on user-defined criteria or relevancy revealed a significant reduction of time spent on the Medline search while maintaining a relatively high relevancy. This might due to individualized and MeSH-based searching strategy. To further enhance the effectiveness and efficiency, more users are to be recruited. Security concerns need to be thoroughly addressed before implementing wireless access or accessing clinical information. Availability and requirement • Project name: personal agent for information search and retrieval. • Project home page: . • Operating system(s): Web-based system, platform independent. • Programming language: C++, JavaScript and html. • Other requirements: none. • License: free to access. • Any restrictions to use by non-academics: none. List of abbreviations AAA: Authentication, Authorization and Accounting. AD: active directory. ASP: active server pages. AP: access point. DNS: domain name system. EAP: Extensible Authentication Protocol. HTML: Hyper Text Markup Language. IIS: internet information server. MAC: Media Access Control. MeSH: medical subject heading. NAS: Network Access Server. ODBC: open database connectivity. PDA: personal digital assistant. PDSV: personal domain specific vocabulary. RADIUS: Remote Access Dial-in User Service. RAS: Remote Access Service. SQL: Structured Query Language. VPN: Virtual Private Network. W2K: Windows 2000. WEP: Wired Equivalent Privacy. WLAN: wireless local area network. Competing interests The author(s) declare that they have no competing interests. Authors' contributions DC, the principal investigator, conducted most of the experiments. HFO was the sponsor of a fellowship awarded to DC by the National Library of Medicine, NIH of the US. SMS was an advisor for genome-related information search. They contributed to the design, coordination, support, and performing of experiments. Pre-publication history The pre-publication history for this paper can be accessed here:

D:\keerthana\PMC001xxxxxx\PMC1079857.xml

1079858

Portals to Wonderland: Health portals lead to confusing information about the effects of health care

Background The Internet offers a seemingly endless amount of health information of varying quality. Health portals, which provide entry points to quality-controlled collections of websites, have been hailed as a solution to this problem. The objective of this study is to assess the extent to which government-run health portals provide access to relevant, valid and understandable information about the effects of health care. Methods We selected eight clinically relevant questions for which there was a systematic review, searched four portals for answers, and compared the answers we found to the results of the systematic reviews. Results Our searches resulted in 3400 hits, 155 of which mentioned both the condition and the intervention in one of the eight questions. Sixty-three of the 155 web pages did not give any information about the effect of the intervention. Seventy-seven qualitatively described the effects of the intervention. Twenty-six of these had information that was too unclear to be categorised; 15 were not consistent with the systematic review; and 36 were consistent with the review, but usually did not mention what happens without the intervention, what outcomes have been measured or when they were measured. Fifteen web pages quantitatively described effects. Four of these were abstracts from the systematic review, nine had information that was incomplete and potentially misleading because of a lack of information about people not receiving the intervention and the length of follow-up; one had information that was consistent with the review, but only referred to three trials whereas the review included six; and one was consistent with the review. Conclusion Information accessible through health portals is unlikely to be based on systematic reviews and is often unclear, incomplete and misleading. Portals are only as good as the websites they lead to. Investments in national health portals are unlikely to benefit consumers without investments in the production and maintenance of relevant, valid and understandable information to which the portals lead.

Background "'Be what you would seem to be' – or, if you'd like it put more simply – 'Never imagine yourself not to be otherwise than what it might appear to others that what you were or might have been was not otherwise than what you had been would have appeared to them to be otherwise."' (Lewis Caroll (1832 – 1898), Alice in Wonderland) The internet offers a seemingly endless amount of health information, but the quality of this information is variable [ 1 ]. A bewildering array of criteria are used to rate the quality of this information, but none of these have been validated and many of them have a short life span [ 2 ]. Guidelines exist to help consumers to critically appraise the relevance and validity of health information [ 3 - 6 ], but few people have the time and skills to apply such guidelines. Health portals, which provide entry points to quality-controlled collections of websites, have been hailed as a solution to these problems. The development of health portals by national governments can be seen as a support of recent legislation and policies establishing the right for individuals to participate in decisions regarding their health care. Information that supports an informed choice about health care should include reliable information about the relative benefits and harms of relevant options [ 7 ]. Moreover, this information should be presented in such a way that it is easily understood. Consistent presentations across various treatment options can make it easier to understand information and makes it easier to make comparisons across treatments. The objectives of this study were to assess the extent to which health portals provide easy access to relevant, valid and understandable information about the effects of health care. We examined four English-language government-run health portals: • Canadian Health Network – Canada [ 8 ] • HealthInsite – Australia [ 9 ] • MEDLINEplus – USA [ 10 ] • NHS Direct Online – England [ 11 ] These four portals lead to similar types of resources including information about health conditions and treatments. They describe their goal as the provision of " appropriate ", " authoritative ", " credible " and " timely " health information for the general public. Table 1 summarises their guidelines for including sites. Table 1 Health portal goals and selection guidelines Portal Goal Selection guidelines for included sites Canadian Health Network "CHN's mission is to support Canadians in making informed choices about their health, by providing access to multiple sources of credible and practical e-health information." • Does the organization have timely and credible information on health promotion and disease prevention? • Is authorship clearly stated? • If the source is not a professionally or legally accredited authority, is there sufficient supporting information to establish an informed perspective. • If a non-professional gives medical information, is this fact clearly stated? • Are original sources clearly referenced? • Are claims relating to the benefits of a specific health promotion/disease prevention services supported by appropriate, balanced evidence? • Is the content original, and of sufficient depth to warrant a link? • Is the resource relevant to the CHN mission statement? • Is the information updated or reviewed on an adequate basis given the content? • If the resource only presents one-side of a controversial issue, is this made explicit? HealthInsite "(Health Insite ) aims to improve the health of Australians by providing easy access to quality information about human health." Sites must have a written policy and procedure that • Includes a policy that each resource is authored by a person or group with appropriate qualifications/experience • Includes a procedure for appropriate attribution of resources • Includes a review process. The policy needs to cite positions/qualifications/names of who reviews • Details the final approval process (including responsibility/qualifications) • The date of publication and, where appropriate, date of previous versions must also be given. Medline Plus "MedlinePlus is designed to help you find appropriate, authoritative health information." • The source of the content is established, respected and dependable. A list of advisory board members or consultants is published on the site. • The information provided is appropriate to the audience level, well-organized and easy to use. • Information is from primary resources (i.e., textual material, abstracts, Web pages). • The primary purpose of the Web page is educational and not to sell a product or service. • The source for the contents of the Web page(s) and the entity responsible for maintaining the Web site is clear. • Information is current or an update date is included. • The site provides unique information to the topic with a minimum of redundancy and overlap between resources. NHS Direct Online To provide high quality health information and advice for the people of England and Wales No information provided. We used each portal to find information about the effects of interventions for eight health problems (table 2 ) and compared the information we found to the results of systematic reviews [ 12 - 20 ]. Table 2 Health care questions Problems Interventions Reference Alzheimer's disease Galantamine 8 Bulimia Antidepressants versus psychological treatment versus both 9 Jet lag Melatonin 10 Lumbar disc prolapse Surgery 11 Malaria prevention Mefloquine 12 Nausea and vomiting in early pregnancy Treatments 13,14* Schizophrenia Haloperidol 15 Vaginal yeast infection Oral versus intravaginal antifungal agents 16 *A substantive update of this review was made in 2004. This was taken into account when comparing website information with the results of the review. Methods Research topics covered by systematic reviews were identified by searching through the journals Evidence Based Mental Health and Evidence Based Medicine 2000–2002. Topics presented in these journals are selected for their presumed clinical relevance. For the sake of consistency, we chose reviews from The Cochrane Library. Topics were chosen to cover a variation in age, sex, mental/physical health, and chronic/acute illness. Search terms for the health care conditions and interventions were developed (See table 3 ). Search terms that lay people were likely to use were preferred, and were also adapted to local spellings and expressions. Where there was a choice between several search terms, the term that gave the most hits was chosen. We also used the portals' lists of health topics and indexes to search for relevant information. Table 3 Search terms Alzheimer's disease alzheimer +alzheimer +galantamine alzheimer and galantamine/reminyl galantamine alzheimer alzheimer galantamine/reminyl galantamine/galanthamine reminyl Bulimia +bulimia +treatment bulimia and treatment bulimia treatment bulimia Jet lag jeg lag and melatonin jet lag melatonin jet lag melatonin Lumbar disc prolapse lumbar disc prolapse herniated disk/disc AND surgery herniated disc/disk slipped disc/disk discectomy Malaria prevention mefloquine and malaria mefloquine malaria mefloquine malaria Nausea and vomiting in early pregnancy morning sickness morning sickness and treatment pregnancy nausea early pregnancy nausea Schizophrenia +haloperidol +schizophrenia schizophrenia and haloperidol schizophrenia haloperidol haloperidol schizophrenia Vaginal yeast infection +yeast +infection +treatment +thrush +treatment yeast infection and treatment yeast infection treatment yeast infection thrush candidiasis Between January and February 2004 two researchers (CG and EJP) independently carried out searches for each topic within each portal. Web pages were included if they referred to both the condition and the intervention. When the systematic review in question covered several interventions, web pages describing any one of these interventions were included. Web pages were only included if the portal linked directly to them or to other pages on the same web site. Web pages were excluded if they were duplicates or if they were clearly not intended as information about the conditions and interventions. Information intended for a professional audience was also excluded when a consumer version of the same information was available. Discrepancies between the researchers' search results were resolved through discussion. Included web pages were then examined to see if they included either qualitative or quantitative information about the effects of the interventions. Qualitative information was categorised as indicating an effect, a probable effect, an unknown effect, probably no effect, no effect, or unclear, if it was not possible to categorise the information (Table 5 ). The results of the systematic reviews were categorised using the same categories to allow comparison of the qualitative information we found to the results of the reviews. Quantitative information was compared directly to the results of the reviews. Table 5 Coding of qualitative information Statements coded as expressing effect You may benefit from the medication It has helped many patients It is said to improve symptoms Statements coded as not expressing effect The treatment can be used Treatment may include Your doctor may recommend the treatment Statements coded as "effective" Is effective/highly effective/(often) very effective Has proven effective/helpful/beneficial Especially helpful Has been found/reported to be helpful/effective Has an effect on the symptoms Improves the symptoms Enjoys a good success rate Works/(usually) works well Usually successful Is the most effective drug X was more effective than Y Was better than other therapies and better than no treatment Strong evidence on the effectiveness of X when compared with Y In clinical trials, some people who took the drug compared to individuals who took a placebo showed some improvement People treated with the drug showed improvements in symptoms Reduces the PANSS scores significantly more than placebo You can protect yourself against X by taking Y Can clear up your symptoms in a couple of days and cure the infection within a week Many studies have shown the treatment to be useful/can ease the symptoms Many people respond to the treatment Most people do very well after treatment These treatments have been beneficial to many women Relieves symptoms in the majority of properly selected patients Have helped many patients Is recommended/should be recommended These drugs all had similar ability to relieve the symptoms Helps some sufferers/some people Of some value in certain patients Statements coded as "probably effective" There have been reports of some benefits Appears to be helpful Results/evidence suggest that the treatment is beneficial/has beneficial effects Statements coded as "unknown effect" Have not been proven Results are variable Some studies suggest this is effective..other studies have found that it doesn't help Effectiveness for many people is unknown Results of studies have been inconsistent The evidence is uncertain The treatment is at present, an experimental approach . Statements coded as "not effective" None found Statements coded as "probably not effective" None found Statements coded as unclear Not all people taking these drugs benefit from them The treatment is not always successful For some people, the treatment may help prevent some symptoms from becoming worse For some people, the treatment is prescribed to possibly delay the worsening of some of their symptoms Does the treatment work? Not always The treatment is of not more benefit than the other treatments May help/relieve pain/improve symptoms/relieve symptoms/be a good option Might help Some studies have shown that (the treatment) may decrease (the symptoms) Can help/improve symptoms/relieve symptoms Is said to improve symptoms The effects of the treatment varies for different people. Some will not notice any effect, while others may find that their condition improves slightly Results Our searches resulted in roughly 3400 hits. Searching was sometimes made difficult by the variety of terms used for the same conditions and interventions. For instance, the use of different terms for vaginal yeast infection, for the same groups of anti-psychotics, and for similar types of back surgery was confusing and resulted in different numbers of hits within each portal. Searches were possibly more extensive than necessary as our goal was to identify as much relevant information as possible, rather than to test the sensitivity and specificity of the search engines. After removing duplicates and excluding web pages that were clearly not relevant, (for instance, minutes of meetings or reports on cost-effectiveness) a total of 155 web pages that mentioned both the condition and the intervention(s) for one of the eight questions remained (figure 1 ). All four portals provided information about each of the eight questions. Figure 1 Flowchart showing the number of web pages identified for each category. Sixty-three of the 155 web pages that mentioned both the condition and the intervention in question did not give any information about the effect of the intervention. The remaining 92 web pages presented information about effect either in qualitative or quantitative terms. Qualitative information Seventy-seven of the included web pages qualitatively described the effects of the interventions. It was frequently difficult to judge what was and was not a statement about the effects of an intervention and what a statement meant in terms of the effectiveness of an intervention. We therefore asked 16 colleagues to categorise a selection of statements. Frequently there was little agreement among them. The use of the word "may" was particularly confusing. For instance, the statement "for some people in the early and middle stages of the disease, galantamine may help prevent some symptoms from becoming worse for a limited time" was categorised as "effective" by one person, "probably effective" by seven, "unknown effectiveness" by five, and "unclear" by three. Statements about what clinicians do were also confusing, for example "your doctor may recommend surgery." Based on the input of our colleagues the three of us agreed on the final code list (Table 5 ) that was used to categorize the qualitative statements of effect. The specific context of a statement was also taken into account in the coding. Twenty-six of the 77 web pages with qualitative statements of effect were categorised as "unclear" because it was not possible to determine what the statement meant about the effectiveness of the intervention. Of the 51 web pages that could be categorised 15 were not consistent with the results of the systematic review and 36 were consistent. However, only four of the 36 that were consistent described what happened to people who did not receive the intervention and only four indicated how long the effect was likely to last. In some cases, portals offered contradictory information. While websites on Health Insite, Canadian Health Network, and NHS Direct Online all support the use of ginger for the treatment of morning sickness, websites on Medline Plus include the following messages: • "Try using ginger, which has proved effective in combating morning sickness." • "Ginger has been studied in pregnant women and appears to be helpful." • "Ginger" is not recommended for morning sickness, especially if you have a history of bleeding disorders or miscarriage. It may cause bleeding or impair fetal development . Quantitative information Fifteen of the 155 included web pages quantitatively described the effects of the intervention. Some of these web pages also gave qualitative information, but in these cases only the quantitative information was categorised. Effects were presented in a variety of ways, including percentages, numbers needed to treat, frequencies and fractions. Four of the 15 web pages were abstracts of Cochrane systematic reviews. Nine had information that was judged to be incomplete and potentially misleading because outcomes for people who did not receive the intervention was not reported. In eight of these nine web pages, the length of follow-up was also not reported, despite the fact that this was relevant to the conditions in question. For example, one web page states that "good results are achieved in 80% to 90% of the cases" for patients receiving surgery for lumbar disc prolapse, implying that 80 to 90% of patients benefit from surgery, without specifying what proportion of similar patients have good results without surgery, what outcome measures "good results" refer to, or how long after surgery. The systematic review found that discectomy appeared to give fast short-term pain relief, but there was no good estimate of this effect, and that the effect on long-term pain relief was unknown. Two of the fifteen web pages were consistent with the systematic review. One of these web pages compared the effect of oral versus vaginal antifungals for yeast infection. The other web page reported the effect of galantamine for Alzheimer's disease and was the only one, besides the Cochrane systematic review abstracts, that provided information about outcomes for people not using the intervention. This information, however, referred to three trials, whereas the systematic review included six trials. Discussion The goal of this study was to evaluate the reliability of information about the effects of health care accessed through health portals that aim to facilitate access to reliable information. In most cases, it was difficult to know whether the information was reliable, in comparison to the results of systematic reviews, because of the incompleteness and vagueness of the information. Generally, websites to which the portals led us did not compare the effects of an intervention with no intervention or an alternative intervention. They also did not give any information about when outcomes were measured or what outcomes were measured in the studies on which the information was based. The evidence on which the information was based was usually not stated. Most of the web pages with information about effectiveness described effects qualitatively. This may reflect an assumption that qualitative presentations are easier to understand, or that information providers do not wish to express more than they know [ 21 ]. However, qualitative descriptions mean different things to different people [ 21 - 23 ]. The difficulties that we and our colleagues had in categorising qualitative descriptions of effects is indicative of the difficulties that others are likely to have understanding the information that the portals lead to. Although most of the information was not as confusing as the quote from Alice in Wonderland at the beginning of this article, it was confusing and often left us wondering what the intended message was and how it related to the available evidence. Quantitative presentations are more precise and less open to misinterpretation, but may be regarded as being more difficult to understand [ 21 ], and may be open to manipulation through framing [ 24 ]. The abstracts from Cochrane systematic reviews, which were included in web pages to which two of the portals led (table 4 ), frequently use research jargon and are likely to be inaccessible for many consumers [ 7 ]. Nine of the 11 other web pages we included that presented quantitative information presented incomplete information. Table 4 Included web pages per portal Portal Included hits Information about effects Qualitative statements about the effect of the intervention Quantitative statements about the effect of the intervention Qualitative statement Statement unclear Statement not consistent with review Statement consistent with review Quantitative statement Cochrane review abstract Statement incomplete and potentially misleading Statement consistent but incomplete Statement consistent with review Canadian Health Network 22 13 12 2 2 8 1 0 1 0 0 Health-Insite 38 21 15 5 2 8 6 3 2 1 0 Medline Plus 77 46 41 17 9 15 5 0 4 0 1 NHS Direct Online 18 12 9 2 2 5 3 1 2 0 0 TOTAL 155 92 77 26 15 36* 15 4 9 1 1 *Only four of the 36 described what happened to people who did not receive the intervention and only four indicated how long the effect was likely to last. None of the health portals report considering how effects are presented in their selection criteria. Included web pages present information about the effects of health care in a variety of ways. Most often this information is presented in ways that may confuse and mislead users. The nature of portals limits any possibility of addressing these problems or providing user support for understanding the effects of health care. By collecting websites from numerous sources, governments can avoid "recreating existing health information" and are able to present information "from multiple perspectives" [ 8 ]. However, to compare the benefits of alternative interventions, it is still necessary for users to search through numerous web sites and piece together information that frequently uses different terminology for the same interventions and conditions, is presented in different formats, and is often vague, incomplete and difficult to understand. Portals depend on having information to which they can link. If reliable and understandable information does not already exist, the portals are of little value. Conclusion The information accessible through these four portals displays the same weaknesses as patient information generally [ 1 , 25 ]. It is seldom based on systematic reviews and is often unclear, incomplete and misleading. People going through these portals to find information that can help them make an informed choice about any of the questions we posed are likely be disappointed with what they find. Portals are only as good as the websites they lead to. Establishing and maintaining national health portals is only worth the investment if an investment is also made in producing and maintaining relevant, valid and understandable information to which the portals lead. Competing interests All three of the authors contribute to the Cochrane Collaboration. CG and AO contribute to the development of Internet-based patient information. Authors' contributions CG and AO conceived the idea for this study. CG and EP developed the protocol with assistance from AO, collected and analyzed the data. CG drafted the manuscript. All three authors contributed to the final manuscript. CG and EP will act as guarantors of the paper. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079859

Family physicians' information seeking behaviors: A survey comparison with other specialties

Background Using technology to access clinical information has become a critical skill for family physicians. The aims of this study were to assess the way family physicians use the Internet to look for clinical information and how their patterns vary from those of specialists. Further, we sought a better understanding of how family physicians used just-in-time information in clinical practice. Methods A fax survey was provided with 17 items. The survey instrument, adapted from two previous studies, was sent to community-based physicians. The questions measured frequency of use and importance of the Internet, palm computers, Internet CME, and email for information seeking and CME. Barriers to use were explored. Demographic data was gathered concerning gender, years since medical school graduation, practice location, practice type, and practice specialty. Results Family physicians found the Internet to be useful and important as an information source. They were more likely to search for patient oriented material than were specialists who more often searched literature, journals and corresponded with colleagues. Hand held computers were used by almost half of family physicians. Conclusion Family physicians consider the Internet important to the practice of medicine, and the majority use it regularly. Their searches differ from colleagues in other specialties with a focus on direct patient care questions. Almost half of family physicians use hand held computers, most often for drug reference.

Background As an information-intensive specialty without patient limits of age, gender, or medical presentation, family physicians require a number of different resources to cover the broad scope of practice. A critical clinical skill for family physicians is timely access to that wide variety of clinical information sources that contribute to patient care decisions. Specific questions about patient management arise in daily practice (about 3.2 questions for every 10 patients seen), with drug-prescribing queries being the most common type of question [ 1 ]. Pursuing answers to questions that arise only occurs about a third of the time [ 1 ]. The most frequent motivation to track questions comes from the belief that a definitive answer exists or the patient's problem is urgent [ 2 ]. It is estimated that about half of questions may readily be answered by information in a clinical record, one-quarter of questions require traditional resources as journals or textbooks, and one-quarter of the questions require synthesis of information about a specific patient with a biomedical knowledge base [ 3 ]. Essential to the use of any information source is the probability of success in quickly and accurately finding the desired information. For the questions they pursued, family physicians spent an average of less than 2 minutes finding an answer using traditional textbooks and journals [ 1 ]. compared to a study of a palmtop drug reference system where it took a group of physicians only 20 seconds to find answers to their questions [ 4 ]. Technologies improve the quality of care family physicians provide by improving access to necessary information. Care may be fragmented or diminished and less evidence based when access is not readily available or available only through specialists [ 5 , 6 ]. As a global information source, the Internet provides extensive options to search for answers, and may influence the way family physicians shape their questions and look for responses. Its importance in clinical practice has been documented [ 5 , 7 - 9 ]. Many physicians have also adopted the use of handheld computers for reference materials and to access necessary information at the point of care [ 10 ]. One study indicates that Personal Digital Assistants (PDAs) were used in 64% of outpatient clinical facilities, with 69% of PDA users accessing pharmaceutical information [ 11 ]. One approach to better understanding family physicians' information needs and how they manage information is to look at how they use resources on the Internet and hand held devices to access information. This study focuses on the ways that family physicians use the Internet to look for information in their practice, and how their information seeking patterns vary from those of specialists. We hypothesized that family physicians would seek a broader array of information resources that were directly linked to patient care when compared to specialists. Further, we wanted to understand more about just-in-time information for use in clinical practice. Methods Patterns of current Internet use by family physicians and specialists were assessed and compared using a survey instrument with 17 items. The survey was adapted from two previous studies [ 8 , 9 ]. Three items measured frequency of use of the Internet, palm computers, Internet CME, and email. Four items measured type of use, including clinical information and specific patient problems, for the Internet, palm computers, and email. In four items, physicians were asked to rank the importance of the Internet for information seeking and CME. Using a Likert scale, three items were used to determine physician beliefs about the Internet. Three additional items measured barriers, type of resources available for searching, and choice of potential CME courses. Demographic data was gathered concerning gender, years since medical school graduation, practice location, practice type, and practice specialty. The survey was sent by facsimile transmission (Fax) during the period of December, 2002-January, 2003. The fax broadcast method of surveying effectively elicits responses from community-based practicing physicians [ 12 ], and avoids the bias of surveying only those physicians currently using the Internet. Previous studies have indicated that virtually all physicians in the United States have access to the Internet [ 8 , 9 , 11 ]. The population of interest for this survey was defined as U.S. physicians of all specialties in active practice, according to the most current American Medical Association physician listing; 518,000 physicians were identified with 69,000 family physicians. A power calculation determined that a sample size of 2200 was needed to generalize to the total population of U.S. physicians in terms of age and gender. Cochran's sampling technique was used to determine the power required for the study, with a margin of error of 5%, and 95% confidence [ 13 ]. In addition, the demographic characteristics of the sample of 2200 were compared to the demographic characteristics of the overall group of 518,000 and tested for differences to further assess the representativeness of the sample. Surveys were faxed to a random sample stratified to include all major specialties drawn from the overall pool of U.S. physicians. Responses were solicited until a usable sample of 2200 surveys had been received. Each survey was personalized with the individual physician's name and fax number before faxing. Directions for returning the survey by fax included an 800-fax number to a designated fax broadcaster. Each returned survey was scanned, and electronic copies were sent by email to the Division of Continuing Medical Education, University of Alabama School of Medicine for data entry. Survey responses were entered into an ACCESS database for analysis. This study represents a subanalysis of data from the overall study, comparing responses of family physicians to those of specialists. Family physicians were classified based on self-identification. Frequency distributions and means were calculated for each survey item. Demographic items and survey items were cross-tabulated and analyzed using Chi-square analysis. Results A total of 2,200 usable responses required by the power calculation to generalize to the overall population of U.S. physicians were received by January 31, 2003; in the month following the solicitation of responses, an additional 194 usable responses were received and were included in the analysis. Of the 457 family physicians that responded, 72.7% were male and 27.4% female; 39.6% practiced in rural areas, and 44.8% reported graduating from medical school more than 20 years earlier. This demographic profile was compared to the American Medical Association profile of U.S. family physicians and no significant differences were found [ 14 ]. The majority (59%) of family physicians regularly use the Internet to access clinical information daily or weekly; they also regularly access the Internet for personal use and for email. Nearly half (47%) reported access by modem. Family physicians' responses differed from survey responses for other specialties in terms of strategies for seeking clinical information, as summarized in Table 1 . Table 1 Physicians' Internet Use Purpose Family Medicine [%] Specialist [%] χ 2 df p-value Personal use 83.0 86.4 3.87 1 0.049 E-mail personal 78.7 81.7 2.16 1 .14 Literature searching 61.5 74.2 31.29 1 < .0001 Accessing online journals 57.6 67.0 14.40 1 .0001 Searching for patient-specific information 66.7 54.4 21.23 1 .0001 Professional association updates 47.3 48.6 0.32 1 .57 Consultation with colleagues 11.7 21.5 21.66 1 <.0001 Prescription/patient orders 6.0 3.4 5.85 1 .02 In comparing the importance of the Internet to other sources of clinical information, family physicians rated journals first, followed by local and national CME meetings, and then websites. However, the majority (73%) believed the Internet was useful and important to physicians. More than half (54%) of family physicians reported confidence in using the Internet to find medical information; however, 14% were not at all confident. Family physicians were more likely to search for information related to a patient problem while other specialists were more likely to search for the latest research on a specific topic (chi square = 10.26, df = 3, p = 0.01). When addressing a specific patient problem, family physicians were more likely to be seeking information on diagnosis/management (73%), patient education materials (58%), followed by guideline summaries (49%). Specialists were similar except for a difference in searching for patient education materials (37%, chi square = 64.54, df = 1, p = < 0.0001). Credibility was ranked as the most important characteristic of the Internet related to clinical information by both family physicians and physicians in other specialties. Family physicians were more likely than other specialists to cite "too much information to scan" as a barrier to clinical information seeking (p =.0004) on the Internet. Other barriers are summarized in Table 2 . Table 2 Physician Internet Barriers Barrier Family Medicine [%] Specialist [%] χ 2 df p-value Too much information to scan 58.6 48.9 12.53 1 .0004 Specific information not available 44.0 48.3 3.40 1 .065 Navigation/searching difficulties 61.3 60.4 0.09 1 .77 Too slow 32.9 28.6 2.83 1 .09 Software incompatibilities 18.3 21.2 1.83 1 .176 Downloading information too difficult 30.6 29.6 0.25 1 .62 Family physicians were also concerned with the relevance of clinical information. They were more likely than specialists to use handheld computers (p =.0004) with nearly half (49%) of family physicians reporting use, most frequently for drug reference. Table 3 summarizes handheld computer usage. Table 3 Hand Held Computer Functions Identified Useful Function Family Medicine [%] Specialist [%] χ 2 df p-value Drug References 94.1 80.3 23.48 1 < .0001 Clinical Guidelines 54.5 45.0 6.27 1 .01 E-mail 5.9 13.2 8.84 1 .003 Patient specific information 20.7 19.3 0.10 1 .75 Web search 5.0 6.9 1.21 1 .27 Discussion Because of the broad scope of family practice and the exponential increase in medical knowledge, mastery of technology is a core skill for family practice. This study looked at the ways in which family physicians make use of technology, and how their information seeking behavior compares to colleagues in other specialties. Family physicians consider the Internet important to the practice of medicine. The majority report regular use as a source of clinical information, driven by clinical questions that arise during the care of specific patients rather than looking for new research findings. This study indicates they differ from colleagues in other specialties in the kinds of clinical information needs that lead to a search for information. Family physicians search for specific material that will benefit a patient or group of patients, often via handheld computers. The use of handheld computers points to interesting questions about whether there is greater use of just-in-time information by family physicians. That is consistent with the findings of family physician use of handheld computers in outpatient settings [ 11 ]. Understanding current best practices and clinical guidelines in order to respond to individual variation and needs among patients is supported by access to technology. Specialists, on the other hand, require in-depth knowledge in a relatively narrow area, with a need to use technology for access to cutting edge research and journals, and contact with a more limited population of colleagues, many of whom may be at a distance. Both groups found problems with navigation and lack of speed in searching. A number of obstacles to using evidence to answer physicians' patient care questions have been identified. This study confirms Internet searching difficulties for both specialists and family physicians, with an extensive amount of information to scan, and lack of specificity for available information. These obstacles appear to be greater for family physicians in their quest for patient-specific information. Specialists find less difficulty in their primary information targets of literature searching, online journal use, and consultation with colleagues via email. As medical information expands and the number of web pages increases, these barriers will grow in the years to come. When family physicians search for clinical information, the Internet may be accessed by desktop/laptop or hand-held computers. Handheld devices allow immediate access to answers to many of the clinical questions family physicians ask, especially at the point of care. Family physicians were significantly more likely than physicians of other specialties to use hand-held computers, and more likely to use them for drug references and clinical practice guidelines. This finding is consistent with the findings for other primary care physicians. A recent survey of internists found that 80% of hand held computer owners used them to access drug information [ 16 ]. Handheld computers may also be used to access electronic medical textbooks, downloadable journals, medical calculators, patient-tracking programs, billing and coding software, word processing and utility software, and web access and content, with future possibilities of dictation of clinical notes and email [ 17 ]. As more physicians integrate hand-held computers into their practices, the number and quality of clinical applications will continue to grow. Gaps in knowledge related to drug prescribing is one of the most common causes of serious medication errors [ 18 ]. A recent study of the clinical use of a hand held drug reference guide demonstrated that physicians felt this technology saved time during information retrieval, was easily incorporated into their workflow, and that it reduced the rate of preventable adverse drug events [ 4 ]. The use of hand held computers for referencing clinical practice guidelines and drug questions by half of the family physicians surveyed indicates that hand held computers are becoming more rapidly integrated into the clinical encounter and provide one step in addressing patient safety issues. Conclusion Family physicians deal with the broadest of clinical knowledge bases, yet two-thirds of their clinical questions go unanswered [ 1 ]. They are most likely to pursue those questions with a high probability of finding an answer or with an urgent clinical application [ 2 ]. Technology is one tool to respond to these problems. Family physicians have access to technology for information and are using it. They consider the Internet an important information source, and are confident in their ability to search for information. Compared to specialists, family physicians direct more attention to patient care questions, perhaps at the point of care. However, when they use the Internet for clinical information, family physicians can be overwhelmed by the amount of clinical information, their inadequate searching skills and their lack of confidence that they will be able to answer a question. The increased use of handheld computers points to more potential use at the point of care clinical encounter; they appear to be particularly useful in accessing drug information and clinical practice guidelines and likely will grow in numbers of users and types of applications. The use of handheld computers may contribute to an effort to increase patient safety. Although technology offers access to information, it also offers a series of challenges to medical educators and researchers, and to those who design technology applications to create bridges for practitioners to the information they need to practice medicine. Competing interests The author(s) declare that they have no competing interests. Authors' contributions NB, LC, RK developed the survey form and study protocol. BC contributed to literature, and supervised administration of the survey. All authors contributed to the writing of the article, and read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079860

Practical considerations for estimating clinical trial accrual periods: application to a multi-center effectiveness study

Background Adequate participant recruitment is vital to the conduct of a clinical trial. Projected recruitment rates are often over-estimated, and the time to recruit the target population (accrual period) is often under-estimated. Methods This report illustrates three approaches to estimating the accrual period and applies the methods to a multi-center, randomized, placebo controlled trial undergoing development. Results Incorporating known sources of accrual variation can yield a more justified estimate of the accrual period. Simulation studies can be incorporated into a clinical trial's planning phase to provide estimates for key accrual summaries including the mean and standard deviation of the accrual period. Conclusion The accrual period of a clinical trial should be carefully considered, and the allocation of sufficient time for participant recruitment is a fundamental aspect of planning a clinical trial.

Background Clinical trials often plan to enroll hundreds to thousands of human subjects, and careful and regulated planning is employed to ensure that the trial's scientific objectives are fulfilled. However, the importance of the feasibility and timeliness of participant accrual is often minimized during the planning stage. This is unfortunate since the recruitment of human subjects into a clinical trial must be timely and is vital to the success of the trial [ 1 , 2 ]. In addition, a slowly progressing trial may expose the human subjects to ineffective treatments for a longer period of time than necessary, and as such, human subject protection may be compromised [ 2 , 3 ]. Therefore, a clinical trial must be planned with an adequate understanding of the potential accrual rate so that the clinical trial's scientific objectives can be evaluated in a timely manner. The first step in estimating the clinical trial's accrual period is to estimate the accrual rate. In the context of a multi-center trial, each clinical center's rate will need to be estimated. One approach for evaluating each clinical center is to solicit information via a questionnaire. Content of the questionnaire could include measurements of the total number of cases currently or recently seen for the targeted condition, a listing of other clinical trials that might compete for the same patients, and a summary of the investigator's (or clinical center's) experience meeting prior accrual expectations. The completed questionnaires can be used to formulate an initial estimate of the accrual rate for each participating clinical center. However, experience has shown the accrual period is longer than planned even when such tangible efforts are made to estimate the accrual rate. Carter [ 1 ] presented a methodological examination of this issue and developed a statistical model to estimate the accrual period of a clinical trial. It was argued that utilizing only the historical mean for the projected accrual period fails to account for the variation in the rate that may occur as the study progresses. In this brief report, we illustrate three approaches to estimating the accrual period of a clinical trial and offer discussion on potential best practices. Methods Unconditional approach A common approach to estimating the accrual period in a clinical trial is to divide the sample size by the expected rate. Often, the expected rate for the clinical trial is estimated by summing the historical trends at each participating research site to create a study-wide estimate of the accrual rate. For example, if subjects are to be recruited from 5 sites at the expected rate of 5 participants per month per site, then one could estimate that it would take 8 months to enroll 200 participants using the combined rate of 25 participants per month. However, since the rate of 25 participants per month is assumed to be fixed in value, it is best to consider that "on average" it may take 8 months to enroll 200 participants. The obvious limitation of this approach is that there is no mechanism to account for known sources of variation in the rate. For example, if the 5 sites were to begin enrolling at different times, then a study-wide estimate of 25 participants per month is no longer tenable. The following approach directly addresses this limitation. Conditional rates A more accurate estimate of the accrual period is possible when additional prior information is incorporated into the model. Two important sources of variation in the overall accrual period are the rates at which individual sites accrue and the length of time in which sites actively recruit participants. For example, suppose that a large research institution (Site A) may be capable of recruiting 15 participants per month and that four additional sites are only capable of recruiting a combined 10 participants per month. Then, the point in time in which the Site A begins enrollment will greatly affect the trial's overall accrual rate. Moreover, the timing of Site A's initiation and subsequent accrual should be critically evaluated by the sponsor. For example, if it is known in advance that Site A will take 4 to 8 weeks longer to initiate recruitment than the other four sites, an estimate of 8 months to enroll 200 participants would seem overly optimistic. The sponsor might be interested in determining the effect of including Site A on key trial markers (e.g., feasibility, cost, and total clinical trial duration) by allowing for up to an 8-week delay in Site A's initiation. However, the calculation of a more refined accrual period, which would drive many of the cost estimates, would require the adjustment for time dependent changes in the overall accrual rate. Fortunately, computational tools such as a spreadsheet aid in the necessary calculations. Poisson process The methodology presented by Carter [ 1 ] allows for a more sophisticated model that can incorporate a variety of sources of variations. In this paper, the exploration of mean accrual time will be examined. As the two previous approaches illustrate, the mean number of participants per month per site was assumed to be fixed, yet the overall accrual time varied. The source of variation introduced was a delay in the initiation of the protocol at a site. There is another source of variation. Just as is well established in economics, historical trends may not adequately reflect future trends especially when short periods of time are considered. One approach to incorporating variation into mean number of participants per month is to assume that participants arrive into the study according to a known probability distribution. Carter [ 1 ] proposed the Poisson distribution since it has widespread applications that often include the statistical modeling of the number of arrivals or observations [ 4 , 5 ]. Essentially, this method simulates the accrual into a trial using a random number generator. At the end of each simulation iteration, the number of days (or months) required for the program to reach the sample size is recorded. When the simulations are repeated many times, an empirical distribution of the accrual period is obtained. Using this empirical distribution, one can answer questions related to the probability of completing accrual within a specified time as well as observe how much variance in accrual time can be expected given the model assumptions. This probabilistic approach is most beneficial when a finite amount of time has been granted for the clinical trial. Thus by conditioning on the predetermined time, one could estimate the number of clinical centers and the rate per month needed to ensure with high probability, such as 80%, that the clinical trial will complete enrollment before the allocated time expires. Results To illustrate these three approaches, a multi-center protocol undergoing development is presented. The primary objective of the study is to measure the effectiveness of a selective serotonin reuptake inhibitor (SSRI) in the treatment of depression in a patient population with comorbid substance dependence. A sample size of 360 participants, enrolled from 10–12 research sites, has been estimated to test the primary hypothesis with 90% power. While the protocol is still being finalized, an initial estimate of the accrual period is of practical interest to the protocol investigators. The final selection of the research sites has not been made, but to be considered for the trial, each site must demonstrate a patient population that would support enrolling two participants per month in the trial. In addition, it is anticipated that the protocol will be rolled out initially at one to two sites with the remaining sites coming online three to eight months later. Table 1 presents the results of the presented models as well as a variation of the Poisson process model that allows for fluctuations in assumed accrual rate over time. For simplicity, each site's accrual rate is assumed to equal two participants per month and ten sites (instead of up to 12) have been chosen so that a conservative estimate of the accrual period is obtained. Using these assumptions and the unconditional approach, it is estimated that 18 months would be required (360 / (2 * 10)) to accrue the participants. However, this calculation does not account for the delayed start of the majority of the sites. Table 1 Estimated accrual periods for a 360-participant, 10-center clinical trial. Unconditional Approach a Conditional Rates b Poisson Process c , d Poisson Process Variable Rates c , e Estimated Accrual Period (in months) Mean 18 23 23.2 42.6 Standard Deviation NA NA 1.0 8.0 Range NA NA [20.1, 25.8] [28.0, 84.0] 75th Percentile NA NA 24.0 46.0 a 10 sites enrolling at a rate of 2 participants per month. b For the conditional rate, the rate per month is illustrated in Figure 1. c Based on 500 simulation iterations d A rate of 2 participants per month with recruitment on 5 days a week e A rate uniformly distributed on [0,2] with recruitment on 5 days a week To account for the delay, the conditional model could be utilized. Suppose the initial rate of accrual is two participants per month for the first two months while the protocol is active at only one site. At the start of month three, one additional site will be released for enrollment to bring the estimated accrual rate to four participants per month. Figure 1 illustrates the expected accrual as additional sites are added to the study and that full accrual potential is not reached until approximately 1/3 of the trial duration has passed. The estimate of 23 months seems much more reasonable given the staggered initiation process; however, additional models could be implemented to examine the effects of fluctuations in the accrual rates. Two separate Poisson models are presented in Table 1 . Both models use the initiation pattern of the conditional model; however, the daily rate, which is the monthly rate divided by the standard number of days in the month, has been adjusted to reflect anticipated enrollment dynamics. Specifically, while the clinical sites treat potential participants every day of the week, research staff are expected to be available only five days per week. Thus, the estimated rate per month is discounted effectively by 5/7 to reflect this anticipated pattern. The second Poisson model further adjusts the estimate of 2 participants per month downward to be uniformly distributed between zero and two. Such an adjustment might be necessary to account for the difference in the number of participants eligible for the trial and the number that will actually consent to participate in the research. In addition, this reduction in the overall rate also adjusts the rate downward for other competing demands that could affect the participant accrual rate but would be difficult to quantify directly. As Table 1 illustrates, a dramatic difference in the expected accrual period is obtained if the rate is allowed to fluctuate uniformly on the interval 0 to 2; however, the conditional approach and the fixed-rate Poisson model provide similar results with the exception that the Poisson-based approach provides additional summary statistics that may be of interest during the planning phase. Figure 1 Expected accrual rate by month assuming an April 2005 start date. Discussion The estimation of the accrual period of a clinical trial has dramatic practical, scientific and economic consequences and warrants greater attention in practice. As our example illustrates, the unconditional approach, while simple to implement, can yield results not consistent with trial assumptions, and may endanger the successful completion of the trial. It is acknowledged that the most complicated aspect pertaining to the estimation of accrual periods is the determination of the expected rate. As our example illustrates, sometimes dramatically different results occur with what are apparently minor modifications to model assumptions. When the potential impact of a poorly accruing clinical trial is considered, spending additional time examining the effects of model assumptions is well justified. In the end, accounting for variation and planning are essential components of good clinical research. For the research study under consideration in this example, an estimate of 24 months to complete study recruitment seems reasonable. An accrual rate of at least two participants per month is the minimum requirement, so the expected rate after adjustment for screening to enrollment dropout percentages should still yield an accrual rate of approximately two per month. However, a more refined estimate of the accrual period can be obtained once final selection of the research sites has been made and all historical information has been analyzed. These models can also be applied after study enrollment has begun, and in the case of the Poisson model, estimating the probability of meeting the established accrual deadline has several practical implications. Although the methods have been presented in the context of a multi-center clinical trial, they are valid in the planning of a single-center clinical trial. To facilitate implementation of the models in practice, a spreadsheet template for the calculation of the conditional model and SAS programs for the Poisson process are posted on the first author's website . Each may be freely downloaded and implemented with very little training. The posted conditional model allows for individual sites to enroll at site-specific rates as well as provides means to quickly adjust model assumptions so that sensitivity analyses can be performed. A more detailed discussion of the Poisson method is available elsewhere [ 1 ]. In summary, the conditional and Poisson accrual estimation methods may be useful to researchers designing a complex, multi-center clinical trial. Conclusion The accrual period of a clinical trial should be carefully considered, and the allocation of sufficient time for participant recruitment is a fundamental aspect of planning a clinical trial. For most multi-center clinical trials, the conditional approach should be implemented. Simulation studies using the Poisson model are recommended if there is uncertainty in the accrual rate or if the accrual rate is expected to change over time. Competing interests The author(s) declare that they have no competing interests. Authors' contributions RC conceptualized the three accrual models and performed the analyses. SS and KB provided critical input into the model assumptions as well as the conceptualization of the referenced research project. All authors participated in the writing and editing of the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079861

Complex Pediatric Elbow Injury: An Uncommon Case

Background There is paucity of literature describing complex elbow trauma in the pediatric population. We described a case of an uncommon pediatric elbow injury comprised of lateral condyle fracture associated with posterolateral dislocation of elbow. Case presentation A 12-year-old boy sustained a direct elbow trauma and presented with Milch type II lateral condyle fracture associated with posterolateral dislocation of elbow. Elbow dislocation was managed by closed reduction. The elbow stability was assessed under general anaesthesia, followed by open K-wiring for the lateral condylar fracture fixation. The patient had an uneventful recovery with an excellent outcome at 39 months follow-up. Conclusion Complex pediatric elbow injuries are quite unusual to encounter, the management of such fractures can be technically demanding. Concomitant elbow dislocation should be managed by closed reduction followed by open reduction and internal fixation (K-wires or cannulated screws) of the lateral condyle fracture.

Background Traumatic elbow dislocation is a rare injury in children constituting 3–6% of all elbow injuries [ 1 ]. It more frequently occurs with medial epicondyle fractures, although, it can infrequently be associated with lateral humeral condyle fracture [ 1 - 3 ]. The complex elbow anatomy with multiple growth centres appearing at different time period in the skeletally immature age can pose a diagnostic dilemma. The management of such unusual injuries can be technically demanding. There is limited evidence available in the literature describing complex elbow fracture dislocation in the pediatric population [ 3 - 7 ]. This report presents a rare condition of a complex elbow injury consisting of lateral condyle fracture in association with posterolateral elbow dislocation. The patient had an excellent outcome after a follow-up of 39 months. Case presentation A 12-year-old boy presented with a grossly swollen and deformed left elbow after sustaining a fall off skateboard and directly landed on the elbow. There was no neurovascular deficit in the extremity. The anteroposterior and lateral radiographs showed posterolateral dislocation of the left elbow in association with Milch type II lateral condyle fracture (Figure 1 and Figure 2 ). Concomitant elbow dislocation was managed by closed reduction. The stability of elbow was assessed under general anaesthesia. Figure 1 Preoperative anteroposterior radiograph of the elbow revealing fracture lateral condyle in association with posterolateral dislocation of elbow. Figure 2 Preoperative lateral radiograph of the elbow revealing elbow dislocation. Open K-wiring of the lateral condylar fracture was carried out on the same day of injury (Figure 3 and Figure 4 ). A standard lateral approach to the distal humerus and elbow joint was used. Under direct visualization, the fracture was anatomically reduced and held in place. Two smooth 0.0625-inch Kirschner wires (K-wires) were inserted from lateral to medial and distal to proximal directions. The positions of the K-wires were verified by fluoroscopic examination in the anteroposterior and lateral planes. The K-wires were then cut and left exposed outside the skin. Figure 3 Postoperative anteroposterior radiograph of the elbow revealing internal fixation of the lateral condyle fracture with the help of 2 K-wires. The elbow dislocation was reduced first by closed technique. Figure 4 Postoperative lateral radiograph of the elbow revealing internal fixation of the lateral condyle fracture with the help of 2 K-wires. The operated elbow was immobilized in an above elbow backslab postoperatively. A close clinico-radiological follow-up at one and two weeks postoperatively was instituted and confirmed no loss of reduction. K-wires were removed under general anaesthesia four and half weeks postoperatively. At 39 months follow up, the elbow had normal appearance and functions with no alteration in the carrying angle and no symptoms. Discussion Complex elbow injury pattern consisting of lateral condyle fracture in association with elbow dislocation has not been well-described in children. We found only a few cases described previously in the English literature. Lateral condyle fracture has been previously described in association with posterolateral elbow dislocation [ 3 , 5 ] and posteromedial elbow dislocation [ 4 , 8 ]. Posteromedial dislocation of the elbow with associated intraarticular entrapment of the lateral epicondyle has also been documented [ 2 ]. Isolated traumatic dislocation of the elbow is a rare injury in children constituting 3–6% of all elbow injuries. The peak incidence occurs in the thirteen to fourteen years of age. Dislocation of the elbow can be classified by the direction of the dislocation of the radius and ulna. Elbow dislocations are rarely associated with lateral condyle fractures, and more frequently occur with medial epicondyle fractures [ 1 - 3 , 9 ]. Lateral condyle fractures represent approximately 15 percent of all elbow fractures in children. It occurs more commonly between five and ten years of age. The lateral condyle is fractured by a varus stress applied to the extended elbow with the forearm supinated, as in falling on an outstretched hand. In addition, it can secondarily be fractured by the pull of the lateral collateral ligament and the extensor muscles [ 8 , 10 ]. There are two classifications which are currently in use to describe lateral condylar fractures. The Milch classification is based on the anatomical position of the fracture line. In Type I fracture, the fracture line courses lateral to the trochlea and passes through the capitello-trochlear groove. In the Type II injury, the fracture line extends into the apex of the trochlea. Milch described the more common Type II injury as a fracture-dislocation and the Type I injury as a simple fracture [ 11 ]. Lateral condyle fractures are also described in relation to the degree of displacement and rotation of the fracture fragment [ 12 ]. Stage I displaced fractures have less than two millimeters of displacement with intact articular surface. In Stage II displaced fractures, there is two to four millimeters of displacement with moderate displacement of the articular surface. Stage III displaced fractures demonstrate significant displacement associated with rotation of the fragment. In this reported case, the lateral condyle fracture was Milch type II and Stage III. The fixation of lateral condyle fracture is of prime importance as it constitutes Salter Harris type IV injury. The evidence supports prompt open reduction and internal stabilisation of the lateral humeral condyle fracture to provide the best results [ 3 , 8 , 10 ]. Growth plate and articular surface should be aligned and restored. Missing or inadequately treated lateral humeral condylar fracture can lead to non-union, malunion, recurrent dislocation, abnormalities in carrying angle, prominence of the lateral humeral condyle, progressive cubitus valgus deformity and tardy ulnar palsy [ 8 , 10 , 11 ]. The main focus has been changed more recently from medial to lateral collateral ligament for traumatic elbow instability in adults. Lateral collateral ligament acts as a major elbow stabilizer and is predominantly responsible for acute recurrent and chronic recurrent dislocations. In children, a lateral capsular pocket defect with an ununited lateral epicondylar fragment has been described contributing to recurrent elbow dislocation and instability. Fixation of this small fragment restores the lateral collateral ligament integrity and improves elbow stability [ 13 ]. Lateral condyle or epicondyle fracture may represent variations of lateral collateral ligament injury. We would like to confirm here that our case had an elbow dislocation variant with lateral condyle fracture instead of avulsion or intrasubstance tear of lateral collateral ligament complex. Van Haaren et al [ 3 ] reported a rare combination of injury in the form of pediatric posterior elbow dislocation with an associated Milch Type II lateral condyle fracture, while Murnaghan et al [ 6 ] and Rovinsky et al [ 7 ] described the Milch Type I lateral condyle fracture-dislocation. These authors recommended oblique, heterolateral and varus stress films to aid in diagnosis. Evidence confirms that if a dislocated elbow is associated with a fracture, it is essential to reduce the dislocation first. After assessing the fracture on the post-reduction films, the fracture can be treated as if the dislocation had not occurred [ 3 ]. In our case, the elbow dislocation was reduced by closed manipulation and subsequently the elbow stability was assessed under general anaesthesia. Open reduction and internal fixation of lateral condyle fracture was carried out with the help of two Kirschner wires. The patient had an uneventful recovery with an excellent outcome as per Hardacre functional rating system for evaluation of the results (i.e. no loss of motions, no alteration in the carrying angle and no symptoms). In cases of elbow fracture-dislocation, prompt reduction of the elbow dislocation is essential. Anatomic reduction and fixation of the lateral condylar fracture fragment can provide stability for the elbow and improve the functional result. This report presents a very rare condition. As these fractures are quite unusual to encounter, the management of such fractures can be technically demanding. In summary, concomitant elbow dislocation should be managed by closed reduction followed by same day or next day anatomical open reduction and cannulated screw fixation or K-wiring of the lateral condyle fixation. Postoperatively, a close clinico-radiologic follow-up helps in early diagnosis of the loss of reduction. Conclusion In summary, complex pediatric elbow injuries are quite unusual to encounter, the management of such fractures can be technically demanding. Concomitant elbow dislocation can be managed by closed reduction followed by open K-wiring or cannulated screw fixation of the lateral condyle fracture. Postoperatively, a close clinico-radiological follow-up helps in early diagnosis of the loss of reduction. We recommend that all patients with a dislocated elbow should have elbow stability assessed under general anaesthesia because of high complication rate associated with a missed lateral condylar injury. Competing interests The author(s) declare that they have no competing interests. Authors' contributions Each author has equally contributed. HS collected the data and written up the manuscript, RA helped in scrutiny of the paper and obtaining illustrations. GRT had the idea and granted permission to use his patient data for preparing the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079862

Assessment of the paraspinal muscles of subjects presenting an idiopathic scoliosis: an EMG pilot study

Background It is known that the back muscles of scoliotic subjects present abnormalities in their fiber type composition. Some researchers have hypothesized that abnormal fiber composition can lead to paraspinal muscle dysfunction such as poor neuromuscular efficiency and muscle fatigue. EMG parameters were used to evaluate these impairments. The purpose of the present study was to examine the clinical potential of different EMG parameters such as amplitude (RMS) and median frequency (MF) of the power spectrum in order to assess the back muscles of patients presenting idiopathic scoliosis in terms of their neuromuscular efficiency and their muscular fatigue. Methods L5/S1 moments during isometric efforts in extension were measured in six subjects with idiopathic scoliosis and ten healthy controls. The subjects performed three 7 s ramp contractions ranging from 0 to 100% maximum voluntary contraction (MVC) and one 30 s sustained contraction at 75% MVC. Surface EMG activity was recorded bilaterally from the paraspinal muscles at L5, L3, L1 and T10. The slope of the EMG RMS/force (neuromuscular efficiency) and MF/force (muscle composition) relationships were computed during the ramp contractions while the slope of the EMG RMS/time and MF/time relationships (muscle fatigue) were computed during the sustained contraction. Comparisons were performed between the two groups and between the left and right sides for the EMG parameters. Results No significant group or side differences between the slopes of the different measures used were found at the level of the apex (around T10) of the major curve of the spine. However, a significant side difference was seen at a lower level (L3, p = 0.01) for the MF/time parameter. Conclusion The EMG parameters used in this study could not discriminate between the back muscles of scoliotic subjects and those of control subject regarding fiber type composition, neuromuscular efficiency and muscle fatigue at the level of the apex. The results of this pilot study indicate that compensatory strategies are potentially seen at lower level of the spine with these EMG parameters.

Background Scoliosis can be biomechanically described as a three-dimensional deformity of the spine, with deviations from the physiologic curves in the sagittal and frontal planes, usually combined with intervertebral rotation [ 1 ]. The disease often occurs during childhood or adolescence. It can be associated with congenital malformation of one or many vertebraes, fracture and/or dislocation of the spine, leg length discrepancy, hormone imbalance, habitual poor posture or by pain and muscle spasms [ 2 ]. When the deformity cannot be associated with any of the aforementioned causes, it is then labelled as "idiopathic scoliosis". Idiopathic scoliosis is the most common diagnosis given to a deviation of the spine [ 2 , 3 ] and despite the fact that a considerable number of studies aimed at explaining its etiology, the cause of idiopathic scoliosis is still unknown. Back muscle composition and idiopathic scoliosis Biopsy studies shows abnormalities in the paraspinal muscles of scoliotic subjects concerning their architecture [ 4 ], their protein synthesis [ 5 ] and their muscle fiber type composition [ 1 , 6 , 7 ]. Even if many authors have investigated the alteration of the muscle fiber characteristics of paraspinal muscles with regards to idiopathic scoliosis, it is still not known if the observed differences are the cause of the disease or a consequence of it [ 1 , 6 , 7 ]. Idiopathic scoliosis affects mostly young women [ 8 ] and the major curve is often located in the thoracic spine. These facts raise a difficulty when one tries to explain the etiology of this disease through the alteration of muscle fibers because there is not much information on healthy backs, especially with regards to women and to muscles of the thoracic spine. Sirca & Kostevc [ 7 ] found that there was more type I than type II muscle fibers in the thoracic spine. However, their study included male subjects only. Mannion et al. [ 6 , 9 ] have investigated the muscle fiber composition in both genders at T10 and L3 levels. In their female subjects group, type I muscle fibers were present in a larger proportion than type II muscle fibers at the level of the thoracic spine. Moreover, type I muscle fibers presented a larger diameter at the thoracic level than at the lumbar level and also had a larger diameter than type II muscle fibers at both levels of the spine. When one looks at the muscle fiber composition of scoliotic subjects, an important factor is whether or not the muscles are located on the concave or the convex side of the major spinal curve. Studies have demonstrated that the concavity of the curve shows alteration of fiber characteristics [ 6 , 10 , 11 ]. In general, in the muscle of scoliotic subjects taken at the same level of the spine (apex around T9, T10), the proportion of type I muscle fibers is smaller on the concave side than on the convex side whereas in normal subjects, a symmetric distribution is observed. However, type I muscle fibers are still more numerous than type II muscle fibers on both sides of the apex [ 6 , 10 ]. The other important difference is that the cross sectional area (CSA) of type II muscle fibers is larger on the concave side than on the convex side. Moreover, this CSA of the type II fibers is larger than in normal subjects [ 6 , 12 ]. Spencer and Eccles [ 11 ] have proposed that the difference on the concave side is due to a disparity in the proportion of each fiber type (type I fibers in a smaller percentage) rather than a difference in their relative size. Surface EMG has been used as a non-invasive correlate to different muscle intrinsic properties such as muscle fiber composition, neuromuscular efficiency (related to weakness) and muscle fatigue. Obviously, the detection of differences in muscle fiber composition would be the primary focus of an EMG study. However, it would also be of interest to determine if scoliotic subjects also show other muscle impairments such as muscle weakness and fatigue. The rationale that justify the use of surface EMG to measure each of these muscle intrinsic property will be developed in the next sections. Assessment of muscle composition with the EMG MF/force relationship Muscle biopsy remains the technique commonly used to study muscle fiber type composition. However it has been suggested that the MF/force relationship could potentially be used as a non-invasive measure of muscle fiber composition and muscle fiber size [ 13 , 14 ]. The conduction velocity of the muscle fibers has been shown to be proportional to the diameter of the recruited muscle fibers [ 15 ]. The central tendency statistics such as the MF and the mean power frequency (MPF) of the EMG power spectrum are highly correlated with the average muscle fiber conduction velocity [ 13 , 16 , 17 ]. Thus, it is expected that the values of the MF or the MPF will increase with the increasing force level due to the recruitment of larger type II muscle fibers [ 14 , 18 , 19 ]. However, with atypical muscles such as the erector spinae where the predominant type I fibres have an equal or larger diameter than type II fibres [ 9 , 20 ], the MF remains usually stable or decreases in some cases across the force levels [ 21 ]. Assessment of neuromuscular efficiency with the EMG RMS/force relationship One EMG measure of interest associated with the functional capacity of a muscle is linked to the "neuromuscular efficiency" concept [ 22 ]. This concept suggests that a weak subject would produce more EMG than a stronger subject to generate a given absolute force, thus less efficient muscle contractions are characterised by steeper RMS/force relationship slopes [ 23 , 24 ]. This concept has been used to study muscles of subjects suffering from neck pain [ 25 ] and cerebro-vascular accident (CVA) [ 26 ]. It appears that with reliable EMG parameters, some differences in efficiency are observed among subjects who are experiencing pain [ 25 ] whereas among CVA subjects, it was only partially demonstrated [ 26 ]. Assessment of muscle fatigue with the EMG RMS/time and MF/time relationships To sustain a sub-maximal contraction for a substantial amount time, the recruitment of new motor units is necessary. While the contraction is ongoing, the muscle produces more EMG [ 27 ]. When the EMG amplitude (RMS) is plotted against time, the slope of the RMS/time relationship is generally positive. However, when one looks at the median frequency (MF) of the power spectrum over time, the MF/time slope presents a negative relationship demonstrating a shift toward lower frequencies. The MF decrease of the EMG power spectrum during a fatiguing muscle contraction is mainly attributed to the decrease of the conduction velocity [ 28 ], which reflects the accumulation of metabolic products on the surface area of the muscle fibers. That could also explain the shift of the MF of the power spectrum towards lower frequencies. The EMG RMS and MF of the power spectrum can thus be considered sensitive measures of muscle fatigue [ 27 , 29 ]. The above EMG measures have been successfully used to study the paravertebral muscle activity in normal and back pain subjects [ 21 , 30 - 32 ]. Before using a similar measurement protocol in a cohort of scoliotic subjects and considering the difficulty of recruiting scoliotic patients before surgery, a pilot study was conducted on a small number of patients. The main objective of this pilot study was to examine the clinical potential of these different EMG parameters for assessing the back muscles of patients presenting idiopathic scoliosis, using a triaxial dynamometer allowing the measurement of asymmetric efforts during extension contractions. More specifically, the sensitivity of these EMG measures (amplitude and MF) to detect muscle weakness and muscle fatigue was assessed among scoliotic subjects and contrasted with normal subjects. Also, the sensitivity of these EMG parameters to the known muscle morphological differences between the concave and convex side in scoliotic subjects was explored. Methods Subjects and tasks The control group (CG) was composed of ten young women with no back problem or physical disability. They were recruited among the children of parents attending a physiotherapy clinic. Subjects were excluded if they had experienced back pain in the last six months prior to the experiment. Six young women with a diagnosis of idiopathic scoliosis formed the scoliotic group (SG) and they were recruited from the Clinique de la Scoliose of Ste-Justine Hospital in Montreal. Any scoliotic subject having experienced episodes of back pain in the last six months prior to the experiment, who was on a specific treatment for the scoliosis, had been wearing a corset for more than three months at the time of the experiment or for more than six months prior to the experiment was excluded. Explanations concerning the experimental protocol were given and a written consent form was signed by all the subjects and their parents. The subjects' characteristics were for the scoliotic group (age: 16 ± 3.5 years; height: 160.6 cm ± 10.2; mass: 51.3 Kg ± 8.5; L5/S1 peak extension moment: 100.2 Nm ± 40.2; handedness: all right; Cobb's angle (all right thoracic curve, apex T8–T10): 35.6° ± 4.2) and for the control group (age: 14.4 ± 2.6 years; height: 154.6 cm ± 10.4; mass 44.8 Kg ± 8; L5/S1 peak extension moment: 100.7 Nm ± 33.3; handedness: all right). After the EMG electrodes were positioned and the subject was stabilized in the static dynamometer, two to three submaximal extension contractions were performed in order to get used to the apparatus. Then, two maximal voluntary contractions (MVC) in extension were performed and the highest value was kept to obtain a reference value for the following tests. The neuromuscular efficiency test consisted of three 7 s ramp extension contractions from 0% to 100% MVC separated by a two minute rest period. The rate of the contraction had to be paced with the speed of a target. If this condition was not respected, the trial was rejected and repeated after an adequate rest period. The ramp demonstrating the best control was kept for data processing. For the muscle fatigue test, the subjects performed a 30 s extension static contraction at 75% MVC as proposed by van Dieen & Heijblom [ 33 ]. Again, visual feedback was given to the subject on a computer screen located in front of her. Dynamometry The subjects stood in a dynamometer which consisted of a triaxial force platform (Advanced Mechanical Technology Inc., model MC6-6-1000) allowing simultaneous measurements of static moment generated around the trunk three axes (flexion-extension, lateral flexion and rotation) at the level of L5-S1 joint during an isometric effort in back extension [ 34 ]. The force platform was fixed on a metal frame and could be adjusted at the superior part of the trunk to be positioned at the level of T4. The pelvis, the knees and the feet were stabilized to minimize movement of the lower body and to isolate back muscle contractions (Figure 1 ). The knees were kept in a slightly flexed position. The L5/S1 extension moments were computed in real time and provided to the subjects as visual feedback on a monitor positioned in front of them. The visual feedback consisted of a vertically moving target with lower and upper bounds corresponding to a tolerance limit of ± 10% MVC. It was used to control the pace of the contraction rate for the neuromuscular efficiency test and to make sure that the isometric contraction of the fatigue test was as steady as possible. Figure 1 Subject positioned in the triaxial dynamometer with the sign convention of the AMTI force platform. Positive My = extension moment, positive Mx = right lateral bending moment and positive Mz = left axial rotation moment. Electromyography Eight active surface electrodes (Model DE-2.3, Delsys Inc., Wellesley, MA) were used to collect the EMG signal. Each pair of electrodes is composed of two silver bars 10 mm long and 1 mm wide encastrated in non-conductive material and separated from each other by 10 mm. The EMG signal was bandpass filtered (20 – 450 Hz), pre-amplified with a gain of 1000, analogue to digital converted at a sampling rate of 4096 Hz and stored on a hard disc for later analysis. A custom Lab View software (National Inst.) was used to collect and process force and EMG data. Electrode sites were identified (details below) and the thickness of subcutaneous tissues at these electrode sites was measured twice on each side with a Harpenden skinfold caliper. Afterwards, the skin at the electrode sites was abraded and cleaned with alcohol. The electrodes were positioned on each side of the following muscles respecting the muscle fibers direction [ 35 ]: Multifidus at L5, Iliocostalis at L3, Longissimus at L1 and T10. One ground electrode (snap-on type) was positioned on the spinous process of T8. Data processing and statistical analyses For the neuromuscular efficiency test, 250 ms windows were selected from the EMG signals at each of the following force levels: 10, 20, 30, 40, 50, 60, 70, 80% MVC. The signal within each window was transformed in RMS and in MF of the EMG power spectrum (fast Fourier transform, Hanning window processing). A spectral resolution of 4 Hz was then possible with respect to MF for the efficiency test (1024 points in a 250 ms window and with a sampling frequency of 4096 Hz). The RMS/force and MF/force relationships of each muscle were determined using linear regression. A linear RMS to force relationship was assumed in the present study based on other studies showing either a linear [ 36 , 37 ] or a quasi linear [ 37 ] relationship depending on the selected back muscle. The slope values for the RMS/force relationship were indicators of neuromuscular efficiency, thus weaker muscles were characterized by steeper slopes. The slope values for the MF/force relationship reflected the average conduction velocity of the muscle and indirectly, muscle fiber type composition. For the fatigue test, both RMS and MF values were calculated on a succession of 21 windows (250 ms) equally placed from the 3 rd second to the 25 th second of a contraction lasting 30 seconds at 75 % MVC. The RMS/time and the MF/time relations were also studied using linear regression techniques and the slope values were indicators of muscular fatigue. To verify if scoliotic subjects produced more asymmetric efforts than controls during the neuromuscular efficiency and fatigue tests, the coupled (lateral bending, axial rotation) L5/S1 moments were computed at the same time-windows as for EMG analyses. For each series of data (efficiency test: 8 values; fatigue test: 21 values) and each subject, the mean, the minimum and the maximum values were determined. Student unpaired t-tests were used to compare groups characteristics such as age, height, body mass, skinfold thickness at each electrode site and to compare L5/S1 peak extension (during MVC tasks) and coupled moments (during the efficiency and fatigue tasks). Two-way ANOVAS (2 Groups × 2 Sides) with one repeated measure (side) were used to compare the slope values between the two groups and between left and right sides for the EMG parameters of both the efficiency and the fatigue tests. The level of statistical significance was set at 0.05. Results Description of subject samples No intergroup differences were observed by the Student t-tests performed for age (p = 0.36), height (p = 0.28) and body mass (p = 0.16). The peak L5/S1 moment produced in extension was equivalent for both groups as disclosed by a Student's t-test (p = 0.98). Concerning the skinfold thickness, although the subjects of the scoliotic group tended to present higher values for all muscle sites, no significant difference was shown when these values were compared with those of the control group. Similarly, regarding the side factor, again no difference was observed within each group (paired Student t-test p > 0.05). During the efficiency tasks, the coupled L5/S1 moments in lateral bending and axial rotation were not significantly different between groups (t-test, p > 0.05). There were no significant intergroup differences either for mean, the minimum and the maximum values in both L5/S1 coupled moments (t-test, p > 0.05). In lateral bending (positive values = right lateral bending), the mean, the minimum and the maximum values were respectively 0.85, -0.56 and 2.5 Nm for the control subjects and 0.34, -3.27 and 1.81 Nm for the scoliotic subjects. In axial rotation (positive values = left axial rotation), the mean, the minimum and the maximum values were respectively 1.86, -0.33 and 4.28 Nm for the control subjects and 1.97, -1.53 and 5.34 Nm for the scoliotic subjects. Similar results were obtained during the fatigue test as no group differences (Student's t-test, p > 0.05) were observed. In lateral bending the mean, the minimum and the maximum values were respectively 1,62, -3,57 and 6,62 Nm for the control subjects and -1.28, -4.37 and 0.71 Nm for the scoliotic subjects. In axial rotation, the mean, the minimum and the maximum values were respectively -2.56, -5.36 and 0.42 Nm for the control subjects and -1.50, -2.84 and 0.82 Nm for the scoliotic subjects. Assessment of muscle composition No significant difference in the MF/force relationships between the two groups were observed for all eight muscles investigated. Regarding side differences, the iliocostalis (L3) showed a nearly significant difference (p = 0.09) (Table 2 ). The MF/force slopes were negative for the right and left multifidus muscles (L5) as well as for the right iliocostalis muscle (L3) in the normal group. In the scoliotic group, the slopes were negative on both sides of the multifidus (L5) and iliocostalis (L3) as well as on the right side of the longissimus (L1). Table 2 Summary of the two way ANOVAs with one repeated measure on the side factor for each muscle investigated for the RMS/force, MF/force, RMS/time and MF/time relationships RMS/Force MF/Force RMS/Time MF/Time F p F p F p F p L5 Group 1.00 0.39 0.66 0.53 0.04 0.96 0.50 0.62 Side 1.78 0.20 0.21 0.65 0.04 0.84 0.21 0.65 Group × side 0.63 0.44 0.82 0.38 0.02 0.89 0.56 0.47 L3 Group 1.72 0.22 1.61 0.23 0.46 0.62 4.59 0.17 Side 2.52 0.14 3.13 0.09 0.00 0.97 8.28 0.01* Group × side 1.75 0.21 0.02 0.89 0.87 0.37 2.69 0.12 L1 Group 0.14 0.87 0.20 0.82 0.34 0.72 0.56 0.59 Side 0.10 0.76 0.41 0.53 0.02 0.88 0.18 0.68 Group × side 0.10 0.75 0.01 0.91 0.56 0.47 1.08 0.32 T10 Group 0.71 0.51 0.16 0.85 0.97 0.40 0.09 0.92 Side 1.15 0.30 0.11 0.75 0.11 0.75 0.13 0.73 0.61 0.45 0.12 0.73 1.95 0.18 0.09 0.77 * : Statistically significant Assessment of neuromuscular efficiency Table 1 presents the mean values of the slopes of the regression line for each measure used for a given muscle for both the control group and the scoliotic group. Table 2 presents the summary of the two-way ANOVAs with one repeated measure for each of the muscle investigated and each of the measures used. Table 1 Descriptive statistics for each measure used (slope of a regression line) for a given muscle for both the control (CG, n = 10) and the scoliotic group (SG, n = 6) Mucles Groups Slope value measures (mean (SD)) RMS/Force MF/Force RMS/Time MF/Time L5 (L) SC 0.80 (0.50) -0.18 (0.38) 0.60 (0.46) -1.04 (0.53) CG 0.98 (0.53) -0.24 (0.41) 0.54 (0.67) -0.75 (0.82) L5 (R) SC 0.81 (0.48) -0.21 (0.26) 0.83 (0.86) -0.92 (0.92) CG 0.99 (0,55) -0.08 (0.59) 0.59 (0.68) -1.12 (0.73) L3 (L) SC 0.83 (0.56) -0.03 (0.33) 0.54 (0.41) -0.31 (0.64) CG 1.29 (0.82) 0.00 (0.17) 0.84 (1.28) -0.28 (0.51) L3 (R) SC 0.67 (0.43) -0.41 (0.24) 1.22 (1.17) -0.70 (0.64) CG 1.44 (1.42) -0.10 (0.27) 0.90 (1.08) -0.60 (0.57) L1 (L) SC 1.32 (0.81) 0.07 (0.31) 0.63 (0.72) -0.52 (0.37) CG 1.08 (0.46) 0.08 (0.19) 0.72 (1.01) -0.57 (0.60) L1 (R) SC 1.26 (1.14) -0.02 (0.22) 0.63 (0.46) -0.38 (0.37) CG 1.18 (0.66) 0.11 (0.18) 0.63 (0.74) -0.48 (0.45) T10 (L) SC 0.68 (0.47) 0.10 (0.24) 0.86 (0.56) -0.49 (0.19) CG 0.87 (0.37) 0.16 (0.33) 0.58 (0.97) -0.33 (0.38) T10 (R) SC 0.79 (0.64) 0.04 (0.30) 0.44 (0.39) -0.49 (0.48) CG 0.80 (0.41) 0.15 (0.22) 0.51 (0.92) -0.23 (0.63) R/L: Right/Left; L5: Multifidus muscle; L3: Iliocostalis muscle; L1: Longissimus muscle; T10: Longissimus muscle For the EMG signals, no significant differences were found in the RMS/force relationships obtained between the scoliotic and control groups or between the left and right sides. This was true for each muscle pair investigated (Table 2 ). Assessment of muscle fatigue As expected, the slopes of the RMS/time relationships were all positive for all muscles in both groups (Table 1 ). There were no significant differences in the RMS/time relationships (fatigue test) between groups and between sides for each muscle pair (Table 2 ). For the MF/time relationships, there was a side difference (p = 0.01) for the iliocostalis (L3) muscles, the right side showing more negative slopes (more fatigue) than the left side. However the Group × Side interaction was not significant (Table 2 ). The slopes were all negative for all muscles in both groups (Figure 2 and Figure 3 ) Figure 2 MF/time relationship for both groups for the left Iliocostalis muscle at the level of L3. Figure 3 MF/time relationship for both groups for the right Iliocostalis muscle at the level of L3. Discussion Control of potential confounders One factor of importance when interpreting EMG signals is the skinfold thickness, since it may act as a biological filter [ 38 ]. Fortunately, no differences were found between subjects, groups and sides. However, the small number of subjects cannot fully support the control of this confounding variable and this will be taken into account in future research protocols developed from those preliminary data. Before contrasting the EMG results of the control and scoliotic subjects, it was also important to verify if the efforts performed in the different planes were similar. Although it cannot be ascertained that the back muscle forces were the same between groups, the analysis of the principal (extension) and coupled (lateral bending and axial rotation) L5/S1 moments demonstrated that the main efforts were comparable. Thus, contrary to what might be expected from their asymmetric deformity of the spine, scoliotic subjects performed coupled L5/S1 moments comparable to those of the control subjects. This is a new finding in this literature. Furthermore, the small coupled moment values, which were either within or close to the maximal measurement errors of the dynamometer (lateral bending: 1 Nm; axial rotation: 8.8 Nm) [ 34 ], were negligible from a physiological point of view. Assessment of muscle composition As seen in the introduction section, there is an alteration of the muscle fiber type composition on the concave side of the thoracic curve in scoliotic subjects. The percentage of type I muscle fibers is reduced in the paraspinal muscles on the concave side relative to the convex side and relative to the homologous muscle of non scoliotic subjects [ 6 , 10 ]. The cross sectional area occupied by type II muscle fibers is larger in the paraspinal muscles on the concave side than in those on the convex side and is also larger than in the paraspinal muscles of normal subjects [ 6 ]. The average muscle conduction velocity should be faster in the muscles on the concave side than in those on the convex side. Considering this, a more pronounced increase of the conduction velocity, and consequently, of MF values, would have been expected in the paraspinal muscles on the concave side of the thoracic curve of scoliotic subjects as the force level increases. This was shown in other studies using the muscles of the extremities [ 23 , 27 , 28 , 39 ]. However, the interpretation of the relationship between muscle fiber composition (proportions, areas) and spectral parameters is non-conclusive in the literature [ 22 , 40 - 42 ] and this section of the study was done on an exploratory basis. The MF/force slope showed no side difference in the present study at the level of the thoracic spine. It is possible that our scoliotic subjects were not affected enough (small Cobb angle, the younger subjects may not have altered muscle fiber type composition yet) by their condition in order to see major alterations in the EMG signals. The lack of difference can also demonstrate that either this measure was not sensitive to the back muscle composition of this population or that there was no difference in the back muscle composition of the subjects evaluated. It is of interest to note that for the muscles at the level of L5, the values of the MF were compressed towards the lower frequencies of the spectrum as the force level was increasing. This could be explained by the recruitment of type II muscle fibers of the multifidus muscle which are known to be smaller than type I muscle fibers [ 29 ]. Assessment of neuromuscular efficiency The peak L5/S1 extension moment showed no group difference. This concurs with other comparative studies contrasting scoliotic and normal subjects [ 43 , 44 ]. Given the same net moment and that the effort was symmetric, we could speculate that the EMG would be the same in both groups and on both sides. This was demonstrated with the results of the present study, where no significant differences were found in the RMS/force relationships obtained between the scoliotic and control groups or between the left and right sides and this, for each muscle pair investigated. Contrary to the present findings, data from other studies demonstrated that muscles of the back on the convex side of the curve of scoliotic subjects produced more EMG than homologous muscles of healthy subjects and that they also produced more EMG than muscles on the concave side [ 45 - 47 ]. However, it cannot be verified from the data of previous studies if the back asymmetric efforts in extension, which could have produced these EMG differences, were similar between groups. Nevertheless, these differences can also be associated with different experimental procedures involving different position of the subject as well as different tasks performed. Assessment of muscle fatigue The RMS/time slopes were all positive indicating that the muscles generated more EMG to maintain the level of the contraction for the required time to perform the task. However, no group or side differences were disclosed (Table 2 ). Likewise, as seen in previous studies on scoliotic subjects [ 46 ] and low back pain subjects [ 32 , 48 ], the slopes of the MF/time relationship were all negative, also indicating the presence of muscle fatigue. In the present study, no between-group differences were shown for this parameter. Thus, since the L5/S1 peak extension moment and the MF/time relationship were equivalent in both groups, the muscle endurance was similar and this for all the muscles evaluated. This is in accordance with the results of Zetterberg [ 46 ], although it is not mentioned if their subjects have performed similar symmetric efforts. The MF/time slope showed a significant side effect in the iliocostalis muscle at the level of L3 but the Group × Side interaction was not significant. A side difference at the lumbar spine was shown in another study on muscles of low back pain subjects [ 32 ] and one possible explanation can be related to side dominance [ 45 ]. The back muscles on the non-dominant side showed less muscle fatigue and this was significant in the right handed population [ 49 ]. This is in accordance with the results of the present study where all subjects were right handed and where the MF/time slopes on the right side were more negative, indicating greater signs of muscle fatigue. Some limitations of the study must be addressed. Group or side discrimination in the muscle efficiency and in the muscle fatigue tasks may not have been possible in the muscles of the thoracic spine due to the small number of subjects. Data obtained from the present study will be used to carry out some power calculations in order to determine the number of subjects needed to demonstrate a clinically significant difference in the main outcome measures between scoliotic subjects and controls in the next step of this research. It is also a possibility that the scoliotic subjects who participated to the study were not impaired enough to show any alteration in their muscle function and or in their muscle fibers. Conclusion The EMG parameters used in this study could not discriminate the back muscles of scoliotic subjects from those of control subject regarding muscle fiber composition, neuromuscular efficiency and muscle fatigue at the level of the apex, where abnormal muscle fiber composition is observed from biopsy data. However, the results of this pilot study showed a side difference for muscles located at lower level of the spine (L3). That might be an indicator that the erector spinae at the thoracic level might rely on compensatory strategies involving muscles located in the lumbar area to maintain a specific torque level during a fatigue task. Competing interests The author(s) declare that they have no competing interests. Authors' contributions NG is a physical therapist and a Ph.D. candidate in the Biomedical Sciences program at University of Montreal. This article is derived from her master thesis. She was involved in all the research processes. AB is the director of the School of Rehabilitation at University of Montreal, Montreal, Québec, Canada. As the research director of Mrs Gaudreault, he was also involved in all the steps of the project, mainly in the conception and the design of the research protocol. CL is a researcher at l'Institut de recherche Robert-Sauvé en santé et sécurité au travail (IRSST), Montréal, Québec. From its expertise in the use of the EMG signal, he was mainly involved during the data acquisition, analysis and interpretation of the results. He also contributed in drafting the article and revising it critically. SD is a professor at the School of Physical and Occupational therapy at McGill University. Her help was precious during the design of the protocol and for the interpretation of the data. She contributed through her comments to the manuscript. CHR is an orthopeadic surgeon working with scoliotic children at Ste-Justine hospital, Montreal, Québec, Canada. He contributed to the project as the medical expert and took part of the recruitment process. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079863

Analyses of the differentiation potential of satellite cells from myoD-/-, mdx, and PMP22 C22 mice

Background Sporadic and sometimes contradictory studies have indicated changes in satellite cell behaviour associated with the progressive nature of human Duchenne muscular dystrophy (DMD). Satellite cell proliferation and number are reportedly altered in DMD and the mdx mouse model. We recently found that satellite cells in MSV ski transgenic mice, a muscle hypertrophy model showing progressive muscle degeneration, display a severe ageing-related differentiation defect in vitro . We tested the hypothesis that similar changes contribute to the gradual loss of muscle function with age in mdx and PMP22 mice, a model of human motor and sensory neuropathy type 1A (HMSN1A). Methods Single extensor digitorum longus muscle fibres were cultured from mdx and PMP22 mice and age- and genetic background-matched controls. Mice at several ages were compared with regard to the differentiation of satellite cells, assayed as the proportion of desmin-expressing cells that accumulated sarcomeric myosin heavy chain. Results Satellite cells of 2 month, 6 month, and 12 month old mdx mice were capable of differentiating to a similar extent to age-matched wild type control animals in an in vitro proliferation/differentiation model. Strikingly, differentiation efficiency in individual 6 month and 12 month old mdx animals varies to a much higher extent than in age-matched controls, younger mdx animals, or PMP22 mice. In contrast, differentiation of myoblasts from all myoD null mice assayed was severely impaired in this assay system. The defect in satellite cell differentiation that occurs in some mdx animals arises from a delay in differentiation that is not overcome by IGF-1 treatment at any phase of cultivation. Conclusion Overall, a defect in satellite cell differentiation above that arising through normal ageing does not occur in mdx or PMP22 mouse models of human disease. Nonetheless, the impaired differentiation of satellite cells from some mdx animals suggests that additional factors, environmental or epigenetic, may lead to deteriorating muscle repair through poor differentiation of satellite cells in genetically predisposed individuals.

Background Like many muscle diseases, Duchenne muscular dystrophy (DMD) is characterised by a gradual loss of muscle function with age. Patients are initially ambulatory and have mild muscle pathology, despite ongoing degeneration and repair. In later stages of DMD, patients experience progressively more severe muscular changes, accompanied by loss of function, physical dependency, and ultimately, death [ 1 ]. DMD patients generally lack the cytoskeletal protein dystrophin, a member of the spectrin-like superfamily of actin binding proteins. Functional dystrophin is localised to the inner face of the sarcolemma and binds to cytoskeletal F-actin and transmembrane beta-dystroglycan as part of multiprotein complex that mediates signalling between the cytoskeleton and the extracellular matrix. The consequences of lack of dystrophin appear to be an enhanced susceptibility to fibre damage and possibly poor signalling between fibres and their environment. A milder form of dystrophin deficiency in humans is the Becker-Kiener type of muscular dystrophy (BMD). Here, dystrophin is not completely absent, but mutations lead to a quantitatively and/or qualitatively reduced gene product which does not accomplish its full function. The onset of BMD is generally later than DMD. In childhood, symptoms are usually very mild and muscle weakness becomes more evident only in the teens or twenties. BMD is non-lethal and patients often achieve normal life span, although the disease can progress in later life [ 2 ]. To date, it is not clear what controls disease progression in either DMD or BMD and no consensus has been reached in the literature. The progressive loss of muscle in DMD and other muscle disorders could be due to a sustained or increasing rate of degeneration above the rate of repair, or a progressive decline in the ability to regenerate the muscle. Some pathological changes appear to be similar to those observed in healthily ageing people, yet premature and exacerbated. A popular, yet challenged view is that in DMD, disease progression is attributable to accumulating deficiencies in the ability of satellite cells resident within the muscle to mediate regeneration and/or their own replacement [ 3 ]. Satellite cell deficiencies could arise because of the excessive demands on repair mechanisms necessitated by the continuous degeneration of unstable muscle that does not express dystrophin. Indeed, symptoms of rapid early turnover of muscle fibre material and cells are apparent before birth in DMD patients [ 4 ], yet serious functional deficits arise only late in the first decade. A commonly used model for studies of DMD is the mdx mouse. These animals, like human patients, show delayed onset of debilitating muscle degeneration. Although a transient burst of frank degeneration occurs in mdx mice during the period of muscle growth around the third postnatal week, degeneration leading to debility only occurs late in life, primarily in specific muscles, such as the diaphragm [ 5 ]. In mdx mice, as in human DMD patients, disease is caused by the absence of functional dystrophin, owing to a nonsense mutation in exon 23. The relatively mild phenotype of mdx mice can, in part, be attributed to the compensatory function of the dystrophin-related protein utrophin, which is highly upregulated in regenerating muscle fibres in adult mdx mutants [ 6 ]. Over time, enhanced muscle turnover and satellite cell numbers are also seen in mdx mice [ 7 ]. Other muscle diseases also show variable clinical progression. Human motor and sensory neuropathy type 1A (HMSN1A, also known as Charcot-Marie-Tooth disease type 1A, CMT1A) is a dominantly inherited demyelinating disorder of the peripheral nervous system. It is most frequently caused by over-expression of the PMP22 gene due to duplication of a 1.5-Mb region on chromosome 17, but it can also result from point mutations in the PMP22 gene [ 8 - 12 ]. The affected individuals typically have distal muscle weakness and atrophy often associated with mild to moderate sensory loss, depressed tendon reflexes, and high-arched feet. Individuals with HMSN1A experience slowly progressive weakness and atrophy of distal muscles in the feet and/or hands. Disease progression is variable for unknown reasons. PMP22 C22 transgenic mice which were modified to harbour seven copies of the human PMP22 gene demonstrate developmental delays in myelination, decreased numbers of myelinated fibres, and abnormally thin myelin similar to HMSN1A [ 13 ]. Being a neuropathy, PMP22 C22 mice can be used as reference animals that display a muscle phenotype without harbouring intrinsic muscle defects. In a recent study, we found that satellite cells of MSV ski transgenic mice display a differentiation defect compared to wildtype control animals and that this defect is exacerbated in ageing animals [ 14 ]. Like mdx mice, hypertrophic MSV ski transgenic mouse muscles have muscle degeneration that is initially efficiently repaired, but which eventually shows defective regeneration and frank muscle defects. In the present paper, we investigate the differentiation potential of satellite cells of single muscle fibres from the hypertrophic mdx and PMP22 mouse models and corresponding wildtype control animals in order to clarify whether ageing-related change in differentiation potential of satellite cells might influence disease progression. Methods Mouse strains mdx mice were obtained from a colony in the lab of T. Partridge (Imperial College, London, UK) and are similar to JAX C57BL/10ScSn-Dmd mdx /J. Control mice were obtained from JAX and were C57BL/10ScSn. MyoD m1 null mice were as reported [ 15 ]. Mice were killed at ages of 6 to 8 weeks (referred to as 2 months), 22 to 25 weeks (referred to as 6 months), or 44 to 55 weeks (referred to as 12 months). Mice of the HMSN1A model PMP22 C22 and the corresponding age matched litter mate controls were received from the lab of C. Huxley at ages of 2 to 3 months (referred to as 2 months), 10 to 12 months (referred to as 11 months), and 15 months. Mice were kept in plastic cages with wire mesh lids in a 12:12-h light-dark cycle and fed ad libitum . Both sexes were used for each experimental time point to test for sex specific effects, although none were observed, and at least 4 mice of each genotype were used at each age. All animal experiments were carried out in accordance with the local ethics committee and UK Home Office approval. Single fibre preparation Single fibres from mouse EDL or soleus muscle were isolated and cultured in order to obtain satellite cells as described recently [ 14 ]. Briefly, muscle tissue was dissected from mice of appropriate age and genotype in a manner that minimised injury, stretch, or other stress factors on the fibres. Connective tissue was removed by incubation in DMEM (Gibco, Paisley, UK) with 0.2% collagenase type I at 35°C for 1 h. Fibres were liberated by trituration in DMEM medium with Pasteur pipettes of different pore sizes. Fibres were fixed in 4% PFA, or placed in isolated wells of 8-well Permanox™ chamber slides (NalgeNunc International, Rochester, USA), coated with Matrigel (1 mg/ml in DMEM, BD Biosciences, Oxford, UK) for satellite cell cultivation and immunocytochemistry. Satellite cell cultivation Single fibres were allowed to adhere to the Matrigel matrix (3–5 mins), before adding 300 μl of plating medium (10% horse serum, HS (Gibco), 0.5% Chick Embryo Extract, CEE (Sera Laboratories International Ltd., Crawley, UK), in DMEM with 1% streptomycin/penicillin and 2% L-glutamine) and incubation in a humidified environment at 37°C and 5% CO 2 . After 3 days incubation to allow satellite cells to migrate off the fibre onto the Matrigel substrate, fibres were removed from the chambers and medium was replaced by proliferation medium (PM; 20% fetal calf serum, FCS, 10% HS, 2% CEE in DMEM). After further two days, PM was replaced by differentiation medium (DM ; 2% FCS in DMEM) and cells were allowed to differentiate for 2 or 5 days. Three days after plating and again after two days in proliferation medium, the total number of cells was analysed for each single fibre culture. To check for the influence of IGF-1 treatment on differentiation efficiency of satellite cells, PM, DM, or both were supplemented with 100 ng/ml of recombinant R 3 -IGF-1 (Sigma, Deisenhofen, Germany). Immunocytochemistry Slides were rinsed in PBS, fixed in cold methanol, blocked in 5% horse serum in PBS, and incubated with mAb against desmin (1/500, clone no. DE-U-10, IgG1; Sigma) and MyHC (1/10, A4.1025, IgG2a; [ 16 ]). Primary antibodies were successively detected with rat anti-mouse IgG1 (1:1,000; Serotec, Oxford, UK), FITC-conjugated goat anti-mouse IgG2a (1:100; Serotec, Oxford, UK), and Cy3-conjugated donkey anti-rat IgG (1:100; Jackson, USA). In the last antibody incubation, the DNA dye DAPI was added. Cells were washed, re-fixed in cold methanol and mounted with antifading agent. Cell differentiation was analysed by monitoring 10 randomly-selected fields of view equivalent to a total area of 5.45 mm 2 of each chamber, representing approximately 15% of the area of each chamber. Differentiation efficiency (labelled 'Myoblast differentiation') was determined as the ratio of nuclei in myosin heavy chain positive (MyHC + ) myocytes and/or myotubes divided by the total number of nuclei in desmin-expressing cells (desmin + cells). Data presented reflect means and standard deviation of fibres from each of ≥ 4 animals unless otherwise stated. Cultures from ≥ 30 fibres were analysed in each case. Results A sensitive assay for satellite cell differentiation Mice wild type or carrying one or two null alleles at the myoD locus were used to establish an in vitro culture system to measure the differentiation potential of satellite cells in culture. In agreement with published data [ 17 ], satellite cells from single EDL muscle fibres from wildtype or heterozygous myoD +/- animals showed high levels of differentiation, as assayed by the fraction of desmin + myogenic cells that express MyHC, whereas satellite cells from myoD deficient animals often failed to differentiate (Fig. 1A,B ). Satellite cells of myoD wildtype mice ( myoD +/+ ) displayed efficient differentiation (80% ± 3) within 2 days of growth factor removal. Comparable numbers of satellite cells from myoD +/- EDL muscles differentiated terminally (75% ± 14), whereas cells obtained from myoD -/- muscle fibres almost completely failed to differentiate (3% ± 1). Thus, single fibre cultures can efficiently quantify myoblast differentiation. Figure 1 A method to quantitate satellite cell differentiation. Single fibres from EDL muscles of 2 month old mice were isolated and cultured for five days, switched to differentiation medium for two days and the fraction of desmin-positive (desmin + ) cells expressing myosin heavy chain determined by immunofluorescence microscopy. A: Satellite cell cultures from myoD heterozygous ( myoD +/- ) and homozygous null ( myoD -/- ) animals. Essentially all cells (blue nuclei: DAPI) express desmin (red: Cy3) in both genotypes, but note the lack of multinucleate myotubes and cells expressing MyHC (green: FITC) in the myoD -/- . B-D: Quantification of satellite cell-derived myoblast growth and differentiation by genotype. Error bar = SD. Numbers above columns indicate number of wells counted (1 fibre/well) from 4 animals of myoD +/+ , myoD +/- , and myoD -/- genotype, respectively. B: Differentiation of myoD -/- myoblasts is poor compared to littermates. C: Increase in cell number after the switch from plating to proliferation medium is independent from the genotype of the animals. myoD +/+ , myoD +/- and myoD -/- cells proliferate to the same extent. D: The fraction of differentiated myogenic cells is unaffected by either the number of myogenic desmin + cells or the number of non-myogenic desmin - cells in each well. In all genotypes ( myoD +/+ , myoD +/- , and myoD -/- ) all fibres yielded myogenic cells. We checked that the myogenic cell yield and proliferation of satellite cells did not differ between myoD +/+ , myoD +/- , and myoD -/- fibres (Fig. 1C ). During the 2 days in PM around a 12- to 14-fold increase in cell numbers occurred, indicating an approximately 12 hour doubling time, independent of genotype. The presence of non-myogenic cells and/or total cell density might affect myogenic differentiation efficiency. Therefore, we used desmin expression to discriminate between myogenic and non-myogenic cells. Differentiation efficiency in wells with different numbers of desmin positive (desmin + ) or desmin negative (desmin - ) cells for all cultures of myoD +/+ , myoD +/ , and myoD -/- fibres was compared (Fig. 1D ). No effect of desmin - cells in the cultures was detected on the differentiation potential of the myogenic cells. On average, there were less than 11% non-myogenic cells in any cultures from mice of any genotype. Similarly, differentiation efficiency did not correlate with the density of either total cells or desmin + cells; satellite cells are capable of differentiating (expressing MyHC) independently of cell fusion. Therefore, in this culture system, density of neither myogenic cells nor non-myogenic cells has a detectable influence on differentiation efficiency. Differentiation potential of satellite cells from PMP22 transgenic mice To test whether reduced muscle motility, altered innervation, or fibre atrophy/regrowth might trigger satellite cell changes in some individuals, we examined PMP22 C22 transgenic mice that all show signs of disease, but have a heterogeneous progression. Mouse models have demonstrated that excess PMP22 protein in Schwann cells leads to repeated focal demyelination lesions in peripheral nerves, decreased conduction velocity, and a muscle histology characteristic of partial denervation, just as in HMSN1A [ 13 ]. Histological analysis of sections of soleus muscles revealed that in some individual animals signs of dramatic fibre atrophy and/or regrowth are present (Fig. 2A ). In addition, significant changes in fibre type occur, as revealed by MyHC isoform expression. In other individuals, however, the alteration in muscle fibre size profile and type was less marked (data not shown). To clarify whether phenotypic variation is reflected in diversity of satellite cell behaviour, we analysed satellite cells of PMP22 C22 transgenic mice at several ages. However, no significant variation in satellite cell differentiation efficiency was observed at any age examined (Fig. 2B ). Again, no influence of myogenic or non-myogenic cell density on differentiation was observed (data not shown). Little inter-animal variation was observed, unlike the case of mdx animals (see below). Thus, PMP22 mice show no loss of satellite cell function. Figure 2 Differentiation of satellite cells of the HMSN1A mouse model PMP22 C22 at three ages. A. Variation in soleus muscle cryosections reacted for slow MyHC reveals variable pathology of individual PMP22 C22 mice. Note fibre atrophy (arrowheads) and large, hypertrophied fibres in the middle compared to the right panel. B. Satellite cells from single fibre culture of PMP22 C22 and littermate controls were analysed for cell yield, proliferation and terminal differentiation. No significant differences were observed. Differentiation potential of satellite cells of mdx mice To test whether satellite cells from EDL muscles of mdx mice display a defect in differentiation, we compared single fibre cultures of mdx with age-matched control animals, on the same genetic background, C57BL/10. In fibre cultures from 2 month old control and mdx animals differentiation was 87% ± 3 and 80% ± 5, respectively (Fig. 3A–C ). Thus, the early phase of significant degeneration/regeneration in mdx mice led to no detectable change in satellite cell differentiation. Figure 3 Differentiation of satellite cells of control and mdx mice of different ages in vitro. A. Box and whisker plot: The box extends from the 25th percentile to the 75th percentile between all animals of one age, with a horizontal line at the median (50th percentile). Whiskers show the range of the data. B. Mean differentiation for each individual animal shows heterogeneity between mdx animals. Individuals showing poor differentiation (8 in total) are significantly more common in mdx (X 2 , P = 0.0076). C. Satellite cell cultures from single fibres of 12 month old control C57BL/10ScSn and mdx animals showing good differentiation in each case. Blue nuclei: DAPI; desmin: red, Cy3; MyHC: green, FITC in both genotypes. We have observed decline in differentiation capacity as satellite cells age in regenerating muscle [ 14 ]. In the present study, satellite cells from control C57BL/10 mice yield high levels of differentiation at all ages examined with only a slight and not significant decrease with age (87% ± 3, 84% ± 5, and 82% ± 6 in fibres from 2, 6, and 12 month old mice, respectively; Fig. 3A ). Similar experiments were performed for mdx animals of corresponding ages. Across all fibres from all mdx animals, no significant difference in differentiation in comparison to wildtype animals was observed. Mean differentiation efficiency was 80% (± 5) at 2 months old, 66% (± 15) at 6 months old, and 72% (± 20) at 12 months old mdx animals. However, as indicated by the high standard deviation, the variance in differentiation was extremely high in older animals. We investigated the reason for the higher variance in differentiation of 6 and 12 month mdx cultures. Whereas numbers of cells per well and desmin + or desmin - cell density showed no apparent contribution to variance, animal to animal differences were striking. Many individual mdx mice yielded satellite cells capable of differentiating as well as age-matched controls. However, some individual animals yielded particularly poorly differentiating cells (Fig. 3B ). This variation could not be accounted for by day to day variation in culture conditions because age-matched mdx and wild type animals were always prepared in parallel. There was no indication of correlation with sex, health status, housing conditions, or season, although numbers of animals were too low to eliminate any of these variables with high confidence. Differentiation defect of affected mdx satellite cells can be overcome by prolonged differentiation time Poor satellite cell differentiation in some older mdx mice could reflect a permanent block or simply a delay. We therefore examined differentiation rate in satellite cells from older animals that showed poor differentiation. Single fibre cultures were allowed to differentiate in low-serum medium for 5 days (5d diff) instead of the normal 2 days (2d diff) employed in all other experiments. Prolonged differentiation did not significantly enhance differentiation of control cultures (78% ± 6 and 83% ± 5 after 2 and 5 days, respectively). However, a prolonged differentiation period did rescue differentiation of mdx cells to levels indistinguishable from control 5 day cultures (Fig. 4 ). A significant increase in differentiation capacity from 63% ± 6 to 87% ± 2 in mdx mice was observed. We conclude from this result that satellite cells from some mdx mice differentiate less efficiently in our experimental system as reflected by a slower rate of MyHC accumulation. Figure 4 Prolongation of differentiation rescues the satellite cell differentiation defect. Satellite cells of 6 months old mdx or control animals were plated and allowed to proliferate as described in Materials and Methods and then cultivated either for 2 days (2d diff) or for 5 days (5d diff) in differentiation medium. Prolongation of the differentiation period to 5 days enhances differentiation efficiency (* P < 0.0028) of affected mdx satellite cells to the level observed in controls. IGF-1 treatment does not influence the differentiation of satellite cells in vitro IGF-1 signals have been shown to enhance both proliferation and differentiation of satellite cells [ 18 ]. To begin to define the nature of the emerging satellite cell defect in muscles of some mdx mice, we exposed satellite cells from 6 month old control or poorly-differentiating mdx animals to IGF-1 (100 ng/ml) either in the proliferation phase, the differentiation phase or both (P+IGF, D+IGF, P+IGF D+IGF, respectively; Fig. 5 ). Controls were cultivated for the same time periods in the appropriate medium without IGF-1 (P+ D+). No significant effect of the IGF-1 treatment on the terminal differentiation of satellite cells was observed in any of the culture stages (Fig. 5A ). Thus, IGF-1 level is not rate limiting for differentiation of mdx cultures that show reduced differentiation rate. Neither can IGF-1 promote differentiation of the undifferentiated ~20% of desmin + cells in control cultures. Figure 5 IGF-1 treatment fails to rescue differentiation of satellite cells in culture. Satellite cells from control or mdx animals at the age of 6 months were treated with IGF-1 (100 ng/ml medium) during either proliferation (P+IGF), differentiation (D+IGF), both (P+IGF D+IGF), or neither (P+D+, control). A . Differentiation was unaltered by IGF-1 addition. B . Non-myogenic cells are more abundant in mdx than control single fibre cultures. As controls, we checked that the proportion of myogenic, desmin + cells in the cultures was not altered significantly by IGF-1 treatment in relation to total cell number in either control or mdx . However, the fraction of non-myogenic cells was over three times greater in mdx (58% ± 6) than in controls (16% +/- 5), independent of IGF-1 treatment (Fig. 5B ). As found in the experiments above, despite this difference in non-myogenic cell numbers, differentiation efficiency was comparable between mdx and controls (Fig. 5A ). Discussion Satellite cells are normally quiescent cells that reside under the basal lamina of muscle fibres. Under conditions of growth and repair, satellite cells become activated and begin a coordinated myogenic program: they initially proliferate and express a range of myogenic genes, including desmin, before aligning and fusing to form terminally differentiated multinucleate syncitia that organise and express the contractile apparatus, which includes the MyHC proteins. We have shown recently that, in MSVski transgenic mice, satellite cell differentiation in vitro was influenced by the age of the study animals and showed a progressive decline with age [ 14 ]. This effect was much less obvious in wildtype control animals. Therefore, we concluded that a satellite cell differentiation defect developed in MSVski mice. We speculated that the defect might be caused by continual degeneration/repair apparent in hypertrophic MSVski mice and that the changes in satellite cells might underlie the worsening of the muscle pathological profile with age. The mdx mouse model of human DMD displays a hypertrophy phenotype in the skeletal musculature reminiscent of MSVski mice. Similarly, DMD patients initially show a mild phenotype that gradually progresses throughout the lifespan, manifesting as muscle loss and fibrosis which culminates in death from respiratory or cardiac failure. As the progression of DMD is not understood and because of similarities between DMD/ mdx pathology and the MSV ski animal phenotype we hypothesised that progressive defect in the differentiation potential of satellite cells might contribute to the pathologic mechanism of the debilitating human disease. We find that satellite-derived cells from ageing mdx mice are, in general, capable of differentiating to the same degree as satellite-derived cells from control animals. We did not assess satellite-derived cells that remained physically juxtaposed to the explanted fibre because such cells could display different behaviour(s) due to variations in the fibre integrity or its surrounding matrix. We chose to measure differentiation as the fraction of desmin-expressing cells co-expressing MyHC rather than fusion for several reasons. Firstly, without using assays that detect syncitia [ 19 ], there is no way of clearly ascertaining whether two cells are truly fused or closely apposed. Secondly, mononucleate cells are capable of terminally differentiating and expressing MyHC; such cells would be missed if assessing fusion. Although we can not eliminate the possibility that the mdx condition leads to an alteration in the cells that express desmin, our study provides evidence against this view. First, yields of desmin + cells are similar between mdx and control. Second, the proliferation rate of desmin + cells appears similar between mdx and control. Third, and as discussed further below, although numbers of desmin - cells are increased in mdx cultures the increase is similar both before and after the differentiation phase suggesting that interconversion of desmin - and desmin + cells is not a significant factor in our experiments. Overall, it is unlikely that a defect in differentiation of satellite-derived cells is a major contributor to disease progression in mdx mice. Despite this lack of significant change in overall differentiation capacity, satellite-derived cells from some individual older mdx animals displayed lower differentiation efficiencies than those from other mdx animals of the same age. Age- and background genotype-matched control mice, young mdx mice, and mice with severe muscle weakness due to the PMP22 transgene did not show this variation. We were unable to correlate this effect to any factor analysed. All animals were held under similar conditions. We consciously used both genders and analysed data in combination of both sexes and separately. We conducted additional experiments using increased or decreased collagenase type I incubation times as well as a more severe collagenase type II digestion to determine if variations in satellite cell activation or yield, basal lamina digestion, or fibre damage could affect differentiation potential, but found no differences (data not shown). Similarly, we performed dilution cloning of satellite cells to assess the effects of proliferation rate on satellite cells and whereas we observed heterogeneity of proliferation rates amongst satellite cells grown from single cells, we again found no ultimate difference in differentiation efficiency (data not shown). For those reasons we can not explain the inter-animal variation of mdx results by differences in the experimental design or genetic background of the animal. We speculate that uncontrolled environmental effects or epigenetic factors affecting other genes in the mdx background explain the variation. It is striking that fibres yielding poorly-differentiating cells are numerous in affected individuals, but nevertheless, some fibres yield cells differentiating as well in controls. This emphasises that relatively heritable heterogeneity in myogenic cells must exist in mdx mice and demands elucidation. Moreover, we can not eliminate the possibility that the mdx individuals showing poor differentiation in our assay would have undergone a worse progression of disease in later life. Additionally, we cannot exclude the possibility that very subtle differences in differentiation behaviour were not detected in our assay system as we have utilized matrigel, a matrix in which growth factors are abundant. Thus, small variations might have been masked that only would be detectable at the application of collagen or gelatine matrices. As shown by others [ 20 ] and in this report, non-myogenic cells, probably fibroblasts, can be obtained from single fibre cultures and are more abundant in mdx samples compared with C57BL/10 controls. These cells probably reside on the fibre surface and migrate away from the fibre onto the substrate as do satellite-derived cells. In vivo , these cells may mediate the fibrotic response to fibre degeneration and could potentially secrete factors such as TGF-β that have been shown to interfere with satellite cell differentiation [ 21 ]. We analysed the proportion of non-myogenic cells in the cultures and whether they influenced the efficiency of differentiation of myogenic cells. We were unable to find a correlation between the contamination of the satellite cell culture with desmin - non-myogenic cells and the differentiation efficiency of the myogenic cells in the same culture well. This confirms what we have observed in wild type, MSVski , PMP22 , and myoD null situations [ 14 ]. There was also no difference in desmin - cell levels between mice showing poor or normal differentiation. Thus, at least in mdx animals up to one year of age, no correlation of fibrosis with poor myoblast differentiation is apparent. Differentiation is delayed, not inhibited in some mdx mice We found that satellite-derived cells from mdx mice showing poor differentiation after two days differentiation, recovered and differentiated as well as controls after three further days in differentiation conditions. No morphological differences in the nature of the differentiated cells were detected at this stage. Thus, the reduction in differentiation observed in some mdx animals is most simply explained as a reduced rate of differentiation. If such a decrease in differentiation rate occurs in vivo , it could have serious consequences for muscle repair, which may require rapid satellite cell mobilisation and can occur within a few days [ 22 ]. IGF-1 treatment has been shown to enhance the efficiency of differentiation of satellite-derived myoblasts and this has been suggested to mediate an autocrine loop triggered by myogenin expression [ 23 ]. However, IGF-1 treatment of poorly-differentiating mdx satellite-derived cells did not enhance their differentiation rate. Moreover, although it has been reported that IGF-treatment of wildtype myogenic cells leads to enhanced proliferation [ 24 , 25 ], in our experimental conditions using recombinant R 3 IGF-I we were not able to confirm those findings. Functional impairment in HMSN1A model mice is not accompanied by satellite cell changes A rodent model for the progressive human neuropathy HMSN1A is the PMP22 C22 transgenic mouse that harbours seven copies of the human PMP22 gene in its genome. These animals manifest symptoms similar to human HMSN1A patients including demyelination of Schwann cells and, with later age, progressive skeletal muscle weakness caused by poor innervation [ 13 ]. Examination of postural soleus muscle from such mice revealed a heterogeneous progression of muscle pathology. Some animals showed severe signs of atrophic fibres and changes in fibre type proportion, probably caused by altered electrical activity consequent to slowed nerve conduction. Other individuals, which also showed poor movement, had relatively healthy muscle histology. A cohort of affected PMP22 C22 mice with altered gait were analysed by single fibre culture and revealed no changes in number, proliferation, or differentiation of desmin + cells compared to age- and genetic background-matched controls. Thus, the HMSN1A model mice show a strong phenotype which seems to involve muscle fibre atrophy after demyelination of its innervating motorneuron followed by satellite cell recruitment in the regrowth phase after myelination is restored. However, these processes have no observable effect on mononucleate cells in single fibre cultures. Differentiation defect in satellite cells lacking myoD There has been controversy surrounding the role of the myogenic transcription factor MyoD in satellite cell differentiation. Early reports suggested the myoD null muscle regenerated poorly and that myoblasts from young myoD null mice differentiated poorly in vitro [ 17 , 26 ]. However, a recent study showed the myoD null satellite cells can differentiate efficiently under some circumstances [ 27 ]. In our single EDL muscle fibre cultures, satellite-derived myoblasts from wild type or heterozygous myoD +/- mice differentiate efficiently. In contrast, few desmin-expressing satellite-derived cells from myoD deficient mice were able express MyHC within two days. Given the rapid repair of entire muscles within several days of toxin-induced injury [ 22 ], the differentiation delay in myoD null would have severe consequences in wild populations if a similar delay occurred in any in vivo setting. An issue not addressed directly by our study is the relationship of the assayed population of desmin-expressing cells from wild type to that from myoD null animals. For example, lack of myoD might cause a change in the numbers of cells expressing desmin. We think this unlikely because the yield of desmin + cells, and the ratio of desmin + to desmin - cells, were unaltered in our single fibre cultures, irrespective of myoD genotype. Whatever the case, our experiment shows that lack of myoD leads to substantial reduction in the capacity of muscle fibre-associated migratory proliferative cells to undergo terminal differentiation into myotubes. Conclusion As there appears to be no distinct differentiation defect in the satellite cells of mdx mice, at least by our assay, the cause of progressive muscle loss in the diseased state remains unclear. The differentiation potential of satellite cells has generated contradictory results in several early studies, where differentiation was measured as myoblast fusion in primary cultures (reviewed in [ 28 , 29 ]). One study concluded no differentiation defect [ 4 ], whereas Jasmin and colleagues found a reduced differentiative capacity in myoblasts derived from DMD patients [ 30 ]. A very recent publication by Schaefer et al. also revealed heterogeneity in the number of satellite cells between individual mdx and C57 animals. The authors observed that this heterogeneity did not correlate with age, gender, or degree of degeneration, but possibly reflected additional genetic factors that influence the maintenance of the satellite cell pool [ 31 ]. Altered myoblast number appears not to explain disease progression. Some studies find an increase in the number of satellite cells associated with DMD/ mdx muscle fibres ([ 32 - 35 ] and our unpublished observations), although others observe little change [ 7 ]. Thus loss of available cells for repair is unlikely to cause disease progression. Despite reports of increased cell death in DMD/ mdx myoblasts [ 36 - 38 ], we found no evidence of differential apoptosis between control and mdx . It has also been suggested that decreased proliferative potential and early senescence of satellite cells is the primary cause of disease progression [ 3 , 39 ]. One group measured this limitation as an accelerated age-related shortening of satellite cell telomere length [ 40 ], a result contradicted by another study using a similar assay [ 41 ]. We observed no change in proliferative capacity of mdx satellite-derived cells. These findings suggest that the proliferative potential of satellite cells or their ability to self renew is not compromised; the number of satellite cells remains higher than controls throughout the lifespan of the animals ([ 32 ]; our observations). In summary, our study is the first to provide data on the differentiation efficiency of satellite cells in older mdx and PMP22 C22 mice. We found that, generally, the age of the individual animal had little impact on the differentiation of satellite cells. The pathological processes in muscle of mice with mdx or PMP22 C22 genotype are not necessarily accompanied by defects in satellite cell differentiation. However, in some mdx individuals, satellite cell differentiation is impaired after the age of two months, in a manner akin to that we observed in MSVski hypertrophic muscle pathology [ 14 ]. It seems clear that there is heterogeneity in the pathology of muscle in various animal models of human disease, probably due to epigenetic/environmental contributions. It remains to be further investigated what causes cases of poor differentiation and whether this impaired differentiation of satellite cells enhances deterioration of the physical condition of the individual. List of abbreviations BMD Becker type muscular dystrophy CEE Chick embryo extract CMT1A Charcot-Marie-Tooth disease type 1A DAPI diamidino-2-phenylindole DM differentiation medium DMD Duchenne muscular dystrophy DMEM Dulbecco's Modified Eagle's Medium EDL Extensor digitorum longus FCS fetal calf serum FITC fluorescein isothiocyanate HMSN1A Hereditary Motor and Sensory Neuropathy type1A HS horse serum IGF-1 insulin-like growth factor 1 MSV Master seed virus MyHC Myosin heavy chain mAb monoclonal antibody PBS phosphate buffered saline PFA paraformaldehyde PM proliferation medium PMP 22 Peripheral myelin protein 22 SD standard deviation Competing interests The author(s) declare that they have no competing interests. Authors' contributions MMS and CJM have contributed equally and have carried out the cellular experiments and drafted the manuscript. HB helped with the animals. CH provided PMP22 C22 animals. SMH conceived the study, participated in its design and coordination, and contributed to the writing. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079864

Surgical revascularization versus amputation for peripheral vascular disease in dialysis patients: a cohort study

Background Surgical treatment of peripheral vascular disease (PVD) in dialysis patients is controversial. Methods We examined the post-operative morbidity and mortality of surgical revascularization or amputation for PVD in a retrospective analysis of United States Renal Data System. Propensity scores for undergoing amputation were derived from a multivariable logistic regression model of amputation. Results Of the Medicare patients initiated on dialysis from Jan 1, 1995 to Dec 31, 1999, patients underwent surgical revascularization (n = 1,896) or amputation (n = 2,046) in the first 6 months following initiation of dialysis were studied. In the logistic regression model, compared to claudication, presence of gangrene had a strong association with amputation [odds ratio (OR) 19.0, 95% CI (confidence interval) 13.86–25.95]. The odds of dying within 30 days and within1 year were higher (30 day OR: 1.85, 95% CI: 1.45–2.36; 1 yr OR: 1.46, 95% CI: 1.25–1.71) in the amputation group in logistic regression model adjusted for propensity scores and other baseline factors. Amputation was associated with increased odds of death in patients with low likelihood of amputation (< 33 rd percentile of propensity score) and moderate likelihood of amputation (33 rd to 66 th percentile) but not in high likelihood group (>66 th percentile). The number of hospital days in the amputation and revascularization groups was not different. Conclusion Amputation might be associated with higher mortality in dialysis patients. Where feasible, revascularization might be preferable over amputation in dialysis patients.

Background Nineteen percent of incident dialysis patients have peripheral vascular disease (PVD) [ 1 ]. Amputation rate among diabetic dialysis patients is ten times that of diabetic population at large [ 2 ]. Over 2-years of follow-up, 2.9% of dialysis patients underwent amputation [ 3 ] and in a surgical series, 23% of patients who underwent amputation were on dialysis [ 4 ]. Thus, peripheral vascular disease is common and carries considerable morbidity and mortality in dialysis patients. However, the surgical management of PVD in the dialysis population remains controversial. It has been suggested by some authors that because of delayed wound healing and prolonged hospitalization, primary amputation is preferred over revascularization for PVD in dialysis patients while others have argued that a careful selection of dialysis patients for revascularization might result in acceptable outcomes [ 5 - 12 ]. In this study, we examined the post-operative morbidity and mortality of surgical revascularization or amputation for PVD in a large cohort of Medicare dialysis patients. Methods The study design was a retrospective analysis of Medicare patients in the United States Renal Data System (USRDS). Study population Of the Medicare patients initiated on dialysis from Jan 1, 1995 to Dec 31, 1999, those who underwent surgical revascularization or amputation within 6 months of initiation of dialysis were studied. Because information on functional status (shown to be an important predictor of post-surgical outcomes in PVD in dialysis patients) [ 6 ] and nutritional status (serum albumin and BMI) were only available at initiation of dialysis, only patients who underwent the surgical procedures in the first 6 months of dialysis were included. The study included only those amputated patients with known PVD, as amputations for diabetic foot are also performed for non-ischemic ulcers. Patients with duplicate entries, previous renal replacement therapies, age < 18 years of age and incomplete follow-up information were excluded. In addition, patients with missing data on serum albumin, height and weight were excluded. Data assembly The Medical Evidence (Form 2728) form is the mandatory form gathered by the Centers for Medicare and Medicaid on patients initiated on chronic maintenance dialysis in the US. The Form 2728 data on demographics (age, gender and race), cause of ESRD (diabetes or others), insurance status (Medicare or non-Medicare), comorbid conditions (coronary artery disease, cerebrovascular disease, peripheral vascular disease, congestive heart failure, malignancy, acquired immunodeficiency syndrome, chronic lung disease), smoking, serum albumin, height, weight and functional ability were used in this analysis [ 13 ]. Body mass index (kg/m 2 ) was calculated from height and weight. Definitions of peripheral vascular disease and procedures A patient with reported PVD in the Medical Evidence form or ICD-9-CM codes 440.20–440.24, 440.29, 440.30–440.32, 443.81, 443.9 or 785.4 from initiation of dialysis to the first hospitalization for amputation or surgical revascularization was considered to have PVD. The clinical indication for the surgical intervention was identified from ICD-9-CM codes as intermittent claudication (440.21), resting pain (440.22), ulcer (440.23, 707.10–707.15, 707.19), gangrene (440.24 or 785.4) or other/ unknown (not any of the above ICD-9-CM codes). The first procedure (amputation or surgical revascularization) that occurred after initiation of dialysis was identified from Medicare billing data. Amputations were defined as ankle or below amputations (procedure codes 84.12 – 84.15), knee or below amputations (procedure codes 84.15 – 84.16) and above knee amputations (procedure codes 84.17 – 84.19). Amputations of toes (84.11) were not included. Proximal bypass procedures involving the aorta-iliac-femoral-popliteal vessels were identified using procedure code 39.29. Distal bypass procedures were identified using procedure code 39.25, "other peripheral vascular bypass surgery, other than aorta-iliac-femoral bypass to the tibial, peroneal or dorsal artery." This code also designates vascular bypass procedures in the upper limb. However, in an earlier study, only three did not have a distal lower-limb bypass procedure in over 300 hospital discharge records with the procedure code 39.25 [ 14 ]. Data on PVD-related procedures before initiation of dialysis were unavailable in the USRDS and were not included in the present study. Outcomes Follow-up duration, mortality and transplantation data were obtained from the United States Renal Data System treatment history, claims and patients files. Patients were tracked until loss to follow-up, transplantation, death, or the administrative censor date of December 31, 1999. Cardiovascular death was defined as cause of death coded in the USRDS death notification form as myocardial infarction, atherosclerotic heart disease, cardiomyopathy, arrhythmia, valvular heart disease, cerebrovascular disease and mesenteric infarction. The number of hospital days (including the index hospitalization) in the first 6 months after the procedure and 30-day and 1-year post-operative mortality were the outcomes of interest. Statistical analysis Baseline characteristics of amputation and revascularization groups were not statistically examined because of the large sample size. The propensity score method was used to account for the confounding that arises because patients whom underwent amputation were not otherwise equivalent to patients in whom surgical revascularization was performed. Stepwise logistic regression model was employed to select patient characteristics independently associated with amputation. Variables examined in the logistic regression model were demographics (age, sex, race), dialysis modality, comorbid conditions (diabetes, coronary artery disease, congestive heart failure, cerebrovascular disease and malignancy), clinical indications (intermittent claudication, resting pain, ulcer, gangrene or other/unknown), hematocrit, nutritional status (BMI and serum albumin) and functional status (required assistance to transfer or ambulate). On the basis of the value of each independent factor multiplied by its beta coefficient, subjects were ranked with respect to their predicted probability (propensity score) of undergoing amputation versus revascularization [ 15 , 16 ]. Thirty-day and one-year mortality in amputation and revascularization groups were examined in a multivariable logistic regression model adjusted for propensity scores, demographics (age, sex, race), dialysis modality, comorbid conditions (diabetes, coronary artery disease, congestive heart failure, cerebrovascular disease and malignancy), clinical indications (intermittent claudication, resting pain, ulcer, gangrene or other/unknown), hematocrit, nutritional status (BMI and serum albumin) and functional status (requires assistance to transfer or ambulate). Similarly, we also examined thirty-day and one-year cardiovascular mortality in amputation versus revascularization groups in a multivariable logistic regression model. Differences in hospital days in the first six months post-procedure were compared between revascularization and amputation groups by analysis of covariance adjusted for variables described in the above mortality analysis. In addition, the duration of follow-up was used as a covariate to account for shorter duration of follow-up in those who died. Hospital days were log transformed to meet normality assumptions. Sensitivity analyses This study relied on ICD-9 CM codes for indications (intermittent claudication, rest pain, ulcer, gangrene or other/unknown) for amputation. As presence of gangrene is a strong indication for amputation and if ICD-9 CM codes reliably identified those patients with gangrene, a logistic regression model that included clinical indications would better predict amputations than another model that did not include clinical indications. This was tested by examining the c statistic of receiver-operator characteristic (ROC) curves of logistic regression models that included and excluded clinical indications. It could also be argued that patients who underwent amputation were treated only at advanced stage (gangrene) of peripheral vascular disease and if amputations were performed at earlier stages, the outcomes post-amputation would be superior. If advanced peripheral vascular disease (gangrene) was associated with higher likelihood of undergoing amputation, patients with lower likelihood of undergoing amputation (higher likelihood for undergoing revascularization) would have had less advanced peripheral vascular disease. Thus, examination of amputation versus revascularization outcomes in patients at low, moderate and high likelihood of undergoing amputation might reveal better outcomes post-amputation in the low likelihood group and worse outcomes in the high likelihood group. These three likelihood groups were created using the 33 rd and 66 th percentiles of propensity scores as cut-points. As procedure code 39.25 might have included upper limb revascularization procedures, analyses were repeated with including only procedure code 39.29 (proximal bypass procedures involving the aorta-iliac-femoral-popliteal vessels). Results Of the 317,533 patients initiated on dialysis from January 1, 1995 to December 31, 1995, there were 5,916 patients who underwent amputation or revascularization in first six months of initiating dialysis. Patients with missing data on demographics or comorbidity (n = 436) and nutritional status (serum albumin or BMI) (n = 1,153) were excluded. There were 279 patients who underwent amputation and revascularization procedures in the same admission. Some of these patients might have undergone revascularization first followed by an amputation in the same limb and others might have undergone amputation in one limb and revascularization in the other limb. Since ICD-9-CM codes do not provide information on which side the procedure was performed, we could not identify the sequence of events and therefore, excluded these patients. Further, amputations are also performed for indications other than peripheral vascular disease and we excluded 106 amputation patients in whom a diagnosis of peripheral vascular disease could not be established based on Medical Evidence forms or ICD-9-CM codes. Thus, 3,942 patients who underwent amputation (n = 2,046) or revasularization (n = 1,896) were studied. The mean age of the entire cohort was 70 ± 9 years, 64.2% had diabetes and 92.6% were on hemodialysis. There were 493 (12.5%) deaths within 30 days of the procedure, of which 390 (79.1%) occurred in patients with gangrene. 1,950 patients (49.5%) died in the first year following the surgical procedure. Table 1 summarizes the baseline clinical characteristics in amputation or revascularization groups. Surprisingly, amputation group was younger than the revascularization group. Compared to revascularization group, amputated group had greater prevalence of diabetes (74% v 53%), congestive heart failure (53% v 49%), cerebrovascular disease (19% v 15%) and required assistance to transfer (7% v 2%) or ambulate (17% v 6%). In multivariable logistic regression model (Table 2 ), presence of gangrene had the strongest association with amputation. Indeed, the area under the ROC curve (Fig 1a ) for the logistic regression model that included all variables except clinical indications was only 0.68 indicating low discrimination of these variables in correctly identifying patients whom underwent amputation. When clinical indication was included in the model, the area under the ROC curve (Fig 1b ) improved to 0.82 indicating excellent discrimination in identifying patients whom underwent amputation. There were 165 (8.7%) deaths within 30 days and 942 (49.7%) deaths within 1 year in the revascularization group compared to 328 (16.0%) deaths within 30 days and 1,275 (62.3%) within 1 year in the amputation group. In unadjusted logistic regression analysis, amputation group had 2 fold higher odds of dying within 30 days of the procedure compared to the revascularization group (Table 3 ). When adjusted for propensity scores and clinical variables, amputated group still had 85% higher odds of dying within 30 days (Table 3 ). In subgroup analyses by likelihood of undergoing amputation, amputation procedure was associated with the highest odds of dying within 30 days in patients with low likelihood of undergoing amputation, reduced but still significant odds in patients with moderate likelihood of undergoing amputation and non-significant increase in patients with high likelihood of undergoing amputation (Table 3 ). Similar results were seen when post-operative mortality was examined using follow-up of 1 year. After adjusting for propensity scores, demographics, dialysis modality, comorbid conditions, clinical indications, hematocrit, nutritional and functional status, the amputation group had 46% higher odds of cardiovascular death within 30 days (OR: 1.46, 95% CI: 1.25–1.71) and 18% higher odds of cardiovascular death within 1 year (OR: 1.18, 95% CI: 1.00–1.39). Clinical indication for the procedure was a predictor of post-operative mortality. There were 6 (6.2%), 12 (10.9%), 16 (6.3%), 390 (15.0%) and 69 (7.9%) deaths associated with intermittent claudication, rest pain, ulcer, gangrene and other/unknown indications respectively. In the above multivariable logistic regression model of mortality in the entire cohort, compared to intermittent claudication, presence of gangrene was associated with 54% higher odds (OR 1.54, 95% CI 1.18 – 2.01, p = 0.002) of death. Presence of rest pain, ulcer or other/unknown indications were not associated with increased risk of death in the multivariable model. The median number of days spent in the hospital were 28 in the amputation group and 26 in the revascularization group. In unadjusted analysis, these differences were significant (p = 0.003, adjusted R 2 = 0.06 for the model). However, these differences were non-significant (p = 0.33, adjusted R 2 = 0.08 for the model) when adjusted for propensity scores, demographics (age, sex, race), dialysis modality, comorbid conditions (diabetes, coronary artery disease, congestive heart failure, cerebrovascular disease and malignancy), clinical indications (intermittent claudication, resting pain, ulcer, gangrene or other/unknown), hematocrit, nutritional status (BMI and serum albumin) and functional status (requires assistance to transfer or ambulate). Among the revascularization group, 1,701 had ICD-9 CM code 39.29, 207 had ICD-9 CM code 39.25 and 12 patients had both codes. When analyses were repeated with revascularization including only ICD-9 CM code 39.29 (proximal bypass procedures involving the aorta-iliac-femoral-popliteal vessels), the results were similar (data not shown). Discussion About 19% of incident dialysis patients have a clinical diagnosis of peripheral vascular disease (PVD)[ 1 ]. In the USRDS Dialysis Morbidity and Mortality Study Waves III and IV, 6.1% of diabetic hemodialysis patients and 0.8% of non-diabetic hemodialysis patients underwent lower extremity amputation [ 3 ] over two years. When compared to general population, outcomes after revascularization or amputation for PVD in dialysis patients were inferior [ 8 , 17 ]. The problems with revascularization in PVD in dialysis patients include high perioperative and 1-year mortality, decreased wound healing, loss of limb despite patent graft and prolonged hospital stay and poor rehabilitation. Therefore, liberal use of primary amputation for peripheral vascular disease was suggested in dialysis patients by some authors [ 5 ]. However, careful patient selection for revascularization in dialysis patients might result in acceptable outcomes. Baele et al [ 6 ] reported in a series of 44 hemodialysis patients who underwent revascularization a two-year survival rate of 48%, perioperative mortality of 9%, primary graft patency at 1 year and 2 years of 71% and 63% and limb salvage at 1 year and 2 years of 70% and 52%. In that study, an aggressive approach to limb salvage was favored when patients were found to be ambulatory or able to use the affected extremity for purposes of weight bearing or transfer. Attempted limb salvage was not advocated for patients who were chronically bedridden or those with uncontrolled infection or tissue necrosis precluding a reasonable expectation of limb salvage. Thus, extensive tissue necrosis in non-weight bearing limb and pre-operative infection might be indications for primary amputation. The results of the current study (Table 2 ) show that while black race, peritoneal dialysis, diabetes and poor functional and nutritional status were associated with amputation, the strong association of presence of gangrene and younger age with amputation are the most striking. As presence of gangrene was a strong indication for amputation rather than revascularization, either by patient preference or surgical determination of high peri-operative mortality risk, older dialysis patients with gangrene might have never started dialysis or withdrawn from dialysis rather than undergo amputation. Thus, amputation might have been performed only in younger patients with gangrene and not in older patients with gangrene. This might explain the association of younger age with amputation. As shown in Figures 1a and 1b , presence of gangrene was the overwhelming factor associated with amputation. In an earlier analysis of prevalent hemodialysis patients in the Dialysis Morbidity and Mortality Study Waves 3 and 4 data, elevated serum phosphorus was also found to be a predictor of amputation [ 3 ]. Because data on serum phosphorus were unavailable in the Medical Evidence form, we could not examine the association of serum phosphorus with amputation in this study. However, a c statistic of 0.82 (Fig 1b ) indicates that the factors considered in this study (Table 2 ) explained most of the variations in the clinical decision of amputation versus revascularization. There were 8.7% deaths within 30 days in the revascularization group compared to 16.0% deaths in the amputation group. These results are comparable to earlier reports on peri-operative mortality of dialysis patients who underwent amputation or revascularization [ 10 , 12 , 18 , 19 ]. In unadjusted logistic regression analysis, amputation group had 2 fold higher odds of dying within 30 days of the procedure compared to the revascularization group (Table 3 ). There are several possible explanations for this observation. First, selection of healthier patients with better nutrition and functional status and without significant tissue damage probably resulted in lower 30-day mortality in the revascularized group. However, adjusting for the above factors did not completely eliminate this association. Second, it might be argued that the clinical indications for amputation versus revascularization could explain the differences in outcomes. However, even after using propensity scores to control for confounding by indication, amputation group still had higher mortality. In this retrospective observational study of Medicare data, unmeasured factors that influenced the decision to perform amputation might invalidate the propensity score approach. However, as noted above, a c statistic of 0.82 indicates that the factors considered in this study explained most of the variations associated with the decision to perform amputation. Thus, it is unlikely that other very significant factors that influenced the decision to perform amputation were not considered in this study. Third, it is possible that amputation was only performed in advanced stages of peripheral vascular disease and amputation in earlier stages might have resulted in superior outcomes. If this were true, as explained in the methods section, amputation would be expected to have better outcomes in patients at low likelihood for amputation. This was not the case as shown in Table 3 . In fact, amputation in patients with low and moderate likelihood for amputation was associated increased mortality and not in the high likelihood group. These results suggest that undergoing amputation rather than revascularization in healthier dialysis patients with lesser tissue damage might actually increase mortality. Thus, our results support the approach suggested by Baele et al that revascularization is feasible and might be the preferred method of treatment of symptomatic PVD in carefully selected dialysis patients. Therefore, symptomatic peripheral vascular disease in dialysis patients should not automatically result in amputation. One of the arguments in favor of amputation which results in loss of limb over revascularization that might save the limb is that revascularization is associated with prolonged hospitalization whereas amputation results in shorter hospital stay, quicker recovery and better rehabilitation. However, in this large study, the median (interquartile range) number of hospital days in amputation and revascularization groups were 28 (6–48) and 26 (14–44) days respectively. Thus, these data do not support the contention that amputation is associated with lower hospital stay. Further, in many studies only about 25% patients post-amputation were ambulatory [ 4 , 20 ]. Thus, it does not appear that amputation would result in shorter hospital stay, quicker recovery and better rehabilitation. A striking feature of the current study is the association of both revascularization and amputation with very high 30-day post-operative mortality in dialysis patients. These results emphasize the importance of preventing or slowing down the development of advanced PVD that requires surgical interventions. Therefore, in the absence of evidence to the contrary, aggressive medical therapy of PVD used in the general population should be adopted to the dialysis population. Thus, in all dialysis patients with PVD (including asymptomatic patients), smoking cessation, maintenance of LDL cholestrol < 100 mg/dl, maintenance of glycosylated HbA1c < 7.0 mg/dl in diabetics, control of blood pressure, use of ACE inhibitors and antiplatelet agents (aspirin or clopidogrel) should be considered [ 21 ]. There are several limitations to our study. First, even though the use of propensity scores might have reduced indication bias, residual bias might still exist. Therefore, all the analyses should be interpreted with caution. Second, clinical data such as the level and extent of arterial stenosis, extent of tissue necrosis, presence or absence of infection were not available. However, it might be reasonable to assume that patients with extensive tissue necrosis and severe infections underwent amputations rather than revascularization. Thus, it could still be inferred that a careful selection for revascularization of patients without severe disease might result in acceptable outcomes and save the limb. On the other hand, information on quality of life pre and post-procedures were also not available. It is possible that major amputation resulted in significant loss in quality of life and successful revascularization improved quality of life. Third, the presence or absence of gangrene was identified from ICD-9 CM codes. It is possible that the strong association of gangrene with amputation was not because gangrene was an indication for amputation rather patients whom underwent amputation were coded to have gangrene. We believe this is unlikely because 79% of all deaths within 30 days of procedure occurred in patients with an ICD-9-CM diagnosis of gangrene and it was also associated with 54% higher odds of death in the multivariable model. Fourth, ICD-9-CM codes on clinical indication were not present in 874 (22.2%) patients and thus this information is unavailable in those patients. However, as the death rate of these patients were not as high of those patients with gangrene, it is quite likely that the ICD-9-CM codes on clinical indication were not entered in patients without gangrene. Fifth, even though small, single center studies could obtain more information on primary graft patency and limb salvage, in the larger national sample of Medicare patients these data are unavailable. Thus, larger studies of national sample of Medicare patients and smaller single center studies complement each other. Finally, the limitations of this study include those of retrospective studies that use existent databases. Since only patients on Medicare defined the study population, it is possible that patients younger than 65 years who were not yet covered by Medicare were excluded introducing some selection bias. Further, the medical evidence form (form 2728) has been shown to have intermediate sensitivity but high specificity for comorbid conditions reported in the form [ 22 ]. Conclusion We conclude that amputation might carry significant morbidity and mortality in dialysis patients compared to revascularization procedures. More importantly, further studies are warranted to determine whether early diagnosis and aggressive medical therapy decrease the need for revascularization or amputation in dialysis patients with PVD. Competing interests The author(s) declare that they have no competing interests. Authors' contributions CL contributed to the conception and design of the study, interpreted the data and drafted the manuscript. NR contributed to the conception of the study and to preparation of the manuscript. LP participated in the design of the study and performed the statistical analysis. SB conceived the study, performed statistical analysis, interpreted the data and drafted the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079865

Enucleation and development of cluster headache: a retrospective study

Background Cluster headache (CH) is a neurovascular, primary headache disorder. There are, however, several case reports about patients whose CH started shortly after a structural brain disease or trauma. Motivated by a patient who developed CH 3 weeks after the removal of an eye and by similar case reports, we tested the hypothesis that the removal of an eye is a risk factor for CH. Methods A detailed headache questionnaire was filled out by 112 patients on average 8 years after enucleation or evisceration of an eye. Results While 21 % of these patients experienced previously unknown headaches after the removal of an eye, no patient fulfilled the diagnostic criteria for CH. Conclusion Our data does not suggest that the removal of an eye is a major risk factor for the development of CH.

Background Cluster headache (CH) is characterized by severe attacks of unilateral pain and cranial autonomic dysfunction [ 1 ]. The pain is usually felt in and around the orbita or adjacent areas of the head (Fig. 1 ). CH attacks last 15–180 minutes and occur, during a cluster period, from once every other day to several times a day. The symptoms of autonomic dysfunction are always ipsilateral to the pain and can involve the eye (conjunctival injection, lacrimation, miosis, ptosis, eyelid edema), the nose (nasal congestion, rhinorrhea), and the face (sweating) [ 1 ]. The exact pathophysiology of CH is, more than 250 years after the first description of episodic CH by Gerhard van Swieten in 1745 [ 2 ], still unresolved. Several convergent lines of evidence suggest that CH is a primary neurovascular disorder (for review see [ 3 ]). The hypothalamus is supposed to be involved in the pathogenesis of CH and might even be pivotal for the timing of cluster periods and single attacks [ 4 ]. Figure 1 Pain in cluster headache . The location of intense pain, usually orbital or supraorbital, in a CH attack, overlayed onto the portrait of Franz Kafka (1924). Kafka suffered from extremely severe headache attacks, possibly CH [39]. Challenging the notion of an exclusively primary headache disorder, several reports described the onset of CH during or shortly after traumatic brain injury [ 5 , 6 ] or structural brain disease (reviewed in [ 7 , 8 ]). In many of these reports, vascular disorders (e.g. arterio-venous malformations [ 9 , 10 ], aneurysms [ 11 ], dissections [ 12 ], or fistulas [ 13 ] of the cranial vasculature) were associated with the onset of CH. In other patients, intracranial neoplasms (e.g. meningeomas [ 14 ], adenomas [ 15 ]) or local inflammatory processes [ 16 , 17 ] were seen in connection with the beginning of CH. All reports on putative secondary CH face the difficult question whether there is a causal relationship or a mere coincidence between CH and the preceding brain injury or disease. The International Classification of Headache Disorders suggests to code a headache with the characteristics of a primary headache (e.g. CH) as a secondary headache if it occurs for the first time and in close temporal relation to another disorder that is a known cause of headache [ 1 ]. We follow the phenomenologic classification of the International Headache Society (IHS) [ 18 ] and regard all headaches with the symptomatology of CH and developing in a close temporal relationship to a brain injury or disease as secondary headaches. In addition to the aforementioned patients, we saw a 37-year-old man who fulfilled the IHS criteria for a secondary CH. He developed strictly right-sided episodic CH 3 weeks after the removal of his right eye bulb [ 19 ]. The patient not only fulfilled the diagnostic criteria for CH [ 1 ], but also responded to standard acute and prophylactic CH therapy. At least another 5 patients, all of them male, could be identified in the literature who developed CH, either episodic or chronic, after the removal of the eye bulb [ 19 ]. Episodic CH originated in a 45-year-old man one year after maxillectomy and exenteration due to a squamous cell carcinoma [ 20 ]. In other patients, however, CH developed up to 18 years after surgery [ 21 ]. Despite the suggestive chronology in our patient with a latency between surgery and first CH attack of only 3 weeks, it remained unclear if the removal of the eye influenced the development of CH or if both events were unrelated. To test the hypothesis that removal of an eye is a risk factor for CH we conducted a retrospective survey among individuals whose eye had to be resected. Methods To identify patients who underwent the removal of an eye, the clinical records of all inpatients admitted to the Department of Ophthalmology, Minister University Hospital between 1986 and 1995 were reviewed. In total, 332 patients had enucleation or evisceration of one eye in the specified time frame. All patients received a detailed questionnaire via mail asking about the occurrence and clinical presentation of headaches before and after the removal of the bulb (33 questions in total). The questionnaire was developed by the authors and included, amongst others, questions about the location, the quality, the duration of individual headache attacks, the occurrence of cluster periods, and the typical autonomic features of CH. The questionnaire included all diagnostic criteria for CH as described in the International Classification of Headache Disorders [ 1 ], but it was not formally validated. The patients were asked to return the completed questionnaire in a pre-paid envelope by mail. Medical records were consulted for personal data, the ophthalmologic diagnosis leading to the removal of the eye, and the operative technique used. A different aspect of this study regarding the prevalence and phenomenology of phantom experiences after removal of the eye was published previously [ 22 ]. The 37-year-old man who developed CH 3 weeks after removal of the ipsilateral eye, reported by us [ 19 ], was not included in the study population presented here because surgery was performed in an external hospital. A post-hoc analysis of statistical power was performed to assess the minimum effect size observable by the available sample size using the statistical package R for Mac OS X [ 23 ]. This analysis was based on the prevalence of CH found in two recent epidemiological studies [ 24 , 25 ]. The required statistical power was set to 0.8 and the significance level to 0.05. Results One hundred twelve patients (78 men and 34 women) completed our questionnaire and were included in the data analysis (response rate, 33.7 %). The average age at the time of eye removal was 48 ± 21 years, and the average latency between eye removal and completing the questionnaire was 8 ± 3 years (range, 3–19 years). The major reasons for removal of the eye bulb were eye trauma (n = 40, 36 %), and malignant (n = 22, 20 %) or non-malignant eye diseases (n = 50, 44 %). Enucleation (removal of the globe with sparing of the extraocular muscles [ 26 ] was performed in 104 patients (93 %), and evisceration (removal of the contents of the globe with the sclera and the extraocular muscles left intact) was done in 8 patients (7 %). After the removal of the bulb, 24 patients (21 %) experienced previously unknown headaches. Although 13 of those patients reported strictly unilateral headaches (usually ipsilateral to the removed eye bulb), the characteristic autonomic symptoms of cluster headache and the typical temporal pattern of headache attacks were absent in all of those patients. None of the patients thus fulfilled the diagnostic criteria for CH [ 1 ]. The power analysis revealed that a CH prevalence of 5.5 or more in our group of 112 patients would have resulted in a significant increase of CH prevalence (based on a population-wide CH prevalence of 56/100,000 [ 25 ]). Based on a CH prevalence of 326/100,000 [ 24 ], a prevalence of 6.2 or more would have been significant. Discussion This retrospective study on 112 patients could not identify individuals who developed CH 3 – 19 years after removal of an eye bulb. During enucleation, the predominant technique used in our patients, the globe has to be separated from all orbital tissue, including the external eye muscles and the optic nerve (Fig. 2 ) [ 26 ]. The eye is innervated by the optic nerve, the nasociliary nerve and the sympathetic and parasympathetic fibers. This led us to the hypothesis that lesions of the autonomic network might induce secondary CH in patients after enucleation [ 19 ]. Similar mechanisms have been discussed in a patient with secondary CH due to an intracranial inflammatory pseudotumor in the posterior fossa [ 27 ]. Our result, however, does not provide evidence for the notion that a first CH attack after the removal of the bulb, as observed previously [ 19 , 20 ], is triggered or at least facilitated by the irritation of trigemino-autonomic nervous structures during surgery and wound healing. The techniques of evisceration and enucleation spare parts of the orbita (especially the external eye muscles) and usually do not affect the cranial nerves III, IV, and VI. During exenteration, on the other hand, the entire orbital content has to be removed. Patients with exenteration were not included in this study because this technique is used less frequently and mainly in cancer patients (which makes longer follow-up periods difficult). Patients with exenteration might be especially prone to develop CH because this procedure results in extensive damage of nervous tissue. As CH pain is often located in or around the orbita and the autonomic symptoms of CH usually involve the eye, it has been speculated if CH can occur without an ipsilateral eye. Patients developing ipsilateral CH after the removal of an eye [ 19 ] irrespective of the temporal relationship between these two events, clearly demonstrate that the eye and the surrounding tissues are not essential for the pathogenesis of CH. Figure 2 Orbital anatomy . High-resolution axial T1-weighted native MRI of a volunteer without structural abnormalities at 3.0 Tesla. Structures removed during enucleation (globe, part of the optic nerve, insertions of the external eye muscles) are highlighted by the red ellipse. CH usually develops between 20 and 45 years [ 28 ], with a mean age of onset of about 29 years [ 29 , 30 ]. CH, however, can start much later in life. Patients with a first CH attack at the age of 83 years and 75 years have been described [ 31 ]. As the mean age in our patients (48 years) is higher than the mean age of onset of CH, one might speculate that, in many of our patients, the removal of the eye was done when they were less susceptible for the development of CH. We do not suppose that the age difference between our sample of patients after eye removal and patients with first CH attack significantly influenced the result of this study. The reports on patients who developed CH in their 70ies and 80ies demonstrate that higher age does not prevent the development of CH. Moreover, many patients with secondary CH were older than the average age of onset of primary CH. The age of onset of the 5 patients with a first CH attack after removal of the eye was 36 years [ 19 ]. The presented study, however, has its limitations. The retrospective, questionnaire-based design might underestimate the prevalence of CH. Descriptions of the main symptoms of CH, however, either in questionnaires or letters, have proven to be useful in several epidemiological studies on CH [ 25 , 32 - 36 ] and take advantage of the distinct diagnostic criteria and the impressive nature of CH. The use of short self-adminstered questionnaires for screening larger populations has also been validated in migraine [ 37 , 38 ]. Our study was designed to screen our population via a questionnaire and then to verify each probable diagnosis of CH with a detailed interview and a comprehensive neurological examination (no patient, however, reported the main features of CH, short attacks of unilateral, severe headaches in bouts). Individuals with infrequent and mild bouts, however, might not recall past CH attacks while responding to a questionnaire. In addition, the relatively low response rate (34 %) might have influenced the present results. On the one hand, patients with CH might be hidden in the group of individuals not responding to our questionnaire. On the other hand, patients with CH are probably more willing to respond to a survey targeted at their symptoms than patients with other forms of headache or without headaches at all. In general, a prospective study which includes patients before the removal of an eye, follows them over years and then compares the outcome to a control group (i.e., a cohort study) would be more effective for the study of a disease's risk factors. In addition, CH is rare with a prevalence of less than 1 %. Investigating possible risk factors for rare disorders requires large sample or effect sizes. Our sample size is large enough to yield statistical significance for a CH prevalence of 5.5 % in our group (or 6.2 %, depending on the assumed population-wide CH prevalence), but might be too small to detect a slight increase of CH among patients after removal of an eye. Conclusion Our data does not suggest that the removal of the globe is a major risk factor for the development of CH. We cannot exclude the possibility that enucleation is associated with a small increase in CH prevalence. Authors' contributions All authors participated in designing the study and constructing the headache questionnaire. PS prepared the manuscript and performed the statistical analyses. OV contacted the patients, collected the data, and assisted with data analysis. HG participated in data collection, interpretation of the results, and drafting the manuscript. IWH and SE participated in interpretation of the results and in preparing the manuscript. All authors have read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079866

Variation in the cortical area map of C57BL/6J and DBA/2J inbred mice predicts strain identity

Background Recent discoveries suggest that arealization of the mammalian cortical sheet develops in a manner consonant with principles established for embryonic patterning of the body. Signaling centers release morphogens that determine regional growth and tissue identity by regulating regional expression of transcription factors. Research on mouse cortex has identified several candidate morphogens that affect anteroposterior or mediolateral cortical regionalization as well as mitogenesis. Inbred strains of laboratory mice can be exploited to study cortical area map formation if there are significant phenotypic differences with which to correlate gene polymorphism or expression data. Here we describe differences in the cortical area map of two commonly used inbred strains of laboratory mice, C57BL/6J and DBA/2J. Complete cortical hemispheres from adult mice were dissected and stained for the cytochrome oxidase enzyme in order to measure histochemically defined cortical areas. Results C57BL/6J has the larger neocortex, relatively larger primary visual cortex (V1), but relatively smaller posterior medial barrel subfield of the primary somatosensory cortex (PMBSF). The sample of C57BL/6J and DBA/2J mice can be discriminated with 90% accuracy on the basis of these three size dimensions. Conclusion C57BL/6J and DBA/2J have markedly different cortical area maps, suggesting that inbred strains harbor enough phenotypic variation to encourage a forward genetic approach to understanding cortical development, complementing other approaches.

Background Species differences in cortical regionalization reflect genetic and epigenetic developmental programs that are presumed adaptations to different ecological niches. For example, Krubitzer and Kahn [ 1 ] review that the mouse, ghost bat, and short-tailed opossum have approximately the same size cortical sheet, but differ substantially in the size of one of three primary sensory cortical areas. The enlarged cortical area in each species reflects a greater behavioral reliance on the represented sensory modality. In the same sensory modality, functional specialization of the sensory periphery is also reflected in the primary cortical area size. Catania and Remple [ 2 ] show that the naked mole-rat, a fossorial species dependent on somatosensation, has a much greater cortical surface area devoted to somatosensation compared to the laboratory rat. As discussed by Krubitzer and Kahn [ 1 ], although differences in genetic determination are well implicated by these species differences, identity of the genes and their developmental actions are not well understood. Recent experimental manipulations in mice have caused striking qualitative and quantitative changes in the cortical area map, identifying several candidate morphogens that affect anteroposterior or mediolateral cortical regionalization as well as mitogenesis [ 3 ]. For example, Fukuchi-Shimogori and Grove [ 4 ] placed an ectopic caudal source of fibroblast growth factor 8 into the developing mouse cortex and caused a caudal duplication of part of the primary somatosensory area. Hamasaki et al. [ 5 ] demonstrated changes in the position and or size of primary sensory visual, somatosensory, and auditory cortical regions in transgenic mice over expressing the transcription factor Emx2 . These and other discoveries (reviewed by [ 1 , 3 ]) suggest that arealization of the mammalian cortical sheet develops in a manner consonant with principles established for embryonic patterning of the body. Signaling centers release morphogens that determine regional growth and tissue identity by regulating regional expression of transcription factors. Grove and Fukuchi-Shimogori [ 3 ] note that such research has provided a starting point for investigating how the cortical area map is generated and modified in a single individual and how maps change in the course of evolution, but that a major step forward would be to identify novel transcription factors involved in cortical area patterning. Along these lines, Funatsu et al. [ 6 ] have employed gene expression array analysis of the dissected embryonic (16.5d) mouse cerebral cortex to expand the list of genes regionally expressed, and noted that regional differences in expression of genes in the cortical plate should eventually convert into functionally distinct cortical areas with anatomically distinguishable borders after birth. Inbred strains of laboratory mice can be exploited to study and understand complex traits of the nervous system if there is significant phenotypic variation with which to correlate gene polymorphism [ 7 - 9 ] or expression data [ 10 ]. As the first step to an integrative and relational discovery program [ 11 , 12 ] in a model system for mammalian cortical area map formation, here we describe significant differences in the cortex of two common inbred strains of laboratory mice, C57BL/6J and DBA/2J. Results Neocortex, visual cortex, and barrel cortex differ between C57BL/6J and DBA/2J Using established histochemical methods to visualize neocortical and primary cortical areas (see Methods and Figure 1 ), we estimate that neocortex (C) is on average 7% larger in C57BL/6J (38.0 mm 2 ) compared to DBA/2J (35.5 mm 2 ) (F 1,28 = 4.25, P = 0.049). While both visual cortex and barrel (PMBSF) cortex areas significantly correlate with total neocortex area (r V 1, C = 0.38, P = 0.039; r PMBSF , C = 0.47, P = 0.009), each also uniquely differs between strains. Using ANCOVA to control for variation in neocortex size, the adjusted mean visual cortex areas for C57BL/6J and DBA/2J are 3.69 and 3.30 mm 2 , respectively, a 12% difference (F 1,27 = 4.51, P = 0.043). Using ANCOVA, the neocortex adjusted mean barrel cortex areas for DBA/2J and C57BL/6J are 2.16 and 1.97 mm 2 , respectively, a 10% difference (F 1,27 = 14.72, P < 0.001). These results are shown graphically in Figures 2 and 3 . In Figure 2 , the ANCOVA plot shows that C57BL/6J mice have more visual cortex; the C57BL/6J linear fit is above the DBA/2J linear fit. In Figure 3 , the ANCOVA plot shows that DBA/2J mice have more barrel cortex; the DBA/2J linear fit is now above the C57BL/6J linear fit, reversed from Figure 2 . There is no significant evidence for heterogeneity of slopes in the fitted lines from Figure 2 or Figure 3 when interaction terms are added to the ANCOVA models. Cortical field size configuration predicts C57BL/6J and DBA/2J strains Given the significant differences in neocortex, visual cortex, and barrel cortex areas between C57BL/6J and DBA/2J, we can ask how well these measures collectively predict or discriminate strain identity. In a logistic regression model predicting strain identity from neocortex, visual cortex, and barrel cortex areas, the overall model is significant (likelihood ratio χ 2 (3 df) = 25.45, P < 0.001; Hosmer-Lemeshow goodness-of-fit χ 2 (8 df) = 1.58, P = 0.99), as is neocortex area (likelihood ratio χ 2 (1 df) = 10.32, P = 0.001), visual cortex area (likelihood ratio χ 2 (1 df) = 9.86, P = 0.002), and barrel cortex area (likelihood ratio χ 2 (1 df) = 17.89, P < 0.001). Table 1 shows the prediction table for this model, revealing 27 out of 30 mice (90%) were correctly classified by strain. Figure 4 and Figure 5 graphically portray these results. Figure 4 plots the predicted probabilities from the logistic regression model by strain. Figure 5 plots a projection from a three dimensional rotating plot for neocortex, visual cortex, and barrel cortex. When rotated to the projection shown, a plane (or line) separates the strains with 90% accuracy. C57BL/6J and DBA/2J strains are not differentiated on other dimensions Neither the area of somatosensory cortex (S1) taken as a whole, nor auditory cortex (A1) area was found to be significantly different between strains. Subjectively, we found the dorsal border of S1 (upper lip, forepaw, hindpaw, and trunk representations) and the borders of A1 to be more difficult to distinguish in our tissue than either V1 or PMBSF. Neither S1 nor A1 area measures added significantly to the ability to predict strain identity. Discussion Grove and Fukuchi-Shimogori [ 3 ] and Krubitzer and Kahn [ 1 ] suggest that the cortical map, at least for primary areas, develops independently of thalamic input, by way of signaling centers releasing morphogens that determine areal identity. This is not a claim that thalamic projections to the cortex do not play a role in cortical area map formation [ 1 , 13 ], but rather that principles of development observed in body embryogenesis are also active in early cortical area map formation. Developmental and genetic manipulations have produced striking evidence for a handful of candidate morphogens in cortical area map formation [ 4 , 5 , 14 ]. Recent gene array expression studies are expanding the list of genes that may act to pattern the mammalian cortex [ 6 ]. To date, there is little evidence these candidate morphogens cause individual or species differences. An approach that ties candidate or novel morphogens to cortical area map development and anatomy within the range of normal individual differences would prove complementary. Complex trait analysis of the mouse central nervous system [ 11 ], allied with gene expression approaches [ 10 ], can be used with recombinant inbred strains of mice [ 15 ] to provide a cumulative, integrative discovery program [ 12 ] that has the potential to tie genomic and transcriptomic variation to variation of the central nervous system and the behaving organism. Here we provide evidence that the cortical area map differs significantly even in two inbred strains of laboratory mice, C57BL/6J and DBA/2J. Importantly, these are the parental strains of the BXD recombinant inbred strains [ 15 ], and this study thus provides an empirical basis for using this mammalian neurogenetic resource to study cortical development. In this paper we have shown that neocortex, visual cortex, and barrel cortex differ in area between C57BL/6J and DBA/2J inbred strains of mice, and that collectively, these measures accurately discriminate these strains. The relatively greater barrel cortex representation in DBA/2J confirms an earlier abstract reporting greater representation of the barrel field in DBA/2J compared to C57BL/6J mice [ 16 ]. The larger neocortex in C57BL/6J is consistent with the larger brain size in this strain [ 8 ]. The dimension of area is one way to measure the cortical map. Other experimental studies have shown changes in field duplication [ 4 ], number [ 17 ], or position [ 5 ]. In future studies of either BXD recombinant inbred lines, or of other commercial inbred strains [ 18 , 19 ] or their derivatives, we suggest that more powerful statistical descriptions of the size and shape of the cortical area map could be used. Borrowing from advances in the field of geometric morphometrics [ 20 ], methods that have been applied to the genetic architecture of the Drosophila wing shape [ 21 ], or mouse mandible shape [ 22 ], could be applied to the mouse cortical area map, with landmarks defined by classical histochemical or immunohistological stains or by other molecular markers. Landmark-based shape statistics are not a panacea for the measurement of biological form [ 23 ], but the point with regard to using isogenic strains is that landmarks can be investigated by replicate measures for reliability and can be correlated with genomic data, or transcriptomic data, at a particular developmental milestone, or across milestones. This is a promising research direction that would complement efforts to answer how the cortex develops, what are the functional or dysfunctional consequences for a particular cortical configuration, and even how the cortex has evolved or can evolve [ 24 , 25 ]. Conclusion Inbred strains of laboratory mice can be used to investigate mammalian cortical area map formation if there is significant phenotypic variation with which to correlate gene polymorphism or expression data. Adult C57BL/6J and DBA/2J mice are markedly different in cortical area maps, suggesting that inbred strains harbor enough phenotypic variation to encourage a forward genetic approach to understanding cortical development, complementing other approaches. Methods Animals Mice were purchased from Jackson Laboratories at 4–6 weeks of age, housed on a 12:12 light:dark cycle in same sex groups in standard laboratory animal cages (5 animals per cage). All experimental procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health (publication 86-23) and the Vanderbilt University Animal Care and Use Committee. Mice were provided a standardized diet and clean water ad libitum until they were killed. The cortices of thirty young adult (6–8 weeks of age) mice were measured in this study (14 C57BL/6J, 16 DBA/2J). Mice at this age are not compromised by known visual and or auditory sensorineural deficits common to older animals of these strains. None of the mice used had visible body or facial wounds. A pilot study of 10 older aged C57BL/6J mice indicated drawings of cytochrome oxidase material were reliable for the cortical measures reported here, in that significant animal differences could be detected within strain, and that neither sex nor hemisphere main effects were significant. In the sample of 30 young adult mice reported here, both sexes were sampled, but sex effects were not detected. Hemisphere measures were averaged by animal when two hemispheres were measured. No age effects were associated within the narrow age span sampled. All measurements were made while blind to strain and animal identity. Cortex Mice were brought to complete anesthesia with a sodium pentobarbital overdose (100 mg/kg) injected intraperitoneally (IP), and then transcardially perfused with 0.1 M phosphate buffered 0.9% saline wash followed by 3% buffered paraformaldehyde fixative. Intact brains were removed from the skull, and the cortex was dissected free of the underlying white matter. Dissected cortices were transferred to 30% sucrose, and flattened between glass slides for 12 hours. Cortices were sectioned parallel to the cortical surface at a thickness of 80 μ m on a freezing, sliding microtome, stained for cytochrome oxidase according to the method of Wong-Riley [ 26 ], mounted on glass slides, air dried, and coverslipped. Measurements The outlines of five regions of interest were drawn under a light microscope with a camera lucida attachment. Regions of interest included neocortex (C), visual cortex (V1), auditory cortex (A1), somatosensory cortex (S1), and the posterior medial barrel subfield (PMBSF) (see Figure 1 ). Barrel rows A, B, C, D, and E were collectively bounded for the PMBSF, and standardized to 5, 4, 6, 7, and 8 barrels, respectively (alpha, beta, gamma, and delta barrels were also included). Digital scans of the drawings were imported into a computer and area measures (mm 2 ) acquired with NIH ImageJ software . Statistics Analysis was done in the Stata/SE 8.2 statistics, graphics, and data management software package , and consisted of graphic plots and descriptive statistics followed by inferential statistics. We used analysis of variance (ANOVA) and analysis of covariance (ANCOVA) to test differences in means between strain, and logistic regression models to predict strain identity. Diagnostic plots and statistics were investigated to validate model assumptions in each case. Alpha was set to 0.05 for statistical significance. Asking if there is a difference in cortical field size between strains when the field correlates with total neocortex size, it is reasonable to consider forming a relative index of size, such as a proportion or percent (ratio) measure. Use of a ratio measure may fail to control brain size [ 27 ]. An alternative to forming a ratio is to use ANCOVA, which adds a covariate to an ANOVA model to statistically control that covariate. For strain prediction, we chose logistic regression rather than discrminant analysis. Both methods produce the same prediction table with our data, while logistic regression carries fewer assumptions and is likely more familiar to the reader. The projection of a rotating plot for neocortex, V1, and PMBSF, was produced in the Data Desk 6.2 statistics, data mining, and visualization software package . Authors' contributions DCA and CEC conceived and designed the study. DCA, CEC, AIR, KME, and FW collected data. DCA analyzed the data and prepared the manuscript.

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1079867

The role of the cytoskeleton in cell body enlargement, increased nuclear eccentricity and chromatolysis in axotomized spinal motor neurons

Background When spinal motor axons are injured, the nucleolus, nucleus and cell body of the injured cell transiently increase in size, the nucleus becomes more eccentrically placed, and the organization of polyribosomes into Nissl bodies is temporarily disrupted. The mechanisms for these classical morphological responses to axotomy have not been satisfactorily explained. Results In this study we address the role of the cell body cytoskeleton in these structural changes. We show that the cytoskeleton of uninjured lumbar motor neuron cell bodies maintains nucleolar, nuclear and cell body size and nuclear position. When isolated, the relatively insoluble cell body cytoskeleton contains Nissl bodies and lipofuscin granules. After axotomy, protein labeling increases markedly and the cytoskeleton enlarges, increasing nucleolar, nuclear and cell body size, as well as nuclear eccentricity. Nearly all of the protein mass that accumulates in the cell body after axotomy appears to be added to the cytoskeleton. Conclusion We conclude that axotomy causes the conjugate enlargement of the nucleolus, nucleus and cell body and increases nuclear eccentricity in spinal motor neurons by adding protein to the cytoskeleton. The change in nuclear position, we propose, occurs when cytoskeletal elements of the axon cannot enter the shortened axon and "dam up" between the nucleus and axon hillock. As a consequence, we suggest that Nissl body-free axonal cytoskeleton accumulates between the nucleus and axon, displaces Nissl body-containing cytoskeleton, and produces central chromatolysis in that region of the cell.

Background Injury to the axon of spinal motor neurons produces major structural changes in the affected cell body. These changes include a transient enlargement of the nucleolus, nucleus and cell body, an increase in nuclear eccentricity and central chromatolysis, which is a centrifugal disappearance of polyribosome-containing Nissl bodies. While known and explored for over a century, these morphological responses to axotomy have not been adequately explained. It has been proposed that enlargement of the motor neuron cell body after axotomy is caused by the entry of water into the cell bodies, possibly the result of an early increase in osmotically-active hydrolytic products of macromolecules within the motor neurons [ 1 ]. On the other hand, the gradual increase in total protein and RNA observed in axotomized motor neurons cell bodies [ 2 , 3 ] could also play a role in their enlargement after injury. That the nucleolus, nucleus and cell body enlarge together after axotomy suggests that their sizes may be coordinated, especially since a similar scaling is observed in normal, non-injured motor neurons of increasing size [ 4 ] and in motor neurons exposed to excess growth hormone [ 5 ]. It is noteworthy that the nucleus, nucleolus and cell body do not each enlarge after axotomy in all types of neurons capable of axon regeneration, implying that an increase in size may not be required for regrowth of the axon [ 6 ]. The repositioning of the nucleus in injured motor neurons was quantified by Barr and Hamilton [ 1 ], who, like others [ 7 , 8 ], found that the direction of increased nuclear eccentricity in motor neurons was usually away from the axon hillock. Suggested mechanisms for increased nuclear eccentricity include a selective influx of water into the area of the axon hillock [ 1 ] and interference with axonal transport, leading to the accumulation of axonal constituents in the injured cell body [ 6 ]. Central chromatolysis also develops progressively in the region between the nucleus and axon hillock after axotomy [ 6 ]. The gradual disappearance of Nissl bodies from the perinuclear region towards the periphery of the cell body and the concomitant loss of basophilic staining of RNA occur even as the radiolabeling and total content of RNA increase [ 6 ]. Neither the molecular basis of chromatolysis nor the reason for its appearance and centrifugal spreading between the nucleus and axon hillock is known. In this study, we first show that the size and shape of normal, non-injured frog motor neuron cell bodies are maintained by a cytoskeleton that can be isolated. We then provide evidence that axotomy increases the synthesis and total content of proteins in the cytoskeleton, which appears to alter its structure and produce nucleolar, nuclear and cell body enlargement. We further show that the cytoskeleton maintains nuclear position in the uninjured cell body and is altered by axotomy to increase nuclear eccentricity. Finally, we propose that both nuclear eccentricity and central chromatolysis result from an accumulation of axonal cytoskeleton that is impeded from entering the truncated axon. Results The cytoskeleton maintains nucleolar, nuclear and cell body size in normal motor neurons Isolated, unfixed motor neurons, like fixed, sectioned motor neurons, vary widely in cell body area [ 5 , 9 ]. The range of cell body areas in motor neurons isolated from normal animals by the method of Sinicropi and McIlwain [ 10 ] is slightly larger than that of their fixed, sectioned counterparts [ 5 ]. As with area measurements on normal motor neurons [ 5 ], nucleolar and nuclear volume increase as cell body volume increases (Fig. 1 ). Since the plasma membrane is damaged during the isolation of motor neuron cell bodies and is no longer semipermeable to most osmolytes [ 11 ], osmotic forces do not appear to be chiefly responsible for maintaining cell body size in isolated motor neurons. Even when intracellular membranes are permeabilized with 1% Triton X-100 and over one-half of the cell body protein is removed from isolated motor neurons by successive extraction and high salt solutions, the mean size of the nucleolus, nucleus and cell body is little affected (Table 1 ). This extraction procedure, which has been used to isolate the nuclear matrix and cytoplasmic skeleton of non-neuronal cells [ 12 ], reduces the cell body protein content in normal frog motor neurons from 4.35 ± 0.87 to 1.88 ± 0.29 ng/cell (a 56.8% decrease). In contrast, cell body and nuclear volume decline only by 15 and 17%, respectively, and nucleolar volume does not change significantly (Table 1 ). The volumes of the nucleolus and nucleus in the extracted motor neurons continue to scale with cell body volume (Fig. 1 ) and the extracted cells have a relatively normal appearance by differential interference contrast microscopy (Fig. 2 ). These findings suggest that nucleolar, nuclear and cell body size in motor neurons are maintained by cytoskeletal structures and led us to examine the ultrastructure of extracted motor neurons. Resinless thin sections [ 12 ] were prepared from frog lumbar spinal cord that had been extracted in situ by the same procedure used for isolated cell bodies. Isolated, extracted cell bodies were not used, because they produce friable thin sections with this embedment procedure. Transmission electron microscopy of motor neurons in unstained thin sections from which the embedment medium – diethyleneglycol distearate (DGD) – had been removed reveals a three-dimensional network of filamentous structures that remains after extraction of soluble proteins (Fig. 3 ). The nuclear skeleton of motor neurons is much less densely organized than the cytoplasmic skeleton, which is a highly cross-linked network of filamentous proteins with electron dense particles – possibly ribosomes [ 13 ] – interspersed throughout the lattice. The filamentous struts of the lattice are tapered, suggesting the possibility of artifactual deposition of some protein onto the lattice. These images, together with the morphometric analyses described above, indicate that the cytoskeleton is largely responsible for maintaining nucleolar, nuclear and cell body size in motor neurons. An interesting aspect of these data is the influence of the embedment medium in thin sections, which is illustrated in Fig. 4 . The three-dimensional lattice seen when the embedding medium is removed is completely invisible when the embedding medium is present (Fig. 4 , middle panel) and differs greatly from the two-dimensional image of stained cells embedded in plastic (Fig. 4 , right panel), which masks much of the underlying filamentous structure. The obscuring influence of the embedding resin has been discussed by Penman [ 14 ]. Nissl bodies and lipofuscin are associated with the isolated cytoskeleton Motor neurons isolated from the grass frog stain with basophilic dyes before and after extraction, indicating the presence of ribosomes. However, they do not contain well-defined Nissl bodies, such as those seen in human motor neurons. To determine whether Nissl bodies are associated with the isolated cytoskeleton, we examined cell body cytoskeletons isolated from human motor neurons and stained with methylene blue. Confocal images of methylene blue-stained cytoskeletons from normal human motor neuron cell bodies showed abundant Nissl bodies in the cell body and proximal dendrites (Fig. 5 ). As with isolated frog motor neurons, the appearance of isolated human cell bodies is relatively unchanged after extraction. Adult human motor neurons, unlike those of frog, also contain large amounts of lipofuscin, and cytoskeletons isolated from human motor neuron cell bodies retain their lipofuscin (Fig. 5 ). The possibility that lipofuscin granules are trapped within the interstices of the cytoskeleton of the cell body and dendrites became apparent when isolated human cell bodies being examined by differential interference contrast microscopy (DIC) were exposed to SDS. When concentrations of up to 1% of the detergent were introduced at the edge of the coverslip, the cell bodies often began to swell and, one by one, individual lipofuscin granules were released and floated away from the enlarged cell body. The granules had average diameters of about 2.5 μm (range 1.8–3.0 μm, n = 16) and appeared to be insoluble in SDS. Even after depletion of most of the cell's lipofuscin, the swollen motor neuron was sometimes still visible by DIC. Lipofuscin was released only from cell bodies that underwent swelling. This sequence of events led us to infer that lipofuscin granules may be caged within the cell body cytoskeleton and can individually escape it as the lattice enlarges to pore sizes that exceed the diameter of each granule. Although the swollen cell bodies eventually became invisible by light microscopy, the cytoskeleton may not be completely soluble in SDS. Approximately one-third of the cell body protein (31.0 ± 15.1%, n = 5) was recovered in the pellet when spun at 13,000 × g for 5 min in the presence of 1% SDS. This may explain our inability thus far to detect and identify any proteins in the isolated cytoskeleton by SDS-polyacrylamide gel electrophoresis. Axotomy enlarges the cell body cytoskeleton Transection of motor axons in the frog lumbar ventral root causes progressive enlargement of the nucleolus, nucleus and cell body of the injured motor neurons [ 4 ]. Given the evidence above that the cytoskeleton largely maintains normal nucleolar, nuclear and cell body size, one would postulate that axotomy increases their size by altering the cytoskeleton. This possibility was tested in morphometric analyses of cytoskeletons isolated from motor neuron cell bodies of frogs 16 days following ventral root transection, when injury-induced enlargement was well underway. As in normal motor neurons, the cytoskeleton of axotomized cells maintains most of nucleolar, nuclear and cell body size (Table 2 ). Scaling of nucleolar, nuclear and cell body sizes after injury was also evident in both unextracted and extracted cell bodies (Fig. 6 ). By postoperative day 16, mean nucleolar, nuclear and cell body volumes had increased 222%, 148% and 145% in the isolated cytoskeletons, respectively, versus 254%, 159% and 164% in unextracted cell bodies (Tables 1 & 2 ). Dividing the injury-induced increases in volume in extracted cells by those in unextracted cells, one finds that the enlargement of the cytoskeleton accounted for 91% of the increase in nucleolar size, 67% of nuclear size, and 60% of cell body size. The injury-induced expansion of the motor neuron cell body is not uniform in all dimensions. In both unextracted and extracted cell bodies, axotomy enlarged the x, y axis much more than the z-axis of the cell. For example, in 5 experiments on a total of 145 unextracted, 16-day axotomized cell bodies, the mean radius increased 26.6 ± 4.5% after injury (p < 0.01; unpaired Student's t-test), while the mean height (z-axis) of the cells increased by only 7.3 ± 8.4% (p = n.s.). Similar differences were found in cytoskeletons isolated from 16-day axotomized cell bodies. Axotomy adds protein selectively to the cell body cytoskeleton Following ventral root transection, the total protein content of the injured motor neuron cell body increases steadily with increasing cell body volume (Fig. 7 ). When we compared the protein content of unextracted cell bodies axotomized 16 days earlier to that of their isolated cytoskeletons, we found that virtually all of the protein added to the cell body after axotomy was recovered in the isolated cytoskeleton (Table 3 ). These data imply that the addition of protein to the cell body cytoskeleton is responsible for its enlargement after axotomy. When normal motor neurons were labeled in situ with 3 H-leucine and their cell bodies isolated and extracted, slightly over one-half of the newly synthesized protein in normal motor neuron cell bodies was recovered in the isolated cytoskeleton (Table 4 ). Axotomy increased protein labeling six-fold in both unextracted cell bodies and their isolated cytoskeletons by postoperative day 16, and again about one-half of the total labeled protein was recovered in the isolated cytoskeleton (Table 4 ). Thus, in normal and axotomized cell bodies about one-half of the newly synthesized protein is not found in the isolated cytoskeleton. However, only new protein added to the cytoskeleton after axotomy is associated with the increase in total protein content of the cell body (Tables 3 and 4 ). Newly synthesized protein not recovered with the cytoskeleton, although elevated by injury, does not appear to contribute measurably to the increase in total protein content of the cell body after axotomy, indicating that it is exported and/or degraded more rapidly than most protein in the isolated cytoskeleton. Axotomy increases nuclear eccentricity by altering the cytoskeleton Based upon evidence that the cytoskeleton maintains nuclear location in non-neuronal cells [ 15 ], we analyzed nuclear position in cytoskeletons isolated from non-injured, extracted motor neuron cell bodies. Extracted frog motor neurons showed the same degree of nuclear eccentricity found in unextracted cell bodies (Table 5 ). Approximately 10% of the normal cell bodies, whether extracted or unextracted, had nuclei that contacted the cell periphery (100% eccentricity). After ventral root transection, nuclear eccentricity in the isolated cytoskeleton increased to the same degree as it did in unextracted cell bodies (Table 5 ). Approximately 60% of the injured cell bodies had nuclei that touched the cell periphery, whether or not they were extracted. Thus, the cytoskeleton maintains nuclear position in normal motor neurons, as it does nuclear volume, and is altered by axotomy to produce an increase in nuclear eccentricity. Discussion The cell body cytoskeleton in normal spinal motor neurons The ability to isolate individual neuronal cell bodies simplifies volumetric measurements of the entire cell body and its cytoskeleton and permits one to quantify their total protein content and radiolabeling. The isolated cell body cytoskeleton retains the shape and most of the size of the unextracted motor neuron. It maintains the size relationships among the nucleolus, nucleus and cell body over a wide range of cell body volumes and fixes the position of the nucleus within the cell. The morphological characteristics of the isolated cytoskeleton are so typical of motor neurons in situ , that it is difficult to conceive that the cytoskeletons we isolate do not exist in the cells from which they are derived. However, our experimental procedures do cause changes in the living cell bodies. The somewhat smaller volumes of the nucleus and cell body in isolated cytoskeletons (Tables 1 and 2 ) and the tapered appearance of its filaments (Fig. 3 ) probably reflect those changes. Nissl bodies might not be associated with the cytoskeleton in vivo , although cytoskeletons isolated in a similar way from non-neuronal mammalian cells also contain polyribosomes [ 13 , 16 , 17 ], and in plant cells, labeled polyribosomes added prior to the extraction procedure were not adsorbed by the cytoskeleton [ 18 ]. Role of the cytoskeleton in the conjugate enlargement of the nucleolus, nucleus and cell body after axotomy The data in this study form the basis for a new explanation for the events leading to the transient enlargement of the nucleolus, nucleus and cell body after axotomy. We find that axonal injury adds protein to the cytoskeleton, likely enlarging the cell body, as well as the nucleus and nucleolus, which are integral parts of its filamentous structure. The injury-induced accumulation of proteins in the isolated cytoskeleton implies that their rate of synthesis exceeded their rate of loss from the cell body. This condition could result from an increase in their rate of synthesis, a decrease in their rate of degradation or export from the cell body or a combination of these factors. The injury-induced increase we observed in labeled protein in the isolated cytoskeleton does not distinguish among these possibilities. However, the rate of total protein synthesis in frog motor neurons is probably increased after axotomy, since we have previously described multiphasic increases in total transcription during this postoperative period [ 4 ]. It is unclear how much of that increase is directed towards proteins in the cytoskeletal and non-cytoskeletal fractions, but the fact that each of these two fractions receives about one-half of the total label both before and after axotomy suggests that the rate of protein synthesis increases in both fractions. We can also infer that the relationships between protein synthesis, degradation and export differ in the cytoskeletal and non-cytoskeletal fractions of the injured motor neuron cell bodies. As noted, the increase in labeled protein in the isolated cytoskeleton is accompanied by an increase in protein mass, indicating that the rate of addition of new protein to the cytoskeleton is not matched by an increase in the rate of protein loss from it. In contrast, the injury-induced increase in labeled protein that is not associated with the isolated cytoskeleton produces no significant protein accumulation in that fraction, indicating equal rates of protein synthesis and loss after axotomy. These observations illustrate once again why it is unsafe to use labeled protein as an indicator of protein mass. Are neurofilaments and peripherin major determinants of cell body size and shape in motor neurons? An intriguing question raised by our study pertains to the identity of the cytoskeletal constituents of the filamentous lattice in spinal motor neurons. On the one hand, there is evidence that cell body size in neurons can be changed by manipulating its intermediate filament content. For example, increased cell body size in transgenic rat motor neurons overexpressing the high molecular weight component of neurofilaments (NF-H) can be reduced by concurrrently increasing the expression of the low molecular weight component (NF-L) [ 19 ]. Likewise, cell body length in differentiated PC-12 cells changes when peripherin is inhibited by transfecting the cells with small interfering mRNAs [ 20 ]. Conversely, axotomy-induced cell body enlargement is accompanied by increases in the cell body content of NF-L in frog motor neurons [ 10 ] and increased peripherin immmunoreactivity in rat spinal motor neurons [ 21 ]. Unlike neurofilament protein, peripherin mRNA also increases after axotomy [ 21 ], a finding consistent with the abovementioned possibility of an increased rate of synthesis of cytoskeletal proteins in our study. However, if neurofilaments or peripherin are constituents of the isolated cytoskeleton, we predict that they are cross-linked (Fig. 3 ) or otherwise modified so as to reduce their solubility. On the other hand, there is evidence that neither neurofilament proteins nor peripherin are responsible for maintaining cell body size and shape in spinal motor neurons. In particular, Jacomy et al. [ 22 ] have reported that spinal motor neuron cell bodies in transgenic mice lacking the high and middle molecular weight neurofilament subunits are incapable of producing neurofilaments, but still retain their normal shape. Likewise, Larivière et al. [ 23 ] found that motor neurons in transgenic mice lacking peripherin also have normal-appearing cell bodies. Although compensations for losses of the individual proteins could conceivably preserve cell body structure, these observations call into question whether neurofilaments or peripherin are essential for much of the shape of normal motor neuron cell bodies. The identification of the major structural proteins in the cytoskeleton of motor neuron cell bodies will depend upon how amenable its proteins are to current modes of protein compositional analysis. Role of the cytoskeleton in increased nuclear eccentricity and central chromatolysis after axotomy We also show that changes in the cell body cytoskeleton appear to be responsible for the increase in nuclear eccentricity following axonal injury. An asymmetric addition of protein to the cytoplasmic skeleton cannot alone account for the change in nuclear position, because the fraction of axotomized cells with nuclei touching the periphery of the cell body (100% eccentricity) is about six times that found in normal cells. Thus, axotomy not only enlarges the cytoskeleton, but also causes dissolution of the lattice on one side of the cell. Additional clues to the mechanism by which the nucleus is relocated in the cell are found in the direction of nuclear displacement and the location of chromatolysis after axotomy. As mentioned, several investigators have reported that the direction of nuclear displacement is away from the axon hillock [ 1 , 7 , 8 ]. We did not test this directionality here, because the axon is routinely lost during cell body isolation. Many others have reported that the loss of Nissl bodies ( i.e., chromatolysis) also appears first in the region of the cell between the nucleus and the axon hillock [ 6 ]. If Nissl bodies are associated with the cell body cytoskeleton in vivo , as they are in the isolated cytoskeleton, then one must ask why Nissl bodies are not present in the cytoskeleton added between the nucleus and axon after axotomy. There is no reason to believe that axotomy causes Nissl bodies to be removed only from the cytoskeleton added to that particular region of the cell. A more plausible explanation is that when the axon, which does not contain Nissl bodies [ 24 ], is drastically shortened by axotomy, it cannot incorporate all of the axonal cytoskeleton being synthesized by the injured cell body. Injury-induced impairment of axonal transport could exacerbate this problem. The excess of Nissl body-free axonal cytoskeleton could then accumulate between the nucleus and axon hillock, causing chromatolysis and further enlargement in that particular region of the cell body, thereby contributing to increased nuclear eccentricity away from the axon hillock. It could also act as a force for disassembly of the cytoplasmic skeleton on the opposite side of the nucleus. Other evidence exists for "damming up" of cytoskeletal proteins destined for the axon. We have previously shown that axotomy increases the content of tubulin and neurofilament protein in the injured cell body, but decreases their content in the axon proximal to the injury site [ 10 ]. That study also showed that the volume of the axon, which is especially dependent upon neurofilaments [ 25 , 26 ], decreases after axotomy, while the cell body volume is increasing. Those observations, together with the data presented here, lead us to propose that axotomy causes newly synthesized axonal cytoskeleton to accumulate between the nucleus and axon hillock, resulting in an increase in nuclear eccentricity away from the axon hillock and chromatolysis between the nucleus and axon hillock. A requirement of this proposal is that it explain the direction in which chromatolysis develops. The loss of basophilic staining after axotomy is termed "central chromatolysis" because in many instances it first appears near the nucleus and spreads outward towards the axon hillock [ 6 ]. If the axonal cytoskeleton dams up in this region of the injured cell body, why does the loss of basophilia spread centrifugally? It is not known exactly where assembly of the axonal cytoskeleton begins in normal or axotomized motor neurons. However, there is some information about the assembly of the cytoskeleton in non-neuronal cells. In her pulse-chase analysis of the incorporation of newly synthesized proteins into the cytoskeletal framework of non-neuronal cells, Fulton [ 27 ] found autoradiographic evidence that new cytoskeleton is first assembled near the nucleus, and then moves towards the periphery of the cell body. If the assembly of the axonal cytoskeleton in motor neurons follows the same pattern after axotomy, then chromatolysis could spread from the nucleus to the cell body periphery. As the movement of newly synthesized axonal cytoskeleton towards the hillock is impeded, chromatolysis would appear first near the nucleus, spreading outward towards the hillock with time and displacing and compressing the basophilic cytoplasmic skeleton. This action could produce a densely basophilic rim in the cell body periphery, as has often been observed [ 6 ]. Further accumulation of the axonal cytoskeleton could ultimately spread to other regions of the cell body. Conclusion We have shown that nucleolar, nuclear and cell body volume in spinal motor neurons, as well as nuclear position, is maintained by the cytoskeleton. The isolated cytoskeleton, whose protein composition has not been determined, contains slightly less than one-half of the total cell body protein. The cytoskeleton is a filamentous lattice that is most clearly seen by transmission electron microscopy in resinless thin sections. Nissl bodies are associated with the isolated cytoskeleton, as are lipofuscin granules, which may be caged within the filamentous lattice. Axotomy increases nucleolar, nuclear and cell body volume, largely by altering the cytoskeleton. The likely cause of the injury-induced enlargement is the addition of new protein to the cell body cytoskeleton. Axotomy also increases nuclear eccentricity by altering the cell body cytoskeleton. We postulate that both increased nuclear eccentricity and central chromatolysis in axotomized spinal motor neurons are consequences of damming up of Nissl body-free axonal cytoskeleton between the nucleus and axon hillock. Methods Surgical procedures All ventral root transections were performed on adult southern grass frogs ( Rana pipiens ) of both sexes, purchased from Carolina Biological Supply, Burlington, NC. The left 9 th and 10 th ventral roots were transected 6–9 mm from the spinal cord via a dorsal laminectomy under general anesthesia (135 mg Finquel/kg body wt.). Animals were allowed to survive for up to 20 days before isolation of motor neuron cell bodies. Pre- and postoperative care and euthanasia by decapitation were conducted under institutional review in accordance with AVMA standards. Cell body isolation Figure 8 illustrates the procedure used to isolate motor neuron cell bodies from unfixed frog lumbar spinal cord [ 10 ]. Small pieces of R. pipiens lumbar spinal cord were first cryoprotected with 70% ethylene glycol and frozen at -80°C. Cryoprotection improves the quality and yield of motor neurons [ 28 ] and permits storage of tissues until needed. After thawing, the tissue was suspended in 0.9 M sucrose in 1.7 mM sodium citrate buffer (pH 5.0) containing 15 mM glucose and expressed through nylon bolting cloth of 202 μm pore size. Nylon cloth with pores of 351 μm on a side was used for the larger human motor neurons. The cell bodies were then centrifuged in a discontinous sucrose gradient to obtain a crude neuronal fraction, from which individual cell bodies were removed with a glass pipette, rinsed twice in 1.5 M sucrose and pooled with other purified cell bodies. Motor neurons were identified by their distinctive size, shape and biochemical profiles [ 29 ]. In each experiment, 100–400 cells were collected from the lumbar spinal cord of three grass frogs. Cytoskeleton isolation Cytoskeletons from isolated motor neuron cell bodies were obtained by serial extraction of soluble proteins, using a modification of the method of He et al . [ 12 ]. The buffer solution used in each step of the extraction procedure contained 50 mM HEPES (pH 7.4), 250 mM sucrose, 250 mM NaCl, 10 mM MgCl 2 , 1.2 mM phenylmethanesulfonyl fluoride, and 1 mM EGTA. The sample was exposed to 100 μl of ice-cold 0.5% Triton X-100 in extraction buffer, 35 μl of DNAase (100 μg/ml) treatment for 30 minutes at 24°C., followed by two rinses in 100 μl of chilled buffer alone, and extraction with 100 μl of chilled 0.25 M (NH 4 ) 2 SO 4 , and then 2 M NaCl. After each step, the cell bodies were centrifuged at 1,000 × g for 3 minutes. Morphometry The volume of isolated motor neuron cell bodies was quantified using images from a Leitz Diavert microscope equipped with a Fairchild CCD 3000 camera and analyzed with a Technology Resources Imagemaster 1000. Cell body volume was calculated as the product of the mean of three measurements of cell body area and the mean of the height at the center and opposite ends of the cell body. The cylindroidal shape of the cell body was determined from measurements of the z-axis in 120 cells suspended in 1.2 M sucrose and viewed between two coverslips with DIC optics. At the center of the cell body, the z-axis averaged 19.3 ± 6.5 μm and was only 102.3 ± 2.2% and 98.6 ± 4.7% of that value, respectively, at opposite ends of the cell body. In five separate experiments, the z-axis was 72 ± 7% as long as the average cell body radius. The flatness of the cell bodies was not induced by pressure from the apposed coverslips, since it was also observed in cells tumbling slowly in a droplet of buffer. Nucleolar and nuclear volumes were calculated as spheres, using the mean radius from three measurements of nucleolar or nuclear area. Nuclear eccentricity was measured by the method of Barr and Hamilton [ 1 ], using the formula [AC÷(AB-CD)] × 100%. A-D are points along a line from the center of the cell body (A) through the center of the nucleus (C) to the cell periphery (B). AC is the distance between the centers of the cell body and nucleus (each computed from their areas by the software program). AB is the distance between the center of the cell body and its periphery and CD is the distance between the center of the nucleus and the nuclear lamina. Nuclear eccentricity of zero means the centers of the nucleus and cell body are identical, while 100% eccentricity refers to nuclei in contact with the cell body perimeter. Confocal microscopy Human motor neuron cell bodies were isolated from lumbar or cervical spinal tissue obtained at autopsy from an individual without neurological disease. Cell body cytoskeletons prepared by the procedure outlined above were stained for 3 min. with 0.0032% methylene blue and examined with a Leica TCS-NT confocal microscope. Electron microscopy Intact, resinless thin sections of cytoskeletal preparations from isolated motor neuron cell bodies are technically difficult to obtain. Thus, lumbar spinal tissue from adult frogs was examined after extraction by the method used for isolated cell bodies with slight modifications. Segments of longitudinally hemisected lumbar spinal tissue 1–2 mm long were exposed sequentially to HEPES-buffered 0.5% Triton X-100, 0.25 M (NH 4 ) 2 SO 4 and 2 M NaCl and fixed in 2.5% glutaraldehyde. To minimize myelin swelling, the segments were kept on ice and DNAase treatment was omitted. Embedment was carried out at 72°C in increasing concentrations of diethyleneglycol distearate (DGD; Electron Microscopy Sciences, Fort Washington, PA, USA) from which 90% of the acetone-soluble contaminants had been removed. Thin sections were then cut on the DGD-embedded tissue, placed on Formvar-coated, carbon-stabilized grids and the DGD removed with dry n -butanol for 1 hour × 3 at 40°C. The resinless sections were then transferred stepwise into absolute ethanol and dried through the CO 2 critical point after 6 wash cycles in a Balzers apparatus. Transmission electron microscopy was performed at 80 kV with a Zeiss EM 10 CA microscope. Negatives were digitized and image contrast and brightness were adjusted electronically. In other experiments, plastic sections were cut from extracted spinal tissue embedded in Spurr's low viscosity Epon following fixation in 2.5% glutaraldehyde and 1% osmium tetroxide and were post-stained with 1% uranyl acetate and Reynolds' lead citrate. Protein analyses After TCA precipitation, using a modification of the method of Bensadoun and Weinstein [ 30 ], total cell body protein content was measured by the micro method of Lowry et al . [ 31 ], standardized to bovine serum albumin. A total of 200 to 300 cell bodies was used for each analysis. Triplicate samples of 1.5 M sucrose from the second rinse step were taken from regions near the purified cell bodies and used as "blanks" for the protein assay. Radiolabeling of motor neuron proteins with 3 H-leucine prior to cell body isolation was carried out on unoperated control and 16-day axotomized frogs, using the method of McIlwain and Hoke [ 32 ]. Briefly, longitudinal sections of lumbar spinal tissue from six unoperated frogs or from the ipsilateral side of six axotomized frogs were incubated in a Yellow Springs Instrument oxygen monitoring apparatus (Yellow Springs, OH, USA) at 17°C for 16 h in frog Ringer solution containing 1 mCi/ml of 3 H-L-leucine. After rinsing and cryoprotecting the radiolabeled tissue, 40–200 cell bodies were isolated as described above and, in some experiments, their cytoskeletons were obtained before their radioactivity was measured by liquid scintillation counting. Authors' contributions DLM designed and supervised the study, participated in parts of the microscopy, and prepared the manuscript. VBH, an experienced research technician, performed most of the experiments.

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1079868

Hyperosmotic stimulus induces reversible angiogenesis within the hypothalamic magnocellular nuclei of the adult rat: a potential role for neuronal vascular endothelial growth factor

Background In mammals, the CNS vasculature is established during the postnatal period via active angiogenesis, providing different brain regions with capillary networks of various densities that locally supply adapted metabolic support to neurons. Thereafter this vasculature remains essentially quiescent excepted for specific pathologies. In the adult rat hypothalamus, a particularly dense network of capillary vessels is associated with the supraoptic (SON) and paraventricular (PVN) nuclei containing the magnocellular neurons secreting vasopressin and oxytocin, two neurohormones involved in the control of the body fluid homoeostasis. In the seventies, it was reported that proliferation of astrocytes and endothelial cells occurs within these hypothalamic nuclei when strong metabolic activation of the vasopressinergic and oxytocinergic neurons was induced by prolonged hyperosmotic stimulation. The aim of the present study was to determine whether such proliferative response to osmotic stimulus is related to local angiogenesis and to elucidate the cellular and molecular mechanisms involved. Results Our results provide evidence that cell proliferation occurring within the SON of osmotically stimulated adult rats corresponds to local angiogenesis. We show that 1) a large majority of the SON proliferative cells is associated with capillary vessels, 2) this proliferative response correlates with a progressive increase in density of the capillary network within the nucleus, and 3) SON capillary vessels exhibit an increased expression of nestin and vimentin, two markers of newly formed vessels. Contrasting with most adult CNS neurons, hypothalamic magnocellular neurons were found to express vascular endothelial growth factor (VEGF), a potent angiogenic factor whose production was increased by osmotic stimulus. When VEGF was inhibited by dexamethasone treatment or by the local application of a blocking antibody, the angiogenic response was strongly inhibited within the hypothalamic magnocellular nuclei of hyperosmotically stimulated rats. Conclusion This study shows that the functional stimulation of hypothalamic magnocellular neurons of adult rats induces reversible angiogenesis via the local secretion of neuronal VEGF. Since many diseases are driven by unregulated angiogenesis, the hypothalamic magnocellular nuclei should provide an interesting model to study the cellular and molecular mechanisms involved in the regulation of angiogenesis processes within the adult CNS.

Background Within the CNS, capillary blood vessels form a network of highly interconnected tubes that direct and maintain blood flow throughout the different regions. In the adult CNS, the vascular supply is not homogenous and marked differences exist in the capillary density present within specific brain regions. Since blood glucose represents the major metabolic support of neurons, it has been proposed that the density of the vasculature network is related to the different levels of the metabolic activity [ 1 ]. It is generally admitted that the adult vasculature is essentially quiescent and that adjustment of blood supply to increased metabolic activity occurs locally via modifications of the diameter of blood vessels [ 2 ]. However previous studies have suggested that chronic stimulation of specific neuronal systems was able to locally modify the blood supply via angiogenesis. For instance, rearing rats in a complex environment was found to increase the capillary density within the visual cortex [ 3 ], whereas prolonged motor activity was reported to induce angiogenesis within the cerebellar cortex [ 4 ] and primary motor cortex [ 5 ]. The magnocellular nuclei of the hypothalamus have long been shown to contain a particularly high density of capillaries [ 6 - 8 ]. These hypothalamic nuclei contain two populations of magnocellular neurons that synthesize two peptidic neurohormones, vasopressin (VP) and oxytocin (OT) that play major roles in the control of body fluid balance. Since these magnocellular neurons synthesize huge amounts of VP and OT throughout life span, it has been admitted that hypervascularization of these nuclei facilitates the supply of circulating glucose needed for sustaining a high metabolic activity [ 9 ]. Moreover, the activity of hypothalamic magnocellular neurons is directly regulated by changes in plasma osmotic pressure and their metabolic activity can be chronically stimulated by prolonged osmotic stimuli [ 10 ]. Interestingly, it has been reported that proliferation of glial and endothelial cells could be observed within the hypothalamic magnocellular nuclei in animals submitted to prolonged osmotic stimulus [ 11 ]. In this context, the aim of our study was to determine whether prolonged metabolic activation of magnocellular neurons was able to modify the vasculature throughout the hypothalamic nuclei via local angiogenesis. Our results show that hyperosmotic stimuli induce local proliferation of SON capillary endothelial cells, leading to a reversible increase in the density of the capillary network within the nuclei. We also show that contrasting with most CNS neurons, magnocellular hypothalamic neurons continue to express high levels of VEGF throughout adulthood and that this endogenous cytokine is at least in part responsible for the local angiogenesis induced by hyperosmotic stimuli. Results In the present study, the observations were restricted to brain sections passing through the hypothalamus that contained the different magnocellular nuclei, including the supraoptic (SON), paraventricular (PVN), and accessory (AN) nuclei. As compared with the other hypothalamic magnocellular nuclei, the SON essentially contains VP and OT neurons and has a clearly defined anatomical localization (along the lateral border of the optic chiasma). For reasons of clarity, the data reported here will therefore mostly concern the SON, although the present findings apply for all the hypothalamic magnocellular nuclei. Hyperosmotic stimulus induces reversible angiogenesis within the SON The kinetics of cell proliferation To determine the kinetics of the proliferative response to hyperosmotic stimulus adult rats were provided with 2 % saline drinking water during 0 (controls), 1, 2, 4, 6 and 9 days. At the end of the stimulation, animals were injected with BrdU and fixed 5 hours later. In control rats, BrdU-labeled nuclei were never detected within the SON (Fig. 1A ), although they were constantly detected within the germinative zones including the dentate gyrus of the hippocampus [ 12 ] and the subventricular zone of the lateral ventricle [ 13 ] (not shown). In all the stimulated rats by contrast, BrdU-labeled nuclei were detected within both the germinative zones and the SON (Fig. 1B–E ) and other magnocellular hypothalamic nuclei including the PVN and accessory nuclei (not shown). The quantification of BrdU-labeled nuclei in the SON indicated that this proliferative response was maximum after to 6 days of osmotic stimulation, and then slightly decreased (Fig. 1G ). The same protocol was used to study the proliferative response within the SON of 6 days osmotically stimulated rats that were rehydrated for 1, 2 and 3 days: in all these animals, only scarce BrdU-labeled nuclei could be detected within the SON after 1 day rehydration (Fig. 1F and 1G ), indicating that cell proliferation was rapidly stopped at the end of the stimulus. Figure 1 Hyperosmotic stimuli induce cell proliferation within the SON . BrdU was administrated to rats drinking 2% saline during various increasing periods, or to rats rehydrated after 6 days of osmotic stimulation, and the animals were fixed 5 hours after the BrdU administration. A-F : Stack confocal images (10 μm-thick) of sections labeled for BrdU. G : Quantitative evaluation of the number of BrdU-labeled nuclei detected within the SON. Cell proliferation is virtually absent in control rats (0 day of stimulation), progressively increases from 2 to 6 days of osmotic stimulation, and is abolished after 1 day of rehydration in 6 days stimulated rats. Data represent means ± SEM of counts made on at least 4 SON areas per rats in 3 rats per experiment. The schematic drawing illustrates the anatomical locations of the SON and other hypothalamic magnocellular nuclei. AN: accessory nucleus; OC: optic chiasma; PVN: paraventricular nucleus; SON: supraoptic nucleus. Scale bar: 100 μm. The nature of proliferating cells In this part of the study, BrdU was injected to stimulated rats provided with 2% saline drinking water during 6 days and the animals were fixed 5 hours later. In order to identify the nature of the cells that proliferate within the SON, we looked for the possible association of BrdU-labeled nuclei with the main cell types contained in the hypothalamic nucleus, including the neurons, glial cells and capillary endothelial cells. Neurons The SON essentially contains magnocellular neurons that were labeled by specific antibodies against VP or OT. A mixing of both antibodies was used to label the whole population of SON neurons. In all sections double immunostained for VP + OT and BrdU, BrdU-labeled nuclei never appeared associated with VP- or OT-labeled neuronal cell bodies (not shown). Glial cells The SON contains three main glial cell types including 1) astrocytes that were labeled by the means of an antibody against GFAP, an intermediate filament protein specifically expressed by this cell type [ 14 ], 2) oligodendrocyte precursor cells that were labeled by an antibody against the proteoglycan NG2 [ 15 , 16 ]), and 3) microglial cells that were labeled by the isolectin B4 from Griffonia simplicifolia (IsoB4, recognizing glycoproteins that are specifically associated with the limiting membrane of resting and activated microglia [ 17 ], and with capillary vessels endothelial cells[ 18 ]). These different glial cells markers produced typical labeling patterns within the SON: 1) GFAP-labeling was associated with astrocytic cell bodies localized to the ventral surface of the nucleus and with their elongated processes extending dorsally throughout the core of the nucleus (Fig. 2A ), 2) NG2-labeling was associated with small cells exhibiting numerous radiating processes that were homogeneously dispersed throughout the SON and the surrounding regions (Fig. 2C ), and 3) IsoB4 labeling was associated with both typical ramified microglial cells dispersed throughout the nucleus and with a number of capillary vessels (Fig. 2E ). In double labeled sections, BrdU-labeled were rarely associated with the GFAP-labeled astrocytic cell bodies located in the ventral portions of the nucleus (Fig. 2B ) and with the IsoB4-labeled isolated microglial cells dispersed throughout the nucleus (Fig. 2D ). On the other hand, labeled nuclei were frequently associated with IsoB4-labeled capillary vessels (Fig. 2D ) and to a lesser extent with NG2-labeled cells bodies dispersed throughout the SON (Fig. 2F ). Figure 2 Cell proliferation concerns a minority of SON glial cells. BrdU was administrated to osmotically stimulated rats drinking 2% saline during 6 days and the animals were fixed 5 hours after the BrdU administration. A, C, E : Stack confocal images (10 μm-thick) of sections labeled for different glial markers. A : GFAP immunostaining is associated with the cell bodies of SON astrocytes localized to the ventral border of the nucleus and with their elongated processes projecting dorsally throughout the core of the nucleus. C : NG2 immunostaining is associated with small ramified cells dispersed throughout the nucleus. E : IsoB4 labeling is associated both with typical ramified microglial cells dispersed within the nucleus and with some tubular, vessel-like structures. Insets show high magnification of the different labeled cell types. B, D, and F : Merged confocal images of sections double labeled for BrdU and for one the different glial markers. Within the SON, BrdU-labeled nuclei are never associated with GFAP-labeled astrocytes ( B ) and IsoB4-labeled isolated microglial cells (arrow heads in F ), whereas they are associated with some NG2-labeled cells (arrows in D ) and with numerous vessel-like structures labeled with the lectin IsoB4 (arrows in F ). GFAP: glial fibrillary acidic protein; IsoB4: isolectin B4; NG2: NG2 proteoglycan; OC: optic chiasma. Scale bars: A, C, E = 100 μm; B, D, F = 25 μm; Insets = 25 μm. Capillary endothelial cells In addition to the isolectin B4 (see above), the endothelial cells composing the SON capillary vessels were labeled with specific antibodies against either nestin (an intermediate filament protein that is expressed by these cells during prenatal and early postnatal periods [ 19 , 20 ] or the endothelial barrier antigen (EBA, a protein that is expressed by endothelial cells in the mature rat brain) [ 21 ]. In all the adult rats examined (2–3-month-old), the phenotypic expression of SON capillary vessels markedly differed from those located in the surrounding brain regions: SON capillaries always exhibited moderate to intense immunoreactivity to both EBA and nestin, whereas all the vessels located in the surrounding regions were EBA-positive but nestin-negative (Fig. 3A and 3C ). The examination of double labeled sections indicated that, a large majority of the BrdU-labeled nuclei detected throughout the SON was associated with capillary-like processes labeled for EBA (Fig. 3B ) or nestin (Fig. 3D ). These data indicate that the cell proliferation occurring within the SON following osmotic stimuli preferentially concern the capillary endothelial cells. Figure 3 Cell proliferation mostly concerns SON capillary endothelial cells . BrdU was administrated to osmotically stimulated rats drinking 2% saline during 6 days, and the animals were fixed 5 hours after the BrdU administration. A, C : Stack confocal images (10 μm-thick) of sections labeled for endothelial cell markers. EBA immunostaining is associated with vessels located within the SON and the surrounding regions ( A ), whereas nestin immunostaining is associated with SON vessels, but only faintly labels those vessels located in the surrounding regions ( C ). B, D : Merged confocal images of sections double labeled for BrdU and for one of the two the endothelial cell markers. Throughout the SON, BrdU-labeled nuclei are frequently associated with vessel structures labeled for EBA (arrows in B ) or nestin (arrows in D ). EBA: endothelial brain antigen; OC: optic chiasma. Scale bars: A, C = 100 μm; B, D = 25 μm. Hyperosmotic stimulus reversibly modifies the organization of SON vessels Since proliferation of endothelial cells generally results in the formation of new vessels, we then examined the consequences of prolonged osmotic stimuli on the SON vascularization. To do this, we compared the anatomical organization and the phenotypic expression of SON capillaries in normally hydrated rats (controls), and in stimulated rats drinking 2% saline for various periods (1 to 9 days). Anatomical organization of SON vessels In this part of the study, the rats were decapitated and brains were incubated in a 4% paraformaldehyde solution in order to fix the serum proteins contained within blood vessels. The organization of capillary vessels was then visualized on 50-μm-thick vibratome sections, by labeling the intra-vascular content by an antibody against Rat IgG conjugated to Alexa-fluor 488. The examination of anti-rat-IgG-labeled sections indicated that 1) in control, normally hydrated adult rats, the density of capillary vessels was significantly higher within the SON area (identified as the area containing the cell bodies of VP and OT neurons) than in the surrounding brain regions (Fig. 4A ), and 2 ) in 6 days osmotically stimulated rats, this density was significantly increased within the SON area but not in the surrounding regions (Fig. 4C–D ). Moreover, during the osmotic stimulation, the size of the individual VP and OT neurons was progressively increased, leading to an extension of the SON area (Fig. 4A–C ). A quantitative analysis of the Rat-IgG-labeled sections indicated that after 6 days of stimulation, the surface occupied by SON capillaries was dramatically increased (× 3 to 4) as compared with the SON of normally hydrated rat. These modifications were reversed by the rehydration of stimulated animals: the density of SON capillaries progressively decreased when 6 days stimulated rats were rehydrated, and was reestablished to control levels in animals rehydrated for 6 days (Fig. 4G and 4H ). Figure 4 Hyperosmotic stimulus reversibly modifies the anatomical organization of SON vessels . Freshly dissected brains of rats normally hydrated ( A-B ); osmotically stimulated during 6 days ( C-D ) and rehydrated during 6 days following 6 days of osmotic stimulation ( E-F ) were fixed by immersion in the fixative and the content of blood vessels was labeled by an antibody against Rat IgG. A-F : Stack confocal images (10 μm-thicks) of sections double immunostained for Rat IgG and VP + OT. Insets show the morphology of VP or OT SON neurons ( A, C, E ), and the organization of Rat IgG-labeled vessels in the lateral hypothalamus ( B, D, F ). G : Quantitative evaluation of the vessels density within the SON and the lateral hypothalamus. For each section analyzed, a 100 point grid was centered on the core of the nucleus or on the overlying lateral hypothalamus (as shown in the schematic drawing), and the vessel density was estimated by scoring vessel contacts with the bars of the grid. The data are expressed as percent of the vessel density determined in the lateral hypothalamus overlying the SON of control rats. H : Quantitative evaluation of the total amount of SON vessels. For each section analyzed the SON surface was first determined by delineating the area containing the neuronal cell bodies labeled for OT or VP (H1 shows the SON area selected from the image C). The surface occupied by vessels was then quantified on binary images of the Rat-IgG-labeled structures within this SON area (H2 shows the binary image of labeled vessels selected from the image D). All the quantifications were performed on at least 4 sections containing the middle portions of the SON (i.e. the largest SON areas), for at least 3 rats per experiment. Note that the size of individual VP or OT immunostained neurons is highly increased in the SON of stimulated rat (inset C) as compared with control (inset A) or rehydrated rat (inset E). Double asterisk: P < 0.01, Mann-Whitney test, statistically different from the control SON. ct: control, normally hydrated; IgG: type G immunoglobulin; LH: lateral hypothalamus; OC: optic chiasma; OT: oxytocin; os 6d: osmotically stimulated during 6 days; reh 6d: rehydrated during 6 days following 6 days stimulation; SON: supraoptic nucleus; VP: vasopressin. Scale bars: A-C = 100 μm. Insets A, C, D = 25 μm. Phenotypic expression of SON vessels In a second step we looked for the expression by SON vessels of nestin or vimentin, two intermediate filament proteins that are expressed by the endothelial cells constituting newly formed capillary vessels in the developing CNS or in brain tumors [ 19 , 20 , 22 , 23 ]. In control adult rats, the large majority of the capillary vessels present throughout the brain exhibited intense EBA immunostaining, but faint if any immunostaining for nestin or vimentin. By contrast, SON capillary vessels always exhibited moderate to intense immunostaining for EBA and nestin, whereas vimentin immunostaining was essentially associated with SON astrocytic cell bodies and processes [ 24 ] (Fig. 5A and 5D ). In osmotically stimulated rats, the SON was found to contain an increased amount of nestin-immunostained vessels, frequently exhibiting higher staining intensity than in the control rats (Fig. 5B ). Contrasting with control rats, vimentin immunostaining was now associated with both SON astrocytes and capillary vessels, whereas vessels located in the surrounding brain regions remained vimentin-negative (Fig. 5E ). As observed for their anatomical organization, rehydration of 6 days osmotically stimulated rats induced progressive modifications of the phenotype of SON vessels, and their expression of nestin and vimentin was reestablished to control levels after 6 days of rehydration (Fig. 5C and 5F ). The examination of sections double immunostained for EBA and either nestin or vimentin further indicated that, as compared with control or rehydrated rats, the SON of osmotically stimulated rats contained an increased amounts of capillary processes that were immunostained for either nestin or vimentin but appeared EBA negative (Fig. 5 G–L ). Figure 5 Hyperosmotic stimulus reversibly increases the expression of nestin and vimentin by SON capillary vessels . A-F : Stack confocal images (10 μm-thick) of sections immunostained for nestin ( A-C ) or vimentin ( D-F ) in rats normally hydrated ( A and D ), osmotically stimulated during 6 days ( B and E ), and rehydrated during 6 days following 6 days of osmotic stimulation ( C and E ). In all the rats, nestin immunostaining of vessels is more intense in the SON than in the surrounding regions, whereas the number and staining intensity of immunostained SON vessels increases in the osmotically stimulated rats ( B vs A ), and return to control levels in the rehydrated rats ( C ). In all the rats, intense vimentin immunostaining is associated with the cell body of astrocytes located along the SON ventral border, and with their elongated processes projecting throughout the nucleus ( D-F ), whereas vimentin-immunostained vessels are only detected in the SON of the stimulated rat ( E ). G-L : Merged confocal images of sections double immunostained for EBA and either nestin ( G-I ) or vimentin ( J-L ) in rats normally hydrated ( G and J ), osmotically stimulated during 6 days ( H and K ), and rehydrated during 6 days following 6 days of osmotic stimulation ( I and L ). In control ( G and I ) and rehydrated ( I and L ) rats, numerous SON vessels are double-immunostained for EBA and nestin (yellow vessels in G and I ), whereas in the stimulated rat, number of capillary vessels labeled for nestin appear EBA-negative (red vessels in H ). As compared with control ( J ) and rehydrated ( L ) rats, the SON of the osmotically stimulated rat contain numerous vessels double immunostained for EBA and vimentin or (yellow vessels in K ), or vimentin-positive but EBA-negative (red vessels in K ). Control: normally hydrated rat; EBA: endothelial brain antigen; OC: optic chiasma; os 6 days: rat osmotically stimulated during 6 days; reh 6 days: rat rehydrated during 6 days following 6 days of osmotic stimulation. Scale bars: A-C = 100 μm; D-F = 100 μm; G-L= 50 μm. SON angiogenesis is related to neuronal vascular endothelial growth factor SON neurons maintain the expression of VEGF throughout adulthood Among a variety of identified angiogenic factors, VEGF is generally considered to play a major role in the processes of angiogenesis, maintenance of cerebral vasculature and permeability [ 25 ]. It has recently been reported that neuronal VEGF plays important role in the process of angiogenesis in the developing CNS [ 26 ]. In full agreement with these data, we observed that throughout extra-hypothalamic regions, VEGF-immunostaining was essentially associated with NeuN-labeled neurons in newborn rats of less that 25 days postnatal (dpn), (Fig. 6A ), and with GFAP-labeled astrocytes in adult (2–3-month-old) rats (Fig. 6B ). In the hypothalamus by contrast, intense to moderate VEGF-immunostaining was associated with magnocellular neurons labeled for VP or OT in both newborn and adult rats (Fig. 6C ). Figure 6 VEGF is expressed by adult SON neurons . A-C : Stack confocal images (10 μm-thick) of sections immunostained for VEGF. In the cortex of newborn ( A , 10-day-old) and adult ( B , 3-month-old) rats, VEGF immunostaining is associated with neuron-like and glial-like structures, respectively. Insets: merged confocal images of double immunostained sections showing that in the cortex of the newborn and adult rats, VEGF immunostaining is localized within NeuN-labeled neurons and GFAP-labeled astrocytes, respectively. In the hypothalamus of an adult control rat ( C ), intense VEGF immunostaining is detected within both neuronal cell bodies located in the core of the SON, and within astrocytic profiles located on the ventral border of the nucleus (arrows in C ), whereas. Insets: merged confocal images of double immunostained sections showing that within the SON, VEGF immunostaining is associated with both OT- and VP-labeled neurons, and with GFAP-labeled astrocytes. Cx: cortex; GFAP: glial fibrillary acidic protein; NeuN: nuclear neuronal antigen; OT: oxytocin; VP: vasopressin; VEGF: vascular endothelial growth factor. Scale bars: A-B = 50 μm; C = 100 μm; insets = 25 μm. Osmotic stimulus increases the expression of VEGF within SON neurons In osmotically stimulated rats, VEGF immunostaining pattern was unchanged within astrocytes located within the SON or the surrounding brain regions whereas it was dramatically modified within SON neurons: whereas faint to moderate VEGF immunostaining was associated with perinuclear, golgi-like structures in SON neurons of control rats, intense VEGF immunostaining was detected throughout the neuronal cell bodies of stimulated rats (Fig. 7A–B ). Figure 7 Hyperosmotic stimulus increases VEGF expression within SON neurons . A-B : Immunostaining for VEGF in control ( A ) and 6 days osmotically stimulated adult rats ( B ). Stack confocal images (10 μm-thick) of SON neurons show that moderate VEGF immunostaining is localized to perinuclear, golgi-like structures in the control rat ( A ), whereas intense immunostaining is dispersed throughout the cytoplasm in the stimulated rat ( B ) (insets show higher magnification of immunostained neurons). C-D : In situ hybridization for VEGF mRNA in control ( C ) and 6 days osmotically stimulated adult rats ( D ). Light microscope micrographs of 20 μm-thick cryostat sections showing that the mRNA labeling detected within the SON is highly increased in the stimulated ( D ) as compared with the control ( C ) rat. OC: optic chiasma; VEGF: vascular endothelial growth factor. Scale bars: A-B = 100 μm; C-D = 100 μm; insets = 50 μm. In situ hybridization experiments for VEGF mRNA further indicated that the mRNA labeling detected within SON neurons was highly increased in 6 days stimulated rats as compared with control rats (Fig. 7C–D ). Inhibition of endogenous VEGF impedes osmotically induced angiogenesis Dexamethasone inhibits both neuronal VEGF expression and SON angiogenesis Dexamethasone, a potent agonist of glucocorticoid receptors, has previously been shown to inhibit the expression of VEGF in various cell types [ 27 - 30 ]. In the present study, we thus evaluated the effects of chronic dexamethasone treatment of adult rats both on the expression of VEGF by magnocellular hypothalamic neurons and on the local angiogenic response induced by osmotic stimulus. Our data clearly show that in all dexamethasone-treated rats, VEGF immunostaining of magnocellular neurons was dramatically decreased as compared with control, untreated rats (Fig. 8A and 8C ). In order to test whether dexamethasone treatment influenced the local proliferative response induced by osmotic stimulus, dexamethasone-treated adult rats were osmotically stimulated during 6 days, injected with BrdU, and sacrificed 5 hours later. Our data indicate that dexamethasone-treatment nearly abolished the angiogenic response to hyperosmotic stimulus within the SON and other magnocellular hypothalamic nuclei (Fig. 8B and 8D ). On the other hand, dexamethasone treatment was found to only poorly modify the cell proliferation detected within the germinative zone of the lateral ventricle (Fig. 8B and 8D ). Figure 8 Dexamethasone inhibits both VEGF expression and SON proliferative response . Untreated ( A-B ) or Dexamethasone treated ( C-F ) rats were osmotically stimulated during 6 days and injected with BrdU 5 hours before their fixation. Stack confocal images (10 μm-thick) of sections double immunostained for VEGF and BrdU ( A-D ). As compared with the control rat ( A-B ), the SON of the dexamethasone treated rat exhibits decreased immunostaining for VEGF ( C ) and a complete disappearance of BrdU-labeled nuclei ( D ). By contrast, dexamethasone treatment has no effect on the BrdU-labeling of proliferative cells within the subventricular zone of the lateral ventricle (insets). Note that in the control rat, BrdU-labeled nuclei are essentially localized to the core of the SON that contains the VEGF-labeled neurons, and are scarce in the ventral portion of the nucleus that contains labeled astrocytes (stars in A and B ). BrdU: bromodeoxyuridine; EBA: endothelial brain antigen; SON: supraoptic nucleus; VEGF: vascular endothelial growth factor. Scale bar: A-D = 100 μm. Local application of a blocking antibody to VEGF inhibits SON angiogenesis In order to inhibit endogenous VEGF, we then used a goat IgG VEGF neutralizing antibody. An intracerebral cannula connected to an osmotic minipump containing the blocking antibody or an IgG isotype was implanted in close vicinity to the SON of rats submitted to prolonged osmotic stimulus. In the implanted rats, the distribution of VEGF neutralizing antibody and of the IgG isotype was determined by the immunocytochemical detection of goat IgG: in all the implanted rats examined, infused IgG was found to diffuse throughout a 1 mm wide area surrounding the lesional cavity induced by the cannula, thus including the whole SON and part of the PVN homolateral to the cannula, whereas no diffusion could be detected within the magnocellular hypothalamic nuclei contralateral to the cannula (Fig. 9B and 9D ). Our data indicated that in rats osmotically stimulated for 6 days, the cell proliferation (evaluated by the administration of BrdU, 5 hours prior to the fixation) was strongly inhibited in the SON adjacent to the cannula and, to a lesser extent, in the homolateral PVN, as compared with the magnocellular nuclei contra-lateral to the cannula (Fig. 9A, C and 9E ). By contrast, a similar high rate of cell proliferation was detected in the hypothalamic magnocellular nuclei of both sides in control rats unilaterally infused with goat IgG (Fig. 9E ). In all the rats infused with either the VEGF neutralizing antibody or the goat IgG, strong cell proliferation was detected throughout the area bordering the lesional cavity (Fig. 9A ). Figure 9 VEGF antibody inhibits the SON proliferative response . Rats were implanted unilaterally with a cannula positioned over the SON and connected to an Alzet osmotic pump containing a blocking VEGF antibody (schematic drawing), osmotically stimulated during 6 days, and injected with BrdU 5 hours before their fixation. A-D : Stack confocal images (10 μm-thick) of sections double immunostained for BrdU and goat-IgG. In the SON homolateral to the cannula, the number of BrdU-labeled nuclei is clearly lower that in the SON contralateral to the cannula ( A vs C ). Immunostaining for goat-IgG indicates that the goat-IgG anti-VEGF antibody has diffused from the cannula to the homolateral SON ( B ), but not to the contralateral SON ( D ). Note that numerous BrdU-labeled nuclei are observed along the border of the lesional cavity formed by the cannula diffusing the anti-VEGF antibody (arrow in A ). E : Quantitative evaluation of the number of BrdU-labeled nuclei detected within the SON of 6 days osmotically stimulated rats implanted with a cannula diffusing either the anti-VEGF antibody or the isotype IgG. Data indicate that cell proliferation is significantly inhibited in the SON homolateral to implanted with cannulas diffusing blocking anti-VEGF antibody, but not with cannulas diffusing goat-IgG. Data represent means ± SEM of counts made on at least 4 SON areas per rats in 4 rats per experiment. Double asterisk: P < 0.01, Mann-Whitney test, statistically different from the SON contra-lateral to the cannula. BrdU: bromodeoxyuridine; cl: contra-lateral to the cannula; hl: homo-lateral to the cannula; IgG: type G immunoglobulin; OC: optic chiasma; SON: supraoptic nucleus; VEGF: vascular endothelial growth factor. Scale bar: A-D = 100 μm. Discussion In mammals, the terminal CNS vascularization occurs postnatally via active angiogenesis, and thereafter this vasculature remains essentially quiescent under basal, non pathological conditions. In the present study we show that 1) the vascularization of hypothalamic magnocellular nuclei can be modified throughout adulthood via local angiogenesis induced by hyperosmotic stimuli and 2) such local angiogenic events are related to the expression of high levels of VEGF by magnocellular VP and OT neurons. Osmotic stimuli induce proliferation of SON capillary endothelial cells More than 28 years ago, it has been established that prolonged stimulation of hypothalamic VP and OT neurons by hyperosmotic stimuli induced substantial proliferation of glial and endothelial cells within the supraoptic nucleus [ 11 ]. Although they fully confirm these previous data, our present data indicate that the cell proliferation occurring locally within hypothalamic magnocellular nuclei mostly involve endothelial cells, whereas proliferation of both endothelial cells and astrocytes was previously reported. A first explanation for such discrepant data is that the proliferation rate of astrocytes has been underestimated in the present study. Proliferative astrocytes were identified here as those cells immunostained for the astrocytic marker GFAP that exhibited a BrdU-labeled nucleus. Since newly formed astrocytes only express very low levels of GFAP[ 31 , 32 ], it is thus possible that proliferative astrocytes appeared GFAP-negative. In all the sections examined however, BrdU-labeled nuclei were preferentially located within the core of the SON, whereas the SON astrocytic cell bodies are localized to the ventral border of the nucleus. The differential evaluation of astrocytic proliferation may rather be related to marked differences in the methods used to label proliferative cells. In the present study, BrdU was injected 5 hours before the fixation of the rats (therefore labeling the cells that have undergone a process of cell division during this short period), while 3 H-thymidine was previously injected twice daily during the whole duration of the hyperosmotic stimulation (14 days). It is thus very likely that under the present conditions, only those cells exhibiting high rate of proliferation were BrdU-labeled, whereas all the cells that have proliferated all along the period of osmotic stimulation were 3 H-thymidine-labeled in the previous study. This clearly indicates that, in the SON of osmotically stimulated rats, the rate of proliferation of SON endothelial cells highly surpasses that of astrocytes. We also show that within the SON of osmotically stimulated rats, high rate proliferation of capillary endothelial cells is accompanied by lower proliferation rate of NG2-labeled cells. Interestingly, the proteoglycan NG2 has been found to be expressed by vascular pericytes surrounding the nascent vessels [ 33 - 35 ]. Although the numerous NG2-labeled cells dispersed throughout the adult rat brain were initially identified as oligodendrocyte precusors [ 15 ], it is now generally admitted that these cells represent multipotent progenitor cells [ 16 ]. It can thus be assumed that at least part of these cells differentiate into vascular pericytes that participate in the formation and/or stabilization of new capillaries [ 36 - 38 ]. SON angiogenesis reversibly modifies the local vasculature The proliferation of endothelial cells, occurring either under physiological or pathological conditions, is generally associated with angiogenesis that leads to the formation of new capillary vessels. The idea that the proliferative response detected within the SON corresponds to local angiogenesis is strongly supported by the present data demonstrating that prolonged osmotic stimulation induced 1) pronounced modifications of the anatomical organization of SON capillary vessels, with both an expansion of a capillary network throughout the increased SON volume and an increase in the network density, and 2) an increased expression by SON capillaries of nestin and vimentin, two intermediate filament proteins highly expressed by newly formed endothelial cells [ 19 , 20 , 22 , 23 ]. Since prolonged osmotic stimuli induce strong activation of the functional activity of VP and OT magnocellular hypothalamic neurons [ 10 ], it can reasonably be assumed that the formation of new vessels within the SON will increase the circulating metabolic support to these neurons. A particularity of magnocellular neurons is that the size of their cell body increases during prolonged osmotic stimulation, leading to a progressive expansion of the volume occupied by the corresponding hypothalamic nuclei [ 11 ]. Newly formed vessels may thus also contribute to the vasularization of the expanded nucleus. An intriguing question raised by the present findings concerns the fate of that SON new vessels formed during the period of osmotic stimulation. Our data clearly show that the density of the capillary network and the phenotypic expression of SON capillary vessels returned to control levels several days after the cessation of the osmotic stimulus. It is thus very likely that newly formed vessels are progressively deleted following rehydration. In control, normally hydrated rats, the SON vascularization however always remains particularly dense as compared with the surrounding regions, and the SON capillary vessels always express high levels of nestin. It can thus be assumed that, even under basal physiological conditions, new vessels are continuously added to the SON vasculature via sparse angiogenic events. Neuronal VEGF is the major stimulus for SON angiogenesis The fact that osmotic stimulus induces reversible angiogenesis within the hypothalamic magnocellular nuclei strongly suggests that local secretion of potent angiogenic factor(s) occurs within these nuclei. Although a variety of angiogenic factors have been identified in the CNS, VEGF is generally recognized as the major factor involved in the processes of angiogenesis [ 25 , 39 , 40 ]. That endogenous VEGF plays a major role in the osmotically induced angiogenesis was suggested by the present findings that 1) contrasting with most CNS neurons, hypothalamic magnocellular neurons continue to express VEGF throughout adulthood, 2) this expression is highly increased by osmotic stimulation, and 3) osmotically induced angiogenic events are impaired when VEGF is inhibited by dexamethasone or by a blocking antibody. Dexamethasone has been shown to inhibit VEGF expression in a large variety of tissue [ 27 - 30 ]. We show here that dexamethasone treatment of osmotically stimulated rats dramatically decreases the VEGF immunostaining associated with magnocellular neurons and, to a lesser extent, astrocytes. Our data further indicate that such decreased VEGF immunostaining of hypothalamic magnocellular neurons correlates with both a potent inhibition of cell proliferation within these nuclei, and a strong decrease of the nestin immunostaining of capillary vessels (not shown). This obviously supports the idea that endogenous VEGF is at least partly responsible for the local angiogenic events occurring within the hypothalamic magnocellular nuclei of hyperosmotically stimulated rats. Since hypothalamic magnocellular neurons of the adult rat express the glucocorticoid receptor [ 41 ], it is very likely that the dexamethasone effect observed here directly results from a direct repression of the VEGF gene. Glucocorticoids are however known to repress a variety of genes [ 42 ], which may also interfere with the angiogenesis described here. In order to demonstrate that endogenous VEGF is directly responsible for SON angiogenesis, we lastly performed a local infusion of a neutralizing antibody to VEGF that has been successfully used in previous studies to block endogenous VEGF activity in the rodent, both in peripheral organs [ 43 , 44 ]and within the brain [ 45 ]. An advantage of this antibody raised in the goat is that its diffusion throughout the brain parenchyma can easily be controlled. In the present study we show that the proliferative response to hyperosmotic stimulus was strongly inhibited within those hypothalamic nuclei that were located within this diffusion zone, but was not affected within the nuclei contra-lateral to the implanted cannula. Moreover, numerous proliferating cells were always detected along the border of lesional cavity induced by the cannula implantation, strongly suggesting that the blocking antibody specifically abolishes the VEGF-dependent cell proliferations, but has only poor effect on the proliferation of glial cells induced by CNS injury [ 46 , 47 ]. Although VEGF was also detected within SON astrocytes, our present observations suggest that the angiogenesis described here is preferentially related to the cytokine of neuronal origin. Within the SON of osmotically stimulated rats, BrdU-labeled proliferating cells were indeed always restricted to the core of the SON containing the VEGF-labeled neuronal cell bodies, and were scarce in the ventral portion of the nucleus containing the labeled astrocytes (Fig. 8 A–B ). Moreover, osmotic stimulus was found to increase the expression of VEGF mRNA in the only SON neurons. In a series of previous studies, astrocytic VEGF has been associated with the angiogenesis occurring in various CNS pathological processes [ 48 - 50 ]. It could thus be assumed that, in the SON as in the other brain regions, neuronal VEGF constitutes the stimulus for physiological angiogenesis, whereas astrocytic VEGF is essentially involved in those angiogenic events occurring under pathological or traumatic conditions. Conclusion In mammals, the final CNS vasculature is established during late postnatal development via active cerebral angiogenesis that is closely correlated with the neuronal expression of VEGF. Thereafter, neuronal VEGF is down-regulated parallely to an increased expression by glial cells, and under basal, non pathological conditions, the vasculature remains essentially quiescent [ 26 ]. In the present study we show that, a simple physiological stimulus can induce reversible angiogenesis within specific hypothalamic nuclei containing neurons that maintain VEGF expression throughout adulthood. Given the importance of angiogenic processes in a number of CNS diseases, these hypothalamic nuclei may thus provide a suitable model to elucidate the cellular and molecular mechanisms that control angiogenesis within the adult CNS. Methods Animals Animals used were adult male Wistar rats of different ages, extending from 10 days to 3 months. They were housed in light (12 h dark and 12 h light) and temperature (21 ± 1°C) controlled rooms. Adult rats (2–3-month-old) were divided into three groups including 1) control rats that had free access to standard dry food and tap water, 2) hyperosmotically stimulated rats that were given 2% NaCl solution as drinking fluid during 1 to 10 days, and 3) rehydrated rats that were in hyperosmotically stimulated during 6 days and were then given tap water as drinking fluid during 1 to 5 days. All these animals were treated in accordance with the principles of laboratory animal care published by the French Ethical Committee. Implantation of intracerebral cannulas After deep anesthesia with equithesine (3 ml/kg), animals were placed in a David Kopf stereotaxic device and a stainless-steel cannula (26 gauge, 10 mm long) was placed dorso-laterally to the right SON according to the stereotaxic atlas of Paxinos and Watson (1982): 1.3 mm posterior to bregma; 2.1 mm lateral; 8.5 mm dorsoventral (Fig. 9 ). The cannula was connected via a polyethylene tubing to a miniature Alzet osmotic pump (model 1007D, delivery rate 0.5 μl per hour during 7 days) filled up with either 1) a goat anti-human VEGF neutralizing antibody (R&D Systems, diluted 30 μg/ml in phosphate buffer saline, PBS) known to successively block rodent VEGF [ 51 ] (n = 5), or 2) normal goat IgG (30 μg/ml) (n = 4). The cannula was cemented in place adjacent to an anchor screw inserted in the skull, and the Alzet pump was inserted into a subcutaneous pocket made with sterile forceps over the rat neck and shoulder blades. Twenty four hours after the surgery, animals were given 2% saline as drinking fluid during 5 days and thereafter fixed by intracardiac perfusion after BrdU administration as described below. Dexamethasone treatment Control rats (cont, n = 4) and dexamethasone-treated rats (dexa, n = 7) received a daily subcutaneous injection of 0.5 ml saline or dexamethasone (5 mg/kg in 0.5 ml saline), respectively, during 6 days. The first day of the treatment, they were given 2% saline as drinking fluid during 6 days and thereafter fixed by intracardiac perfusion after BrdU administration as described below. Administration of bromodeoxyuridine (BrdU) BrdU (Sigma) was administered intraperitoneally (150 mg/kg in 0.5 ml of 0.01N hydroxy-chloride solution) 5 hours before the fixation of the animals. In situ hybridization Adult rats either normally hydrated (control, n = 3) or drinking 2% saline during 6 days (osmotically stimulated, n = 3) were killed by decapitation. Brains were rapidly dissected, frozen at -40°C in isopentane and stored at -80°C. Coronal sections of hypothalamus (20 μm) were cut with a cryostat (Leica CM3000), thaw mounted on polylysin coated slides, fixed in 4% paraformaldehyde in PBS buffer (in mM: NaCl 130, Na 2 HPO 4 7, NaH 2 PO 4 3) for 5 min at 4°C, rinsed in PBS, dehydrated in increasing concentrations of ethanol (70 and 95%) and air dried. In situ hybridization was performed with oligonucleotide probes complementary to VEGF (482–529; Genbank accession n°AF215725) that did not match any known sequence in Genbank except those of the intended genes. Oligonucleotide probes were labeled at their 3' end with digoxigenin 11-dUTP using terminal transferase (Roche). Sections were incubated in a humid chamber overnight at 43°C with the primers (10 nM) in hybridization buffer. They were rinsed at 45°C in decreasing concentrations of saline-sodium citrate, followed by a final 30 min wash in the buffer supplemented with 5% BSA and 0.1% N-Lauroylsarcosine. Slides were then incubated 2–3 h with a sheep antidigoxigenin antibody coupled to alkaline phosphatase (Roche) diluted 1/500 in buffer supplemented with 5% BSA, carefully rinsed, and incubated 12 hours with nitroblue tetrazolium and 5-bromo 4-chloro 3-indolylphosphate (Roche). After mounting in Mowiol (Calbiochem, La Jolla, CA), slides were viewed under a light microscope. No hybridization signal was detected in control sections hybridized with a 50-mer random probe not complementary to any sequence in Genbank [ 52 ]. Immunocytochemistry After deep anesthesia with Equithesin, animals were either 1) decapitated and, after rapid dissection, the brain was fixed by immerssion in a solution of 4% paraformaldehyde 0.1 M phosphate buffer, pH 7, or 2) perfused through the ascending aorta with phosphate-buffered saline (PBS), pH 7.4, followed by 100 to 500 ml of fixative. The forebrain was dissected and fixed by immersion overnight in the same fixative. It was then cut frontally with a vibratome into 40–50-μm thick sections. These were carefully rinsed in PBS and subsequently treated for multiple fluorescence labeling. The vibratome sections were incubated for 48 hours at 4°C with one or two primary antibodies including polyclonal rabbit IgG, polyclonal goat IgG, monoclonal mouse IgG or IgM, and monoclonal rat IgG antibodies (The different markers, vendors and concentrations used in this study are listed in Table 1 ). After rinsing in PBS, they were incubated for 4 hours at 4°C with corresponding secondary antibodies conjugated with Alexa-488 (Molecular Probes, Eugene, USA) or with Cy3 (Jackson Laboratories, USA). The primary and secondary antibodies were diluted in PBS containing 1% normal goat or donkey serum and 0.1% Triton X-100. Sections treated for the immunodetection of BrdU were incubated within 2N HCL for 30 minutes at room temperature, carefully rinsed in PBS, and incubated 48 h at 4°C with a mouse or rat monoclonal antibody anti-BrdU, either alone or in combination with another primary antibody. Table 1 Antibodies used Marker Species Working dilution Vendor or productor Proliferation BrdU Mouse IgG 1:500 Novo Castra (Newcastle, UK) BrdU Rat IgG 1:500 Accurate Chemicals (New York, USA) Magnocellular neurons OT Rabbit IgG 1:2000 Produced by G. Alonso VP Rabbit IgG 1:2000 Produced by G. Alonso Astrocytes GFAP Rabbit IgG 1:5000 Dakko (Glostrup, Denmark) Microglia and endothelial cells Lectin from bandeirae 1:2000 Sigma Simplifolia (IsoB4) Oligodendrocyte progenitors NG2 Rabbit IgG 1:500 Chemicon (Temecula, USA) Vessels Rat IgG-Alexa 488 conjugated Goat IgG 1:2000 Molecular Probes (Eugene, USA) EBA Mouse IGM 1:5000 Sternberger Monoclonals (Lutherville, USA) Nestin Mouse IgG 1:100 Hybridoma Bank (Iowa city, USA) Vimentin Mouse IgG 1:5000 Sigma BrdU: bromodeoxy uridine; EBA: endothelial brain antigen; GFAP: glial fibrillary acidic protein; IgG-IgM: immunoglobulins type G and M; OT: oxytocin; VP: vasopressin. The specificity of the vasopressin, oxytocin and of commercial antibodies has been assessed by absorption tests. Additional controls consisted of (1) omitting the primary antibodies and applying the secondary antibodies alone, (2) applying each primary antibody sequentially and then reacting them with an inappropriate secondary antibody, and (3) exciting each fluorochrome by the inappropriate illumination. This allowed us to confirm that the two secondary antibodies used in double immunostaining experiments did not induce artifactual fluorescent labelling and that there was no overlap of the emission spectra of the two fluorochromes. Imaging and quantification After rinsing in PBS, labeled sections were mounted in Mowiol and observed under a Biorad MRC 1024 confocal laser scanning microscope equipped with a krypton/argon mixed gas laser. Two laser lines emitting at 488 nm and 568 nm were used for exciting GFP and the Alexa-488 or Cy3-conjugated secondary markers. The background noise of each confocal image was reduced by averaging four image inputs. The organization of the immunostained structures was studied 1) on single confocal images or 2) on bi-dimensional reconstructed images obtained by collecting 5 to 10 consecutive confocal images 1 μm apart through the whole vibratome section thickness, and by projecting on the same plane. Unaltered digitalized images were transferred to a PC type computer and Adobe Photoshop was used to prepare final figures. Quantitative analysis was performed on series of sections passing through the middle portions of the SON (i.e. the largest SON areas): 1) The cell proliferation was quantified by counting the BrdU-labeled nuclei detected in four SON areas per rat, in at least three rats per experiment, 2) The nature of proliferative cells was evaluated on sections double-labeled for BrdU and a specific cell type marker. The number of double-labeled cells was pooled among at least 4 SON areas per rat, in 4 rats, and 3) The anatomical organization of SON vessels was evaluated on sections of rat brains fixed by immerssion and double immunostained for Rat-IgG and for VP + OT. The density of SON vessels was quantified according to the point-counting method [ 53 ], by scoring the number of point intersections of Rat-IgG-labeled vessels with a grid centered either on the SON area containing the neurons labeled for VP or OT or on the lateral hypothalamus overlying the SON. The total amount of SON vessels was evaluated by measuring the surface occupied by Rat-IgG-labeled vessels (% of fluorescent pixels on binary images) within the SON area containing the VP + OT-labeled cell bodies (with the Image Tool image analysis system, University of Texas Health Science Center, San Antonio, USA). All quantifications were performed on at least 4 SON areas per rat, and for at least 3 rats per experiment. Data were statistically compared by using the non parametric test of Mann and Whitney. List of abbreviations used AN: accessory magnocellular nuclei BrdU: bromodeoxyuridine CNS: central nervous system EBA: endothelial brain antigen GFAP: glial fibrillary protein IgG: immunoglobulin type G IsoB4: isolectin B4 NeuN: neuronal nuclear antigen NG2: NG2 chondroitin sulfate proteoglycan OC: optic chiasma OT: oxytocin PVN: paraventricular nucleus SON: supraoptic nucleus VP: vasopressin VEGF: vascular endothelial growth factor Authors' contributions GA conceived the study, conducted most experiments and wrote the paper; EG and AG conducted the immunocytochemical experiments; AV conducted the in situ hybridization experiments. Table 2 Quantitative evaluation of the nature of SON proliferative cells % of BrdU-labeled nuclei associated with specific cell markers Cell type Marker Neurons VP + OT 0 Astrocytes GFAP 6 Oligo. Precursors NG2 18 Microglia IsoB4 8 Vessels IsoB4 60 Vessels Nestin 67 Vessels EBA 55 BrdU was administrated to osmotically stimulated rats drinking 2% saline during 6 days, and the animals were fixed 5 hours after the BrdU administration. The relative proportion of each BrdU-labeled cell type was calculated from at least 4 sections per rat in 4 stimulated rats. More than 500 BrdU-labeled nuclei were scored for most markers. GFAP: glial fibrillary acidic protein; EBA: endothelial brain antigen; NG2: proteoglycan NG2; OT: oxytocin; VP: vasopressin.

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1079869

Functional modulation of human delta opioid receptor by neuropeptide FF

Background Neuropeptide FF (NPFF) plays a role in physiological pain sensation and opioid analgesia. For example, NPFF potentiates opiate-induced analgesia and the delta opioid receptor antagonist naltrindole inhibits NPFF-induced antinociception. The nature of the interactions between NPFF and opioid receptors seems to be complex and the molecular mechanisms behind the observed physiological effects are not known. Results We used a stable Chinese hamster ovary cell line expressing c-MYC-tagged human delta opioid receptor to study the interactions at the molecular level. Our results imply that NPFF can directly modulate the activation of delta opioid receptor in the absence of NPFF receptors. The modulatory effect, though only moderate, was consistently detected with several methods. The agonist-induced receptor trafficking was changed in the presence of (1DMe)NPYF, a stable NPFF-analogue. (1DMe)NPYF enhanced the receptor activation and recovery; opioid antagonists inhibited the effects, indicating that they were delta opioid receptor-mediated. The binding experiments with a novel ligand, Terbium-labeled deltorphin I, showed that (1DMe)NPYF modulated the binding of delta opioid receptor ligands. The levels of phosphorylated mitogen-activated protein kinase and intracellular cAMP were studied to clarify the effects of NPFF on the opioid signaling mechanisms. Application of (1DMe)NPYF together with a delta opioid receptor agonist enhanced the signaling via both pathways studied. Concomitantly to the receptor trafficking, the time-course of the activation of the signaling was altered. Conclusion In addition to working via indirect mechanisms on the opioid systems, NPFF may exert a direct modulatory effect on the delta opioid receptor. NPFF may be a multi-functional neuropeptide that regulates several neuronal systems depending on the site of action.

Background Neuropeptide FF (NPFF) belongs to a family of RFamide peptides and was originally isolated from bovine brain [ 1 - 3 ]. It has a wide range of functions, including effects on pain mechanisms [ 1 , 4 ], opioid tolerance [ 5 ], cardiovascular regulation [ 6 ] and neuroendocrinological function [ 7 ]. At the physiological level NPFF seems to have both a direct analgesic effect and a modulatory effect on the opioid system. Some of the effects may be mediated via the NPFF receptors as two such receptors, NPFF1R and NPFF2R, have been identified [ 17 - 19 ]. Both NPFF1R and NPFF2R are expressed in the central nervous system and NPFF binds to both of them [ 17 - 20 ]. Also the other RFamide peptides bind to the NPFF receptors with varying affinities [ 21 ], and therefore the exact nature of the receptor-ligand interactions between RFamides and their receptors is still unclear. The interaction between NPFF and opioid system in pain and analgesia seems to be complex in nature and the molecular mechanisms behind the observed physiological effects are not known. Binding studies have shown that NPFF does not displace opioid receptor ligands from any of the opioid receptor subtypes and opiates do not bind to NPFF binding sites [ 16 ]. However, many studies suggest that NPFF mechanisms are functionally coupled to the opioid system [for a review see ref. [ 8 ]]. In the rat spinal cord, the highest NPFF-like immunoreactivity is found in the superficial layers of the dorsal horn, an area involved in the nociceptive processes and pain mechanisms [ 9 - 11 ]. NPFF has been designated as a morphine modulatory peptide since it is able to influence the actions of opioid peptides within spinal cord and brain [ 8 , 12 , 13 ]. NPFF displays both anti-opioid and opioid-like effects depending on the route of administration. Supraspinal administration of NPFF attenuates opioid antinociception [ 1 ] and precipitates opioid withdrawal syndrome [ 5 ]. Intrathecally administered NPFF causes long-lasting analgesia, which is reduced by both naloxone and naltrindole [ 4 ]. NPFF in the periaqueductal grey produces a selective attenuation of tactile allodynia in neuropathic rats [ 14 ] that could be mediated indirectly by naloxone-sensitive opioid mechanisms [ 15 ]. In pontine parabrachial nucleus NPFF modulates synaptic transmission through interaction with presynaptic DOR, providing evidence for the cellular mechanisms of the analgesic action of NPFF at the supraspinal level [ 12 ]. Delta opioid receptor (DOR) belongs to the family of G-protein coupled, seven trans-membrane receptors [ 22 , 23 ]. DOR couples to the pertussis toxin -sensitive G i/o -type of heterotrimeric G-proteins. The receptor can regulate several effector systems, including adenylyl cyclase activity [ 22 , 24 ], the phosphorylation of mitogen activated protein kinases (MAPK) [ 25 ], voltage-gated calcium and potassium channels [ 26 ] and phospholipase C [ 27 ]. In CHO-cells the DOR-induced activation of MAPK-pathway is predominantly mediated by the Gβγ-subunit of G i/o [ 28 ] whereas adenylate cyclase response is mediated by the Gα i/o -subunit [ 24 ]. The involvement of DOR in analgesia has been shown using many pain models [ 29 ] and agonists acting at DOR have a strong antinociceptive effect [ 30 ]. The agonist stimulation causes rapid desensitization of the receptor by phosphorylation [ 31 ], which in turn produces the uncoupling of the receptor from its G-protein. The phosphorylation can be followed by endocytosis of the ligand-receptor complex [ 32 ] but the desensitization may occur also without receptor internalization [ 31 ]. The internalized receptor is either degraded or recycled back to the cell membrane [ 32 , 33 ]. We constructed a stable Chinese hamster ovary cell line expressing c-MYC-tagged human DOR (MYChDOR). The cell line was used as a model system to learn about the mechanisms involved in the interactions between NPFF and hDOR at the cellular and molecular level. Results Characterization of the cell line The cellular localization of the expressed c-MYC-tagged delta opioid receptors in the stable CHO-K1 clone was analyzed with immunocytochemistry. The cells were stained with an anti-c-MYC antibody, detected with a fluorescent secondary antibody and analyzed with a laser scanning confocal microscope. The untransfected wild type cells did not show any fluorescence when stained similarly as the CHO/MYChDOR cells and the pre-absorption of the antibody solution with the c-MYC-peptide before immunocytochemistry removed all signal in the CHO/MYChDOR cells, implying that the antibody bound specifically to the MYC-tagged receptor (data not shown). In the CHO/MYChDOR cells a clear fluorescence signal was seen on the cell surface (Figure 1A ) and DOR agonists induced the internalization of the receptor (Figure 1B & 1C ). The receptor density and ligand binding characteristics of the expressed receptor were determined with 3 H-diprenorphine binding assay. The observed values for K D and B MAX were 0.203 ± 0.090 nM and 0.530 ± 0.042 pmol/mg protein (318000 receptors/cell), respectively. The functionality of cells expressing MYC-tagged DOR was shown with the inhibition of forskolin-stimulated accumulation of cAMP and phosphorylation of ERK2. DPDPE dose-dependently inhibited the forskolin-induced accumulation of cAMP and similarly increased the phosphorylation of the ERK2 (data not shown and see below). The possible presence of NPFF1R and NPFF2R in the CHO-K1 cells was studied with several methods. First the cells were studied with immunocytochemistry and confocal microscopy. Several antibodies against NPFF receptors were used. In the Figure 2A–D the results obtained with two antibodies are shown. The pre-absorption of the antibody solutions with the antigenic peptide before immunocytochemistry removed all immunoreactivity from CHO-K1/hNPFF2R cells in the case of each antibody tested, implying that the antibodies recognized specifically the antigen. No immunoreactivity was found in the untransfected CHO-K1 cells (Figure 1B & 1D ), which further suggested that the antibodies bound specifically to the hNPFF2R. Next the cells were analyzed with RT-PCR. cDNA made of total RNA from human medial hypothalamus and human placenta were used as positive controls for NPFF1R and NPFF2R, respectively, since a relatively high level of receptor expression has been shown in these tissues [ 17 - 20 ]. NPFF1R and NPFF2R specific primers based on human and mouse receptor sequences were used in the analysis. No PCR products were obtained from the CHO-K1 cDNA using conditions where the controls tested positive (Figure 1E & 1F ). The binding of NPFF receptor specific radioligand 125 I-(1DMe)NPYF to the CHO-K1 cells was tested. The cells did not bind the 125 I-(1DMe)NPYF (Figure 2G ). Cell membranes of a commercial hNPFF2R expressing CHO-K1 cell line (Euroscreen S.A., Brussels, Belgium) were used as a positive control in the experiment (Figure 2H ). 125 I-(1DMe)NPYF bound to these cell membranes with the same affinity as reported by the supplier and as observed previously in our laboratory [ 21 ]. In addition, the NPFF-analogue alone did not affect any of the tested parameters, such as cAMP or MAPK (see below), which further supported the absence of NPFF receptors in CHO-K1 cells. Effect of (1DMe)NPYF on the agonist activated DOR trafficking The CHO-K1 cells expressing MYC-tagged DOR were treated with 100 nM DPDPE (or DeltI) with or without 100 nM (1DMe)NPYF for 0, 5, 10 or 30 min at 37°C. The receptor trafficking was analyzed with fluorescence associated cell sorting, FACS. After the drug treatments, the cell surface receptors were detected with an anti-MYC-antibody and a fluorescent secondary antibody. At the basal state the amount of the cell surface fluorescence, which should reflect the amount of DORs located on the cell membrane, was observed to be variable between runs. To control the variation the cells were cultured for a fixed period of time after the last passage prior to internalization studies and grown into 80–90 % confluence. The cells were let to recover at 37°C after they were collected from the culture plates and before they were used for the experiments, to ensure that the receptor number on the cell membrane was balanced. Some constitutive receptor trafficking was still observed and therefore the treated cells at each time-point were compared to time-matched untreated control cells in order to distinguish between constitutive and ligand-induced receptor trafficking. As seen in the FACS analysis, (1DMe)NPYF (100 nM) alone did not induce receptor internalization at any time-point tested (Figure 3 ). However, after 30 min treatment with the NPFF analogue somewhat higher fluorescence was detected on the cell surface as in the time-matched control cells. Already after 5 min treatment with 100 nM DPDPE or with 100 nM DPDPE and 100 nM (1DMe)NPYF the cell surface fluorescence was significantly different from the untreated control cells, indicative of ligand-induced internalization of the receptor. There was no significant difference between the treatments at this time-point although the double treatment with 100 nM DPDPE and 100 nM (1DMe)NPYF showed a slight tendency to decrease the surface fluorescence more. At 10 min time-point the treatment with 100 nM DPDPE together with 100 nM (1DMe)NPYF (57.9 % decrease in the cell surface fluorescence) caused significantly greater internalization of DOR than the treatment with 100 nM DPDPE alone (32.7 % decrease in the cell surface fluorescence) (Figure 3 ). At the 30 min time-point the cell surface fluorescence was at a significantly reduced level after both treatments but no significant difference was detected between the treatments (DPDPE alone and DPDPE together with (1DMe)NPYF). Characterization of Tb-DeltI and its binding on whole cells To further investigate the effects of (1DMe)NPYF on DOR function a binding assay based on time-resolved fluorometry was developed. Tb-labeled DeltI was custom-made at Perkin Elmer Wallac. DeltI was labeled with a Tb-chelate to the Cys-modified N-terminus of the nascent peptide. To check whether the modification changed the affinity of the peptide, Tb-DeltI was tested in a classical radioligand displacement assay. Tb-DeltI displaced the 3 H-diprenorphine from CHO/MYChDOR -cell membranes with similar affinity to the unlabeled DeltI (Figure 4A ). Tb-DeltI bound to CHO/MYChDOR cell membranes dose-dependently and the binding was saturable at a nanomolar range (Figure 4B ). The results with fixed whole cells showed similar binding characteristics as with the cell membranes, although the absolute values were below the ones observed with the cell membrane preparations. It was considered that the whole cell protocol can be used to study the effects of (1DMe)NPYF on the binding of Tb-DeltI on CHO/MYChDOR since only relative data between the treatments was desired. (1DMe)NPYF alone could not significantly displace Tb-DeltI. Next the effect of (1DMe)NPYF on the specific binding of Tb-DeltI was studied. The total binding of Tb-DeltI on the CHO/MYChDOR cells was determined in the presence of different concentrations of (1DMe)NPYF. The unspecific binding of Tb-DeltI was determined by 1 μM DeltI. (1DMe)NPYF modified the specific binding of Tb-DeltI to the CHO/MYChDOR cells. The K D for Tb-DeltI was only modestly affected by (1DMe)NPYF whereas 1 nM and 10 nM (1DMe)NPYF significantly decreased the B MAX for Tb-DeltI (Table 1 ). The higher concentrations of (1DMe)NPYF affected the B MAX only modestly. Inhibition of the forskolin induced accumulation of the cAMP DOR inhibits adenylate cyclase activity in numerous tissues and cell lines via coupling to G i/o -protein. We examined the ability of the ligand-activated MYC-tagged DOR to inhibit the forskolin stimulated cAMP accumulation in CHO-K1 cells. As expected, already 5 min treatment of the cells with the DOR agonist DPDPE (100 nM) reduced the level of forskolin-stimulated cAMP significantly (Figure 5A ). Similarly, the treatment with DPDPE together with 100 nM (1DMe)NPYF at this time-point and at 10 min time-point inhibited the accumulation of cAMP. Although there was a slight tendency for (1DMe)NPYF to enhance the effect of DPDPE already at 5 min and 10 min time-points, the difference between the treatments was not significant. It was only after 30 min treatment that (1DMe)NPYF had a robust effect on the inhibition of cAMP induced by DPDPE (Figure 5A ). At 30 min time-point the double-treatment with DPDPE + (1DMe)NPYF resulted in 76 % inhibition of forskolin stimulated cAMP accumulation in comparison to 57 % inhibition when treating the cells with DPDPE alone. (1DMe)NPYF alone did not have any significant effect on the amount of cAMP. Both the effect of DPDPE and DPDPE + (1DMe)NPYF was completely blocked by the pre-treatment with pertussis toxin (Figure 5A ). (1DMe)NPYF was also able to enhance the inhibitory effect of other DOR agonists, e.g. DeltI, on the cAMP accumulation (data not shown), indicating that the modulatory effect of (1DMe)NPYF on the inhibition of cAMP accumulation is not agonist specific. The effect of (1DMe)NPYF dose on the inhibitory action of DPDPE on the cAMP accumulation was also studied. The cAMP accumulation was measured at 30 min time-point. Above 100 nM DPDPE the cAMP inhibition reached saturation and addition of higher concentration of DPDPE did not result in further inhibition of forskolin stimulated cAMP accumulation (Figure 5B & 5C ). (1DMe)NPYF had a significant effect on the DPDPE-induced cAMP inhibition (two-way Anova, DPDPE p < 0.001; (1DMe)NPYF p < 0.001, [DPDPE] × [(1DMe)NPYF p < 0.01, n = 3). More specifically, 10 nM and 100 nM (1DMe)NPYF together with DPDPE enhanced the DOR agonist induced inhibition of cAMP accumulation significantly at all tested DPDPE concentrations (Figure 5B ) whereas the lowest and highest tested concentrations of (1DMe)NPYF had hardly an effect on the cAMP accumulation (Figure 5C ). Activation of MAP-kinase signaling pathway The activation of MAP-kinase p42 MAPK (ERK2) is characterized by the appearance of the phosphorylated form of the ERK2. The phosphorylated ERK2 was detected on the immunoblots after the cells were treated with DPDPE, (1DMe)NPYF or both for 0, 5, 10 and 30 minutes at 37°C. The basal level (t = 0 min) of phosphorylated ERK2 was at the detection limit of the antibody and only a very faint band could be detected (Figure 6A , left-most lane). When quantified, this band did not markedly differ from the background. The treatment of the cells with (1DMe)NPYF alone did not result in the activation of ERK2 (Figure 6A , right panel). Neither did any of the treatments significantly affect the total amount of ERK2. The total ERK2 was controlled from the immunoblots with an antibody detecting the unphosphorylated form of the kinase, which was compared to the amount of tubulin in each sample. DPDPE (100 nM) activated the MAPK pathway as expected (Figure 6A and 6B ). The peak in activation was observed after 10 minutes DPDPE-treatment followed by a decrease in the amount of the phosphorylated kinase. The addition of (1DMe)NPYF (100 nM) together with DPDPE (100 nM) significantly promoted the activation induced by DPDPE. The peak in activation with DPDPE + (1DMe)NPYF was detected already after 5 min in contrast to 10 min peak-activation time for DPDPE alone. At 10 min time-point there was no significant difference in the amount of phosphorylated ERK2 between DPDPE and DPDPE + (1DMe)NPYF-treated cells. However, at this time-point the level of phosphorylated ERK2 in the DPDPE + (1DMe)NPYF-treated cells had already started to decline whereas in the DPDPE-treated cells the level of phosphorylated ERK2 was still increasing when compared to the earlier and later time-points. In both DPDPE-treated cells and DPDPE + (1DMe)NPYF-treated cells after 30 minutes agonist stimulation the level of activated ERK2 had almost decreased back to basal values. Discussion NPFF's role in physiological pain sensation and opioid analgesia is well characterized. Some of the NPFF's pain-related effects are mediated by the delta opioid receptor system [for review see ref. [ 8 ]]. In this study we examined the role of NPFF in the modulation of DOR-mediated cellular responses. We wanted to establish a simple model system to study the effect of NPFF analogue, (1DMe)NPYF, on the DOR function in the absence of NPFF receptors. We generated a cell line expressing N-terminally c-MYC-epitope tagged human DOR at close to physiological expression levels (see below). We also verified that the cells do not express functional NPFF receptors. The expression level and binding affinity of the selected CHO/MYChDOR clone was within the same range as that reported for the well-studied NG108-15 neuroblastoma cell line [ 34 ] and for some native neuronal tissues e.g. rat striatum [ 35 ]. At this expression level the receptor trafficking and cellular signaling of the cells are expected to be normal but extreme over expression of a receptor might disrupt the functional properties of the cells. The cells responded to DOR ligands as expected in two different functional assays and the ligands also induced the internalization of DOR. In the basal state, some receptors were found in the internal parts of the cells, which is consistent with the previous findings for the delta opioid receptor [ 36 ]. Taken together, the pharmacological studies and confocal microscopic analysis together with functional assays suggested that the N-terminal modification of hDOR did not affect the ligand selectivity, functional coupling or trafficking of the expressed receptors. DOR belongs to the large family 1 of GPCRs. We hypothesized that instead of, or in addition to operating via indirect mechanisms, NPFF could have a direct modulatory effect on DOR. Compounds that modulate the receptor activation have been described for other members of the receptor family [for a review see ref. [ 37 ]]. Modulators can potentate or block the agonist stimulation of the receptor in many ways, such as by changing the agonist affinity or stability of receptor-G-protein interaction [ 38 - 40 ]. Our FACS studies showed that in the presence of (1DMe)NPYF the agonist-induced internalization of DOR was enhanced. The increase in the receptor internalization by (1DMe)NPYF was most obvious after 10 min agonist exposure. The DOR antagonist naltrindole could completely block, not only the receptor activation by DOR agonists alone, but also the enhanced activation resulting from the co-application of (1DMe)NPYF with DOR agonists. This provides evidence that the observed effects were solely DOR-mediated. In vivo many of the analgesic effects of NPFF are blocked by both the non-specific opioid-antagonist naloxone and the DOR-specific antagonists naltrindole [ 4 , 44 ]. The differences between time-points and treatments in the FACS analysis were consistent and reproducible as assessed with statistical methods. To study the effect of NPFF in DOR systems, we introduced a new tool to study the interactions between receptors and their ligands. A Tb-labeled DOR-ligand was used in a time-resolved fluorescence based binding assay and it proved comparable to the traditional radiolabel-based methods. Tb-DeltI could be applied to both cell membrane and whole cell assays. This new ligand and other related compounds open interesting possibilities to further study the ligand-receptor interactions, by using methods such as time-resolved FRET. Our results from the time-resolved fluorescence based binding assays indicate that NPFF can modulate the binding of DOR ligands to the receptor. Consistent with the earlier reports with spinal cord membranes [ 16 ], (1DMe)NPYF did not markedly bind to the classical DOR-binding site in the CHO/MYChDOR cells. Only micromolar concentration of (1DMe)NPYF could displace Tb-DeltI in whole cell and membrane binding assays to some extent. The binding experiments showed that (1DMe)NPYF affected the K D of Tb-DeltI only slightly, whereas the B MAX values were significantly decreased by low to modest concentrations of (1DMe)NPYF. It should be noted that the effects of (1DMe)NPYF on ligand binding observed in assays using a labeled agonist Tb-DeltI were partly verified by using a labeled antagonist, 3 H-diprenorphine, as the ligand. In addition, while most of the experiments were performed with whole cells, a protocol that mimics the cellular environment for the natural receptor binding, the results were also to some extent confirmed with cell membrane preparations. This was to avoid the contribution of possible endogenous effector molecules present in whole cells. The lack of change in the K D for opioid ligands implies that especially at low concentrations (1DMe)NPYF does not work through the classical opioid binding site of DOR. The dose-dependence of NPFF-agonist on the B MAX was bell-shaped. This could imply that the NPFF-analogue affects only some conformations of the receptor. Interestingly, also the effect of NPFF on prolactin release from rat pituitary cells show a bell-shaped dose-response curve [ 41 ]. However, the receptor involved in the prolactin release mechanism was not characterized. The NPFF-analogue had an effect on the B MAX at lower concentrations (1–10 nM) than on the signaling cascades studied. This could be related to the mechanism of the functional modulation of DOR by the NPFF-analogue. One possible mechanism could involve a modulatory binding site for NPFF on DOR. Modulatory binding sites have been described for other members of the GPCR family [ 37 - 40 ]. In line with our results, the GPCR modulators exert their effects commonly only in the presence of the receptor ligand [ 37 ]. In the future, the exact nature of interactions between DOR and NPFF will be further studied. The modulatory effect of (1DMe)NPYF on DOR-mediated signaling was studied by measuring the activation of two differently regulated signaling cascades. The signaling studies suggested that (1DMe)NPYF alone did not affect DOR function but when applied together with a DOR agonist it significantly promoted the response of the activated signaling cascade. (1DMe)NPYF could enhance the DOR agonist induced inhibition of forskolin-stimulated cAMP accumulation, a signaling cascade primarily regulated by the G i/o α-subunit. The signaling via DOR was most strongly affected by (1DMe)NPYF at low to modest concentrations of DOR agonist. The cAMP inhibition resulting from double treatment with DPDPE + (1DMe)NPYF was greater than inhibition obtained by treatment with DPDPE alone at a broad DPDPE concentration range with various concentrations of the NPFF analogue. Also the maximal cAMP inhibition obtained with the double treatment was greater than that observed after treating the cells with a DOR agonist alone. In addition to the regulation of cAMP levels, (1DMe)NPYF enhanced the DOR agonist-induced phosphorylation of ERK2, a member of MAP-kinase family. The activation ERK2 phosphorylation follows a series of activation steps initiated by the Gβγ-subunit. Interestingly, in the cells subjected to (1DMe)NPYF together with a DOR agonist an earlier peak in activation of ERK2 was observed than in cells treated with a DOR agonist alone. The finding that NPFF affects two differently regulated signaling cascades downstream from G-protein activation gives support for our hypothesis that NPFF works at the receptor level, prior to the G-protein activation. Both the DOR agonist-induced activation of receptor-mediated signaling and the enhanced activation caused by the addition of (1DMe)NPYF together with DOR agonist were completely blocked by PTX, giving evidence that NPFF delivers its effects via the same G-proteins as the DOR agonists binding to the classical binding site. The enhancement of the DOR-mediated signaling could be due to the increased receptor trafficking. The accelerated internalization of the membrane-bound receptors could account for the enhanced signaling observed. The effect of (1DMe)NPYF on the internalization of DOR was transient and relatively small. There is some evidence that DOR ligands can affect the balance between receptors located on the cell membrane and receptors residing in the internal parts of the cells [ 45 , 46 ]. Petäjä-Repo et al. [ 45 ] have shown that DOR ligands can work as molecular chaperones, i.e. ligand binding to the receptor induces the transport of receptors to the membrane. It is likely that also the increased recruitment of the receptors from the internal pools to the cell membrane could account for the observed effects. Cahill et al. [ 46 ] showed that enhanced antinociceptive activity of DeltI is correlated with the recruitment of spinal opioid receptors from intracellular stores to the plasma membrane and that inflammation, a state that activates DOR -mechanisms, produces outward movement of intracellular DORs towards the plasma membrane. Therefore, the observed rate of internalization may be masked to some extent by the recruitment of the receptors from the internal pools. It should be noted that all the phenomena described above were observed with both the peptidic agonist DeltI and the cyclic agonist DPDPE. This implies that the effect is not between the agonists and (1DMe)NPYF but rather that (1DMe)NPYF modifies the receptor and alters its characteristics. Conclusion Our results suggest that some of the NPFF's antinociceptive effects and interactions with opioid systems could at least in part be mediated directly via DOR. The agonist-induced internalization of DOR and the DOR mediated signaling are enhanced by NPFF, the specific binding of ligands to DOR is modified by NPFF. All changes observed in the different assays were in agreement with each other, although they had a bit different time-courses, and they all support the suggested direct interaction between NPFF and the DOR-ligand complex. The different time-course of events could be due to the various regulatory mechanisms involved. For example, the inhibition of the adenylate cyclase activity is directly regulated by the G i/o -protein α-subunit and the phosphorylation of ERK2 is mainly activated by the Gβγ subunit. The observed effects are also physiologically relevant, as published data suggest that opiate-induced analgesia is potentiated by NPFF [ 44 ], and the DOR antagonist naltrindole inhibits the NPFF-induced antinociception [ 4 ]. Although the modulatory effect of (1DMe)NPYF was moderate or in some cases quite small, it was consistently observed with all methods used in this study. As mentioned earlier, CHO-K1 cells do not express detectable levels of NPFF receptors. Therefore, DOR alone could be responsible for the modulatory effects of (1DMe)NPYF observed in this study. The results were obtained using a heterologous expression system and therefore they cannot be directly extended to in vivo conditions. Recent data based on opioid knockout mice show that DOR is mainly involved in the inflammatory and mechanical nociception [ 47 ]. NPFF expression is also significantly increased in inflammatory pain [ 3 ], and NPFF modulates descending inhibitory input to the wide-dynamic range neurons [ 8 ]. Some of the effects of NPFF in pathological pain and interactions with morphine may be mediated via DOR. This does not exclude e.g. the possible involvement of NPFF2R in pain mechanisms [ 18 ]. As two receptors for NPFF have been characterized, it seems likely that NPFF can have several functions depending on the site of action and availability of receptors. NPFF may be a multi-functional neuropeptide capable of affecting several neuronal systems through different receptors and signaling pathways, the effects depending on the other players present at the site action. Methods Materials Cell culture medium components were from BioWhittaker (East Rutherford, NJ, USA), Sigma-Aldrich (St. Louis, MO, USA) or Gibco BRL/Invitrogen (Carlsbad, CA, USA). Blasticidin S HCl was from Invitrogen and FuGene 6 from Roche (Basel, Switzerland). [D-Pen(2,5)]enkephalin (DPDPE) and naloxone were purchased from Sigma. Deltorphin I (Y-D-AFDVVG-NH 2 ) was from Bachem (Bubendorf, Switzerland). (1DMe)NPYF (D-YL-(NMe)-FQPQRF-NH 2 ) was either custom-synthesized (Peptide Technologies, Gaithersburg, MD, USA) or from Bachem. Tb-labeled DeltI was custom made at Wallac (Perkin Elmer Wallac, Turku Finland) and all the reagents for the time-resolved fluorometry were purchased from Wallac. Mouse monoclonal anti-c-MYC antibody 9E10 and the c-MYC blocking peptide were from Sigma-Aldrich, rabbit polyclonal anti-c-MYC 9E10 antibody was from Santa Cruz Biotechnologies (Santa Cruz, California, USA), rabbit polyclonal C-terminal and N-terminal anti-NPFF2R antibodies and blocking peptides from Novus Biologicals (Littleton, CO, USA), rabbit anti-phospho-ERK1/2 antibody from Cell Signaling Technology (Beverly, MA, USA), mouse anti-ERK2 antibody from BD Transduction Laboratories (Becton Dickinson and Company, Franklin Lakes, NJ, USA), mouse anti-tubulin antibody from NeoMarkers (Fremont, CA, USA), goat anti-mouse Alexa 488 and goat anti-rabbit Alexa 488 antibodies from Molecular Probes (Eugene, Oregon, USA), goat anti-mouse horse radish peroxidase (HRP) and goat anti-rabbit-HRP conjugates from Bio-Rad (Hercules, California, USA). Nitrocellulose membrane was obtained from Schleicher & Schuell (Keene NH, USA). Enhanced chemiluminescence (ECL) reagents, 125 I-(1DMe)NPYF and 3 H-diprenorphine were from Amersham Pharmacia Biotech (Uppsala, Sweden). Restriction enzymes and other molecular biology products were purchased from Promega (Madison, WI, USA) or New England Biolabs (Beverly, MA, USA), PCR-primers were from Sigma Genosys (St. Louis, MO, USA). The CHO-K1/hNPFF2 cell membranes were from EuroScreen S.A. (Brussels, Belgium). The AcroWell filter plates were from Pall Life Sciences (NY, USA). General laboratory reagents were from Sigma-Aldrich or Merck (Darmstadt, Germany). Plasmid construction, transfection and cell culture The human delta opioid receptor (a kind gift from Dr. Brigitte Kieffer) was N-terminally tagged with c-MYC-epitope (EQKLISEEDL). The coding sequence was amplified with primers that added the c-Myc epitope tag, a Kozak-sequence and an Nhe I-site immediately upstream of the hDOR and a Bst XI-site downstream of the receptor sequence. The PCR-product was inserted into Nhe I/ Bst XI-site of the vector pcDNA6.0A (Invitrogen) and the construct was verified by sequencing. The human NPFF2R gene was cloned from human placental total RNA by RT-PCR. The full- length coding sequence was further subcloned into the pcDNA6.0A (Invitrogen) with similar methodology as that used for hDOR (instead of c-MYC tag, a FLAG tag was inserted N-terminally) and the construct was verified by sequencing. The pcDNA6.0A-FLAGhNPFF2R construct was used for transient transfection of Chinese hamster ovary cells deficient in dihydrofolate reductase (CHO-K1, dhfr -). The plasmid was transfected into CHO-K1 cells with FuGene 6 reagent according to the supplier's instructions and 24 h post-transfection the cells were used for immunocytochemistry. To create a stable cell line expressing c-MYC tagged human DOR, the pcDNA6.0A-MYChDOR vector construct was linearized with Ssp I and the linear DNA was used to transfect CHO-K1 cells. The stable transfection was performed using FuGene 6 reagent according to supplier's instructions. A stable clone expressing the receptor at the desired level was selected using FACS analysis. The cells were incubated with rabbit-anti-MYC in PBS supplemented with 1 % normal goat serum (v/v) on ice, washed with ice-cold PBS and further incubated with the goat anti-rabbit Alexa 488 antibody in PBS on ice. After washes with ice-cold PBS the cells were suspended in PBS supplemented with 10 % fetal calf serum (v/v) and sorted. After expansion the obtained cells were plated onto 96-well plate and clones originating from a single cell were selected. The clones were analyzed for the receptor expression with immunocytochemistry and radioligand binding and the functionality of the heterologously expressed receptor was tested with MAPK and cAMP -assays. The expression of NPFF receptors in CHO-K1 cells was tested with reverse-transcription polymerase chain reaction (RT-PCR). Total RNAs from wild type CHO-K1 cells and the CHO-K1 clone expressing MYC-tagged DOR were isolated with RNAwiz protocol (Ambion). Total RNA from human placenta and human medial hypothalamus were isolated and were used as positive controls in RT-PCR. cDNA from the total RNA was produced with The Superscript First-Strand Synthesis System for RT-PCR (Invitrogen). The expression of NPFF receptors was analyzed with several primer pairs specific for either human or mouse receptors (the receptors from hamster have not been cloned). Sequences for the primers specific for the human NPFF2R were: 5'-GAT TGG TCC AGG GAA TAT CTG TC-3' and 5'-CAG TGT GCA AAA GGG TAG ATG TAG-3' or 5'-GGA TGG CCA TTT GGA AAC-3' and 5'-CCA ATC CTT CCA TAC ATG-3'. Sequences for the primers specific for the human NPFF1R were 5'-CGA CAA TGC CAC ATG CAA GAT GAG-3' or 5'-CGC AAC CGC TCC TAC CCT CTC TAC-3' together with 5'-AGG GGA AGG CGT AGA CGG TGA C-3'. Corresponding mouse specific primers were also used in the analysis. CHO-K1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % (v/v) FBS, 4.5 g/litre glucose, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 1 mM glutamine and 1 × HT-supplement. For the transfected cells the selection pressure was maintained by adding 0.5 μg/ml Blasticidin S HCl to the culture media. The cells were kept at 37°C in 5 % CO 2 /humidified air and the culture medium was changed every 3–4 days. The cells were sub cultured in ratio of 1:5–1:10 and only cells undergone fewer than 20 passages were used for the experiments. Immunocytochemistry and confocal microscopy The cells were collected, plated onto 18 mm × 18 mm cover slips and cultured o/n. After o/n incubation the cells were rapidly cooled on ice, the medium was removed and the cells were washed two times with ice-cold PBS. The cells were fixed with 4 % PFA (w/v) for 30 min at +4°C and washed twice with PBS. The fixed cells were first incubated with 1 % normal goat serum (v/v); with or without 0.05 % saponin (w/v) in PBS for 30 h at +4°C and then o/n with 1:5000 dilution of mouse anti-c-MYC/1:2000 dilution of rabbit anti-NPFF2R antibody in 1 % normal goat serum (v/v) in PBS. After washes with PBS the cells were incubated with 1:1000 dilution of goat anti-mouse/anti-rabbit Alexa 488 antibody in PBS for 1 h at +4°C. They were then washed with PBS and the cover slips were mounted onto objective glasses. The slides were analyzed with a Leica TCP-SC laser scanning microscope system with Ar-Kr laser (Omnichrome, Melles Griot, Carlsbad, CA, USA). The images were acquired and processed with Leica TCS NT/SP Scanware software. The excitation and emission wavelengths used were for the Alexa 488 secondary antibody excitation 488 nm and emission 492–540 nm. The samples were scanned with identical settings taking images every 0.5 μm. The PMT values for the laser scanner were set with a negative control slide, i.e. autofluorescence was eliminated. The image representing the mid-nuclear level of the scanned sample was taken from the image stack for a comparative analysis between time-points and treatments. The scanned images were further analyzed and the figures for the publication were produced with Adobe Photoshop 6.0 (Adobe Systems, San Jose, CA, USA) and/or Corel Draw 9.0 software (Corel, Ottawa, Canada). Each image was again treated similarly and the figures were not manipulated in any way. Representative images were chosen for the Figures 1 and 2 . Flow cytometry The CHO/MYChDOR cells were collected and suspended into the culture medium. The cells were treated with drugs in suspension as for the immunocytochemistry for the indicated times. After the incubation, the membrane trafficking was stopped by rapidly chilling the cells on ice and removing the medium. After this the cells were kept on ice. The cells were washed with PBS and stained in PBS-suspension with rabbit anti-MYC antibody (1 % normal goat serum (v/v) used to block unspecific binding), followed by the detection with a fluorescent secondary antibody. Dead cells were detected with isopropidium iodide. Cells were analyzed with FACScan flow cytometer (Becton Dickinson and Co., Mountain View, CA, USA). Data from 10000 cells were collected for each run and each sample was run in replicate or triplicate. The internalization percent was calculated as: where F(ctrl) in the fluorescence of the time-matched control cells and the F(sample) is the fluorescence measured from the drug-treated cells. Determination of intracellular cAMP levels 50000 cells/well seeded onto 96-well plates and incubated o/n. In the case of pertussis toxin pre-treatment, the cells were incubated 16–20 h with 100 ng/ml PTX before the exposure for drugs. The cells were incubated for one hour with serum-free medium at 37°C; then 0.3 mM 1-Methyl-3-isobutylxanthine (IBMX) and 0.3 mM indomethazine were added and the cells were further incubated for 10 min. After this 10 μM forskolin was added with or without the drugs (DPDPE, (1DMe)NPYF or DPDPE + (1DMe)NPYF). The cells were incubated for the indicated times at 37°C, the reaction was stopped by adding lysis buffer (supplied by the kit) and cooling the cells on ice. The amount of cAMP was determined from the cell lysates with Delfia cAMP kit (Perkin Elmer Wallac, Turku, Finland). The time-resolved fluorescence was measured with Victor 2 Multilabel counter (Perkin Elemer Wallac, Turku, Finland). Phosphorylation of MAPK 25000 cells/well were seeded onto 24-well plates and incubated o/n. The medium was replaced with serum-free medium and the cells were incubated for one hour at 37°C. Serum-free medium containing 100 nM DPDPE or 100 nM (1DMe)NPYF or 100 nM DPDPE + 100 nM (1DMe)NPYF (end concentrations) was added and the incubation was continued for the times indicated. The reaction was stopped by removing the medium and adding modified RIPA-lysis buffer (PBS, pH 7.6; 1 % Nonidet P-40 (v/v); 0.5 % sodium deoxycholate (w/v); 0.1 % SDS (w/v), 10 mM NaF; 0.4 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 mM Na 2 VO 5 ; 0.2 mM bestatin). Cells were lysed on ice for 15 min and the lysates were collected. The protein content was determined with the Bradford method. 10 μg of protein was analyzed on 10 % SDS-PAGE gel and the gel was blotted onto nitrocellulose membrane. The membrane was blocked with 5 % fat-free dry milk (w/v) in TBS-T (10 mM Tris/HCl pH 7.6; 150 mM NaCl; 0.05 % Tween 20 (v/v)). It was then incubated with 1:2000 dilution of rabbit anti-phospho-ERK1/2 antibody in the blocking buffer o/n at 4°C, washed with TBS-T, incubated for one hour at room temperature with 1:3000 dilution of goat anti-rabbit-IgG conjugated to HRP in the blocking buffer. After washes, the membrane was detected with the ECL-protocol. The band intensities were measured with AlphaDigiDoc1000 documentation system (Alpha Innotech, San Leandro, CA, USA). After stripping with 0.2 M glycine; 0.5 M NaCl, pH 2.5 the membrane was similarly stained with mouse anti-ERK2 (1:5000 dilution) and mouse anti-tubulin (1:1000 dilution) antibodies to determine the total amount of ERK2 and to control the equal loading of samples, respectively. Goat anti-mouse-IgG conjugated to HRP (1:3000 dilution) was used as the secondary antibody. Preparation of cell membranes The cells were collected with PBS + 0.2 % EDTA (w/v) and washed with cold PBS. The CHO/MYChDOR or CHO-K1 cells were suspended into ice-cold 50 mM Tris/HCl, 5 mM EDTA, pH 7.5 and homogenized with Potter S-homogenisator on ice. The homogenate was centrifuged 47800 × g at 4°C and the pellet was suspended into the Tris/EDTA buffer and the homogenization/centrifugation was repeated. The pellet was suspended into cold binding assay incubation buffer and the protein content was measured with the Bradford method. Radioligand binding on cell membranes The binding experiments were performed as described in [ 21 ]. 25 μg or 50 μg of crude membrane preparation prepared as described above was used per assay. As positive control membrane preparation from EuroScreen S.A. (Brussels, Belgium) was used. For the saturation binding assay the membranes were incubated for one hour at room temperature with 0.2–10 nM 3 H-diprenorphine in 50 mM Tris/HCl, pH 7.5 or 0.002 – 0.2 nM 125 I-(1DMe)NPYF in the binding buffer described in [ 21 ]. The non-specific binding was determined with 10 μM naloxone for 3 H-diprenorphine and with 1 μM (1DMe)NPYF for 125 I-(1DMe)NPYF. In the displacement assays the effect of 0.1 nM – 10 μM DPDPE and/or (1DMe)NPYF on 6 nM 3 H-diprenorphine binding was determined. Otherwise, the assay conditions were as for the saturation assay. Tb-DeltI binding on cell membranes and whole cells 6 μg of crude membrane preparations or whole cells (25 000 cell/well) that were plated one day before experiment and fixed with 4 % PFA for 10 min at room temperature, were used in the saturation binding experiments. The whole cell assays were carried out on normal cell culture plates and the assays with membrane preparations on special filter plates (Pall Life Sciences). The cells or membranes were incubated in 50 mM Tris/HCl pH 7.5; 2.5 mM MgCl 2 ; 60 mM NaCl; 25 μM EDTA, 0.2 % BSA (w/v) for 90 min at room temperature with 0.01 – 781.25 nM Tb-DeltI in the presence or absence of 1 or 5 μM unlabeled DeltI. To study the effect of (1DMe)NPYF on the Tb-DeltI binding, 1–1000 nM (1DMe)NPYF was added together with unlabeled DeltI. After the incubation the unbound Tb-DeltI was removed by washing the plates four times with 50 mM Tris/HCl pH 7.5; 2.5 mM MgCl 2 . Vacuum pump system was used to wash the filter plates. Since the chelate-bound Tb-ion is nonfluorescent, it needed to be released from the complex. In order to release Tb-ion from the chelate 150 μl Delfia enhancement solution was added to the wells and the plates were incubated on a shaker for 15 min. Thereafter, 40 μl Delfia enhancer that changes the pH of the assay solution appropriate for Tb-fluorescence was added, and the plates were further incubated for 5 min on a shaker. The time-resolved fluorescence of the released Tb-ion was measured with Victor 2 Multilabel counter (Perkin Elemer Wallac, Turku, Finland). Statistics All the experiments were repeated at least three times with three replicates in all assays. Prism GraphPad Softwear version 4.0 (GraphPad Softwear, San Diego, CA, USA) was used for all statistical analysis. Two-way Anova with Bonferroni's post-test or one-way Anova with Bonferroni's multiple comparison test was used to assess the statistical significance of the binding assay, cAMP-assay, MAPK-assay and FACS-analysis results. When a parametrical test was used the analysis was done on the raw, non-normalized data. List of abbreviations (1DMe)NPYF, D-YL-(NMe)-FQPQRF-NH 2 ; CHO, Chinese hamster ovary; DeltI, deltorphin I; DOR, delta opiate receptor; DPDPE, [D-Pen(2,5)]enkephalin; ERK, extra cellular signal-regulated kinase; GPCR, G-protein coupled receptor; IBMX, 1-Methyl-3-isobutylxanthine; NPFF, neuropeptide FF; MAPK, mitogen-activated protein kinase; PFA, paraformaldehyde; PTX, pertussis toxin Authors' contributions M-LÄ participated in planning the experiments, executed them, and wrote the manuscript. PP participated in planning of the experiments, and interpretation of the results, and obtained funding. Both authors revised and accepted the manuscript.

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1079870

The Incidence Of Prescribing Errors In An Eye Hospital

Background Relatively little is known about the incidence of prescribing errors and there has been no work on this in a single specialty ophthalmic hospital. Knowing where and when errors are most likely to occur is generally felt to be the first step in trying to prevent these errors. This study is an attempt in, the setting of an eye hospital, to try to identify and attribute these medication errors. Methods The study setting was a single specialty eye hospital geographically separated from the main general hospital. Pharmacists prospectively recorded the number of errors of prescribing during a 4 week period at an eye hospital in UK. The errors were categorised as error of prescription writing or drug error. Potential significance of the errors was not addressed. Results Overall 144/1952 (8%)prescription sheets had errors. 7% of the total errors were errors of prescription writing while 1% were drug errors. The majority of errors were made by junior doctors and no drug errors were made by senior doctors. The outpatients department had by far the highest prevalence of errors. Conclusion Certain areas within the hospital and certain grades of staff are more prone to drug errors. Further study is required to look at the reasons why this is so and what systems can be put in place to reduce these errors.

Background Medication errors are some of the commonest types of medical errors.[ 1 ] Although the true incidence of medication errors for UK is not known, a pilot study identified errors of prescribing (rather than of dispensing) as the commonest cause of medication errors in the study hospital.[ 2 ] The Department of Health has recommended that by 2005 serious errors in prescribed drugs should be reduced by 40%.[ 3 ] Since errors of prescribing are the commonest form of avoidable medication errors, it is the most important target for improvement.[ 4 , 5 ] One drug or dosage, which may be harmless in one patient, may cause serious harm to another. Consequently, it is important that all branches of medicine try to identify and study their own sources of prescribing errors. To the best of our knowledge, a study identifying medication errors arising in an eye hospital has not been previously published. The aim of this study was to estimate the number of medication errors, where they most commonly occur and the doctors most likely to commit them. Methods The study was conducted at Sunderland Eye Infirmary. This is a single specialty hospital situated two miles from the main general hospital. It contains its own self-contained medical records, theatres, accident and emergency department (which is walk-in), in-patient ward and pharmacy. The outpatients department has clinics that cover all the main specialties within ophthalmology. For the purpose of the study, the hospital was divided into the following areas • The out patients department (OPD) • Accident and Emergency (A&E) • Day case centres (including day-case theatres and laser rooms) • In patient ward From this latter group only discharge summaries were assessed for errors. In patient drug charts were not assessed The study was conducted prospectively over a four week period starting on 1 st July 2002. It was performed during the working hours of the pharmacy (9 am to 5 pm) and therefore evenings and weekends were excluded. In order to replicate normal working conditions as much as possible, prescribing doctors were not informed of the study. The participating pharmacists were introduced to the study protocol over two half-day sessions. Errors on the prescription were identified by three dispensing pharmacists (all prescriptions at the time of the study were hand written). The pharmacists recorded on sheets the errors as either; 1) errors of prescription writing or 2) drug related errors. Prescription writing errors consisted of :- • Incorrect patient details e.g. patient name, hospital number or date of birth • Illegibility • Incorrect format • Scripts where the prescribing doctor could not be identified. Once a prescription writing error was identified, the script was not further assessed for drug related errors. If there was no prescription writing error identified then the script was assessed for drug related errors and these were defined as:- • Incorrect drug dose or timing • Incorrect route of administration. Since the dispensing pharmacists did not have access to patient notes from A&E, OPD and day case centres, they were unable to comment on the correctness of prescribing decision in each situation. For the purpose of this study, senior doctors included consultants, staff grade and associate specialists. Junior doctors were senior house officers, specialist registrars and research fellows. Results Over the study period, a total of 1952 prescription orders were received by the pharmacy with a total of 3402 medication orders – although the majority of prescriptions only had a single medication order. The contribution to the total prescriptions from the different parts of the hospital and the breakdown of errors is shown in Table 1 . Overall 159 (8%) of the 1952 prescriptions had at least one error of writing or a drug error. The breakdown of these errors was as follows:- Errors of prescription writing 144/1952 of the scripts had incorrect formats or were illegible. They constituted 7% of the total errors in this study. In 18/144 (13%) of them the prescribing doctor could not be identified. Of the remaining 126 prescriptions, senior doctors made 46% of the errors compared to 54% made by the junior doctors. The inpatient discharge scripts did not have any errors of prescription writing. A&E, OPD and day case contributed to 56(39%), 64(45%) and 24(17%) of the errors respectively. Drug related errors were identified in 15/1808 (this being the total number of prescriptions without written errors). This represented 1% of the prescription orders. 8/15 of the errors (53%) were of incorrect dosage while the remainder were either incorrect route of administration or incorrect frequency. All the errors, in this category, were made by junior doctors at the hospital. The distribution of writing and drug errors between senior and junior doctors is shown in Table 2 . Distribution of the prescription writing errors and drug related errors with respect to part of the hospital in which they occurred are illustrated in Figure 1 . It can be seen that no errors were identified from the in-patient discharge summaries while 25%, 64% and 14% were the percentage distribution of these prescriptions from A&E, OPD and day case areas respectively. As discharge summaries (which include discharge ophthalmic medications only and not the patients' non-ophthalmic drugs) were found not to have any errors of prescription writing or drug related errors, it was felt necessary to double-check this finding. Discharge summaries were then further analysed by one of the investigators (KM) and they were also cross-checked with the in-patient notes. There were found to be a total of 130 discharge summaries during the study period and it was confirmed that there were no prescription writing or drug related prescribing errors in theses summaries. It as also noted that 103 (79%), of the 130 discharge summaries, were written by senior doctors. Discussion The prescribing of medicines is an integral part of the provision of health and represents a relatively safe, effective and inexpensive mode of treatment.[ 6 ] However a study in the US has identified medication errors as the cause of harm in 1–2% of all hospital admissions.[ 7 ] The end point of a patient receiving a medication involves a series of steps (illustrated in figure 2 ) and errors can be made at any point in this process. This study addresses only two links in the chain i.e. correct completion of the prescription and drug errors. We did not assess treatment decisions or dispensing errors. However, if prescribing errors are to be reduced (the Department of Health target is by 40% by 2005) it is important to know where they are occurring, who is committing them and in what situations. In effect it is necessary for each specialty, hospital or team to know where their 'weak points' are in order that they can put in the appropriate defences. This study is an attempt to identify some of the weak points in a single specialty ophthalmic hospital. The overall rate of errors in our study was 8%, but as only one error was counted per script, the actual true number of errors may have been greater. The literature suggests that between 1 and 38% of all medications are administered in error.[ 8 ] Dean et al,[ 1 ] found a prescribing error rate of 1.5% in one UK general hospital. This wide variation is partly due to differences in definition of drug errors and partly due to problems of reporting (Osborne at al suggest that 25% of medication errors are not reported).[ 9 ] A standardised definition such as that suggested by Dean and Barber[ 10 ] is essential if different units, specialties and countries are to be compared. Similarly a rigorous and reasonably standardised collecting system is necessary – although no system will be foolproof, it would at least achieve a level of consistency. Ophthalmology represents a specialty with a wide range of differing medications and different routes of administration. Most drugs are given as drops or ointment into the conjunctival sac but many are given orally and intravenously. Medications range from the very safe e.g. artificial tears to the potentially serious e.g. intravenous steroids and oral cytotoxics. Ophthalmic medications also have the potential, unlike in most other specialties, to cause sight threatening side effects e.g. topical steroids. Even topical medications can have serious side-effects because of absorption from the highly vascular nasolacrimal system and the lack of first-pass liver metabolism. A good example of this is topical β-blockers used to lower the intraocular pressure in glaucoma but which can exacerbate or even cause bronchospasm.[ 11 ] All medication errors are, of course, potentially serious in that they may indicate flaws in a system that may allow more serious errors to occur. Our study shows that the clinical area with the highest incidence of prescribing error rates was the out-patients department. This finding is somewhat surprising both in its scale (50% of all scripts) and its location. We had felt, prior to commencing the study, that the accident and emergency department would be the most likely area to produce prescribing errors as it has a high throughput, a rapid turnover of patients and is generally staffed by the more junior doctors in the department. The actual prescribing error rate in A&E was only 5% and we now suspect this relatively low rate of error was for two reasons:- 1. There is only a narrow range of drugs that are usually used in the ophthalmic A&E setting 2. Because of the set-up of the A&E department, many patients give their prescriptions to an A&E nurse before going to pharmacy and it may be that some errors are being corrected at this stage. Interestingly, the day case prescription sheets are also for a small range of medications and many are checked by the nurses prior to the patient being sent to pharmacy. This gives the same proportion of errors (5%) as in A&E. The in-patient ward discharge prescriptions had no errors in this study. There may be two reasons for this: 1. All the ward discharge prescriptions are checked by the nursing staff prior to going to the pharmacy 2. All the ward discharge were written by the senior medical staff The out-patient setting seems to differ from the other areas in two main respects:- 1. there is a much wider range of drugs being prescribed – both topical and systemic 2. it would be unusual for a nurse to handle the prescription prior to the patient presenting it to the pharmacy. We feel that it is these differences that, at least partly, explain the differences between the rates of error between OPD and the other areas. However a 50% error rate seems very high and we are currently addressing other possible reasons for this. The figures indicate that errors of prescription writing were much more common than drug errors. This is as we would expect – not only from the fact that the study did not count drug errors if writing errors had occurred. There are a number of ways to write a prescription wrongly but only one or two ways to commit a drug error. There also seems, from our results, to be a tendency to get drug doses/timings correct but to cut as many corners as possible to speed up writing prescriptions. This is also shown by the fact that both junior and senior doctors made far more writing errors than drug errors. Seniors made no drug errors -perhaps reflecting their greater experience of using the drugs but made just as many writing errors as the juniors. Prescription writing errors are a rich source of medication errors and one that could be almost completely removed with electronic prescribing.[ 12 , 13 ] This study is an attempt to represent a 'snap-shot' of medication errors at a single institution over a period of four weeks. It is therefore important to be cautious about the generalisability of the results – the study hospital may have unique features or the study time period may have been an exceptional one. The literature indicates a wide variation in estimates of medication errors and further work is needed to compare different specialties, hospitals and countries for a true picture to emerge. We have not addressed the potential clinical significance of the errors made in this study but again this needs further study including agreement over definitions of serious and less serious errors with ophthalmic medications. It is possible that our study has underestimated the total number of drug errors by not looking for any further errors in the scripts that had transcription errors. These prescriptions with writing errors were probably filled in haste and may therefore, have been more likely to contain drug errors resulting in an underestimate of this category. In conclusion, this study found an overall medication error rate of 8%. Most of these errors occurred during the writing of the prescriptions. Junior doctors were more likely to make errors than were senior doctors. The out-patients department had a very high rate of medication errors and this is a finding that needs further study. It is important to look into the reasons why these discrepancies exist and to address them in a non-punative way and to look for ways in which the prescribing systems can be changed to take account of these weak points. Electronic prescribing is soon to be used in the study hospital and it is likely that this will significantly reduce the number of prescription writing errors and drug errors.[ 14 ] We will repeat this study, using a similar format, after the introduction of electronic prescribing to see if this has occurred. Conclusion Some areas of the hospital and certain grades of doctors are more prone to medication errors compared to others. These specific areas need to be targeted when planning and implementing any changes to improve services. Competing interests The author(s) declare that they have no competing interests. Authors' contributions SGF conceived the study, and participated in its design and co-ordination. KM carried out the data collection and statistical analysis. All authors have read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079871

A polymorphism at codon 31 of gene p21 is not associated with primary open angle glaucoma in Caucasians

Background Primary open angle glaucoma (POAG) is considered to be a neurodegenerative optic neuropathy, in which cell death occurs by apoptosis. p21 , is an important protective component of the apoptotic pathway, regulating cellular arrest in the presence of DNA damage. An unstable or altered p21 protein could modify the cellular response to genomic injury and abolish the effect of p21 . A previous study on a Chinese cohort suggested that the p21 codon 31 polymorphism may alter the state of apoptosis in glaucomatous optic neuropathy, failing to protect the ganglion cells. The aim of this study was to test the hypothesis that a p21 codon 31 polymorphism is associated with POAG on a Caucasian cohort. Methods 140 POAG patients and a control group of 73 healthy individuals were included in the study. All the subjects were of Caucasian origin. Genomic DNA was amplified by polymerase chain reaction, followed by enzymatic restriction fragment length polymorphism technique (PCR-RFLP). Patients and controls were genotyped for a single nucleotide polymorphism (C/A transversion) in the third base of codon 31 of p21, which leads to a serine (Ser)/arginine (Arg) substitution. Results The distribution of the genotypes in the POAG patients showed 128 (91.4%) Ser homozygotes, 10 (7.1%) Ser/Arg heterozygotes and 2 (1.5%) Arg homozygotes. In the control cohort, there were 61 (83.6%) Ser homozygotes and 12 (16.4%) Ser/Arg heterozygotes. No Arg homozygotes were present amongst the control group. Both the allelic and genotypic frequencies of the Ser or Arg residues at codon 31 were not significantly different between POAG patients and controls (Fisher's exact test, P = 0.20 for alleles and P = 0.0561 for genotypes). Conclusion This study suggests that the p21 codon 31 polymorphism does not contribute to the risk of POAG in the Caucasian population.

Background Primary open angle glaucoma (POAG [MIM 137760]) is a multifactorial neurodegenerative disease, causing blindness to approximately 70 million individuals worldwide [ 1 , 2 ]. The aetiology of POAG is poorly understood, with both genetic and environmental factors contributing to the pathophysiology [ 3 , 4 ] Irrespectively of the causative factors leading to visual loss, most of which are still not understood, the final common pathway in POAG is retinal ganglion cell death, mediated by apoptosis, a genetically regulated process [ 5 ]. The apoptotic pathway consists of multiple interacting pathways, some of which are still unclear. It appears that, following DNA damage, cells can either proceed to apoptosis or enter a transient arrest cycle, allowing time for DNA repair. p21 gene, also known as WAF1 or CIP1 , is a key component of this pathway. It can be up-regulated either by activated wild type p53, which acts as a transcription factor [ 6 ], or independently, by various factors such as TGF β , vitamin D, TPA and nerve growth factor. p21 expression results in inhibition of the cyclin-dependent kinases (Cdks), that are essential for cell division. Consequently, cell cycle is arrested at the G1 phase, until genomic repair is established. An unstable or altered p21 protein, therefore, could significantly affect the activity of Cdks, modifying the cellular response to genomic injury and abolishing the protective effect of p21. Such an outcome can derive from a single nucleotide polymorphism in the third base of codon 31 of the p21 gene, following a C to A transverse change, which results in a Serine/Arginine amino acid substitution. This polymorphism probably encodes a probable DNA-binding zinc-finger domain, causing functional changes to the p21 protein. There is evidence from a recent case-control study on a Chinese cohort that the Arg allele of the p21 codon 31 polymorphism is more common amongst POAG patients [ 7 ]. In this study, we tested the hypothesis of a possible association between the p21 codon 31 polymorphism and POAG on a Caucasian population. Methods Case selection Having obtained ethical approval from our local research ethics committee, blood samples were analysed from an unrelated Caucasian cohort of 140 POAG patients and 73 controls from the north east of England. The definition for POAG included characteristic cupping of the optic disc, open iridocorneal angle and typical glaucomatous visual field defects. An experienced glaucoma specialist clinically examined both patients and control groups (M.B.). Patients with intraocular pressure (IOP) higher than 30 mmHg at first presentation and secondary types of glaucoma (pseudoexfoliative, pigment dispersion syndrome, trauma or steroid induced) were excluded. The control group consisted of the spouses of our POAG patients, who were free from any coexisting ocular pathology and had normal visual acuity, IOP, visual fields and optic discs. Molecular genetic analysis Purified genomic DNA was amplified by polymerase chain reaction (PCR) for exon 2 of the p21 gene. For each subject, 1 μl of DNA was mixed with 1 unit Taq DNA polymerase (Promega, Madison, WI, USA), 10x Taq polymerase buffer (Promega, Madison, WI, USA), 2 mmol dNTP, 0.25 μM of each oligonucleotide (primer) and H 2 O to total volume of 30 μl. The primers used were: Forward (5'-GTC AGA ACC GGC TGG GGA TG -3') and reverse (5'- CTC CTC CCA ACT CAT CCC GG -3'). Reactions were treated in a thermal cycle machine to incubation at 94°C for 5 min followed by 35 cycles of: 94°C for 30 sec, 57.2°C for 30 sec, 72°C for 30 sec and a final incubation of 72°C for 7 min. For each sample, the amplified PCR product was digested with the restriction enzyme Blp I (New England Biolabs, Beverly, Massachusetts, USA). The Blp I digestion mixture contained 10 μl PCR product, 6 units of enzyme, 3 μl buffer (NEB Buffer 4) and H 2 O to total volume of 20 μl. The reactions were allowed to proceed for 12 hours at 37°C. The resulting fragments were separated by electrophoresis on a 3% agarose gel and visualised by ethidium bromide staining with a digital camera. The Ser allele has a single Blp I restriction site (GCTNAGC), resulting in two fragments of 89 bp and 183 bp, where the Arg allele remains undigested, producing a single band of 272 bp. The molecular genetic analysis was performed in the same laboratory by two investigators who were masked to the phenotype of the samples studied. Fisher's exact test was used to compare the genotype and allele frequencies in cases and controls. Results Our cohort consisted of 140 POAG patients and 73 controls. The median age was 73 years for the POAG patients (range 51–87, S.D. = 8.01) and 78 years for the controls (range 68–90, S.D. = 4.4). Mean IOP was 20.8 mmHg for the patients (S.D. = 2.6) and 16.2 mmHg for the controls (S.D. = 3.4). Median cup/disc ratio was 0.8 and 0.3 for patients and controls respectively. The frequency distribution and the allelic frequencies of p21 codon 31 polymorphisms in POAG patients and healthy subjects are illustrated in Table 1 and 2 respectively. The allele frequencies follow Hardy-Weinberg proportions. The distribution of the genotypes in the POAG patients was 128 (91.4%) Ser homozygotes, 10 (7.1%) Ser/Arg heterozygotes and 2 (1.5%) Arg homozygotes. In the control cohort, there were 61 (83.6%) Ser homozygotes and 12 (16.4%) Ser/Arg heterozygotes. No Arg homozygotes were present amongst the control group. We therefore pooled the Arg/Arg groups with the Ser/Arg group for the genotype analysis. Both the allelic and genotypic frequencies of the Ser or Arg residues at codon 31 were not significantly different between POAG patients and controls (Fisher's exact test, P = 0.20 for alleles and P = 0.0561 for genotypes). Discussion Apoptosis and cell-cycle arrest are regulated through various interacting pathways [ 8 ]. DNA damage leads to activation of transcription factors, such as the p53 gene, which can induce apoptosis by up-regulating the protein Bax and down-regulating the protein Bcl-2. Alternatively, DNA insult can cause activation of the p21 gene, a tumour suppressor gene, either directly or through transactivation by wild type p53. p21 is a cyclin – dependent kinase inhibitor resulting in cell-cycle arrest by inhibiting the G1 to S and S to G2 phases [ 9 ]. Cancer research has revealed that mutations of p21 are very rare and that single nucleotide polymorphisms are more likely to have a functional effect [ 10 ]. The Ser/Arg polymorphism at codon 31 is located in a highly conserved region of the p21 [ 11 ] and has been associated with cancer of the lungs, breast [ 12 ], bladder [ 13 ], and colorectal tumours [ 14 ]. A recent study on a Chinese population showed an association between the Arg form of the p21 codon 31 polymorphism and POAG, suggesting that this allele may alter the state of apoptosis in glaucomatous optic neuropathy, failing to protect the ganglion cells [ 7 ]. In our cohort, we have not detected any statistically significant difference of the Ser and Arg allelic frequencies between the POAG group and the healthy individuals. This difference may reflect sampling bias, as the Chinese cohorts were smaller (58 POAG patients and 59 control subjects), or it could be attributed to ethnic disparity. The Arg allele is considerably more common in Chinese, with reported frequency of 0.50. Our findings correlate well with previous studies on Swedish and French populations, which identified a low Arg allele frequency of 0.05 [ 15 ]. However, we cannot exclude the possibility of p21 codon 31 polymorphism being associated with POAG in other ethnic groups or the likelihood of an association between POAG and another p21 polymorphism. This might be a worthwhile future strategy to elucidate the issue. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079872

Is post-trabeculectomy hypotony a risk factor for subsequent failure? A case control study

Background Ocular hypotony results in an increased break down of the blood-aqueous barrier and an increase in inflammatory mediator release. We postulate that this release may lead to an increased risk of trabeculectomy failure through increased bleb scarring. This study was designed to try to address the question if hypotony within one month of trabeculectomy for Primary Open Angle Glaucoma (POAG), is a risk factor for future failure of the filter. Methods We performed a retrospective, case notes review, of patients who underwent trabeculectomy for POAG between Jan 1995 and Jan 1996 at our hospital. We identified those with postoperative hypotony within 1 month of surgery. Hypotony was defined as an intraocular pressure (IOP) < 8 mmHg or an IOP of less than 10 mmHg with choroidal detachment or a shallow anterior chamber. We compared the survival times of the surgery in this group with a control group (who did not suffer hypotony as described above), over a 5 year period. Failure of trabeculectomy was defined as IOP > 21 mmHg, or commencement of topical antihypertensives or repeat surgery. Results 97 cases matched our inclusion criteria, of these 38 (39%) experienced hypotony within 1 month of surgery. We compared the survival times in those patients who developed hypotony with those who did not using the log-rank test. This data provided evidence of a difference (P = 0.0492) with patients in the hypotony group failing more rapidly than the control group. Conclusion Early post-trabeculectomy hypotony (within 1 month) is associated with reduced survival time of blebs.

Background Glaucoma is one of the leading causes of blindness in the world [ 1 , 2 ]. Medical therapies are the mainstay of treatment for primary open angle glaucoma, however resistant cases may require surgical intervention [ 3 ]. Trabeculectomy has been the preferred surgical procedure for uncontrolled glaucoma since its introduction in 1967 by Cairns. Although the procedure has undergone various modifications, the principle behind trabeculectomy remains the same. It is a filtering procedure that provides a fistula for drainage of aqueous humour via the sub-conjunctival space, into the conjunctiva and subsequent absorption by the surrounding tissues [ 4 ]. Ocular hypotony due to over-filtration is a well documented and sight-threatening complication of trabeculectomy [ 5 , 6 ]. To our knowledge there has been no publications looking at the long-term outcome of eyes that have had trabeculectomy with early (less than one month) hypotony. We hypothesise that hypotony in this early postoperative period could result in an increased breakdown of the blood aqueous barrier compared to non- hypotonous post trabeculectomy eyes. This could allow release of inflammatory mediators into the aqueous and through the filtering bleb into the surrounding tissues. These mediators could cause scarring thereby resulting in failure of the trabeculectomy. The purpose of our investigation was to compare the survival times of trabeculectomy in two groups of patients, those who experienced hypotony in the early postoperative period and those who did not. Methods The notes of patients undergoing trabeculectomy for primary open angle glaucoma (POAG) from Jan 95 to Jan 96 at Sunderland Eye Infirmary were reviewed. All surgeons (8) were Consultant grade, and none (at the time of the study) had a special interest in glaucoma. Exclusion criteria included normal pressure glaucoma, secondary glaucomas, combined phacotrabeculectomies, and augmented surgery (with Mitomycin-C and 5-Fluorouracil). All cases with pre-operative shallow anterior chambers were also excluded, although anterior chamber depth was not formally measured. To minimise confounding factors and make as homogenous a study group as possible we also excluded previous intraocular surgery, any previous intraocular inflammation and prior argon laser trabeculoplasty. For the purpose of our study, hypotony was defined as an IOP of < 8 mmHg or an IOP < 10 mmHg with choroidal detachment or shallow anterior chamber (with either iris-cornea touch, or lens-cornea touch). Two distinct groups of patients were identified 1) Control group were those who did not experience hypotony within a month of their operation or at any stage postoperatively. 2) Study group included patients who had IOP < 8 mmHg alone or an IOP < 10 mmHg with choroidal detachment or a shallow anterior chamber within one month after trabeculectomy. We defined failure of trabeculectomy as IOP > 21 mmHg which was persistent (recorded at more than 3 occasions) at or after 6 months postoperatively, or commencement of topical antihypertensive agents, or repeat surgery. Results 97 cases matched the inclusion criteria. These patients were from the population of Sunderland and its surrounding areas. The cases in both the control and hypotonous groups were exclusively Caucasian and there was no evidence of difference in mean age, gender or median IOP between the two groups. 39% (38/97) cases experienced hypotony within the 1 st month postoperative whilst 61% (59/97) did not. The notes of patients in both groups were reviewed for each visit until 5 years postoperatively. The cases that failed and the duration of survival of their filters were noted for the control and study group. We compared the survival times in those patients who developed hypotony with those who did not using the log-rank test. A Kaplan Meier curve illustrates their 5 year survival (Fig 1 ). This data provided evidence of a statistically significant difference (P = 0.0492) with patients in the hypotony group failing more rapidly than the control group. Figure 2 indicates the number of trabeculectomies that failed in each year of the study and it can be seen that most failed within the first year – especially in the hypotonous group. Discussion Postoperative hypotony, flat anterior chamber and choroidal haemorrhage are known complications of full thickness filtration procedures [ 7 - 10 ]. Trabeculectomy was introduced to limit these. Scarring of the filtering bleb by fibrous tissue is the most common cause of long term failure in glaucoma filtration surgery [ 11 ]. This is commoner in younger patients [ 12 ] and in African Caribbean races [ 13 , 14 ]. Use of adjunctive antimitotic agents such as 5-FU and Mitomicin C maintain the filtration bleb by preventing fibrosis thereby achieving a lower intraocular pressure compared to those trabeculectomies performed without the use of these agents. They frequently cause transient hypotony in the initial 4–6 weeks [ 15 - 17 ] and were excluded from our study. Eyes with previous uveitis or multiple intraocular surgeries can develop hypotony secondary to growth of a cyclitic membrane in the ciliary body[ 18 ]. Inflammation in the anterior chamber angle could precipitate ciliary body shutdown thereby resulting in reduced aqueous production and ultimately hypotony in these cases. Trabeculectomy in uveitic eyes are also at a higher risk of failure[ 19 ]. In order to try to reduce the variables in our investigation, eyes with previous uveitis and intraocular surgery were excluded. Early post-trabeculectomy hypotony can result from various factors such as over-filtration caused by loose closure of the scleral flap, conjunctival bleb leak, ciliochoroidal detachment [ 16 , 17 ], hyposecretion of aqueous humour accompanying ocular inflammation [ 15 ] and cyclodialysis cleft [ 18 , 20 ]. Trabeculectomy can result in ciliary body shutdown [ 21 ] that leads to intraocular inflammation and breakdown of the blood aqueous barrier thereby resulting in hypotony in the early postoperative period. 18 Rarer causes of early postoperative hypotony include retinal detachment and globe perforation[ 18 ]. Post-trabeculectomy hypotony has been associated with many complications such as accelerated cataract progression [ 18 , 22 ], ciliochoroidal effusion, choroidal haemorrhage, corneal changes [ 19 ], inflammation, prolonged recovery of visual acuity [ 5 ] and hypotony maculopathy with marked visual loss[ 18 ]. Dellaporta [ 23 ] and later Gass [ 24 ] described the retinal manifestations of hypotony such as optic disc swelling, chorioretinal folds and changes in retinal pigment epithelium. Gass described these findings as 'hypotony chorioretinopathy'. Our series also suggests, as a further complication of post-trabeculectomy hypotony, reduced survival time of non-augmented filtering blebs. Hypotony is associated with an alteration in aqueous flow and its composition – partly because it initiates an inflammatory response[ 25 ]. This means that in the hypotonous eye not only will there be a reduced flow of aqueous through the sclerostomy and into the bleb, but the aqueous that does pass into the bleb will have even more inflammatory mediators than normally occurs post-operatively. This reduction in flow and increase in inflammatory mediators are likely to be potent causes of sub-conjunctival scarring and subsequent failure[ 26 ]. This is the mechanism that we propose for our finding that post-trabeculectomy hypotony results in increased chance of failure of the filter and is summarised in figure 3 . In our investigation, a statistically significantly higher proportion of trabeculectomies experiencing postoperative hypotony failed within 5 years compared to the control group. These findings have implications for surgical practice such as tight suturing of scleral flaps to limit egress of aqueous into the bleb and meticulous suturing of the overlying conjunctiva to prevent bleb leakage. As far as literature is concerned Schwenn et al [ 5 ] conducted a prospective 1 year follow-up of 43 eyes undergoing trabeculectomy. Contrary to our findings, their results suggested that early hypotony was more likely to result in better IOP control. However, our population base was larger and follow-up period longer. Stewart and Shields [ 27 ] followed 36 eyes that had undergone trabeculectomy, they studied the postoperative anterior chamber depth and complication rate. They concluded that a flat anterior chamber with cornea-lens touch resulted in a significantly higher rate of complications, including corneal oedema, cataract, and bleb failure. Those eyes with iris-cornea touch were allowed to reform their anterior chambers spontaneously, and had a similar outcome to eyes that maintained an anterior chamber throughout the follow up. These findings may seem contradictory to our results, however our definition of hypotony does not rely on anterior chamber depth alone. To satisfy this group an eye in our series required an IOP of less than 10 mmHg along with a shallow anterior chamber or choroidal detachment or IOP less than 8 mmHg. Our patient group may have had more severe post-operative hypotony. Stewart and Shields patient group had a diverse range of glaucoma diagnoses as opposed to our homogenous group of primary open angle glaucoma, therefore direct comparisons are inappropriate. In addition their follow up was for 3 months only and their sample size only 36. A randomised controlled trial, usually the best way to reduce potential biases, would be ethically inappropriate in this clinical situation. The limitations of our study include consecutive patients who underwent trabeculectomy for POAG in the one year period could not be included as 24 were lost to follow-up, 2 developed endophthalmitis and 2 required vitreoretinal surgery. Those 24 patients for whom we had incomplete data were fairly evenly distributed for the hypotonous (n = 10) and the non-hypotonous (n = 14) groups. We did not collect data on the pre-operative topical drug regime. The use of two or more antiglaucoma medications is a risk factor for failure of filtration surgery [ 19 ]. Broadway et al have shown that not only does the number of prior topical antiglaucoma medications, but also the duration of use of these agents affect the success of trabeculectomy [ 28 - 31 ]. Conclusion Hypotony results in the break down of blood-aqueous barrier causing the release of inflammatory mediators. Persistent conjunctival inflammation induces enhanced bleb scarring [ 26 , 32 ]. Excessive conjunctival scarring significantly reduces the success of surgery [ 33 - 35 ]. In our series, early post-trabeculectomy hypotony (within 1 month) is associated with reduced bleb survival time over 5 years. Most of the cases of failure in the hypotonous group occurred in the first year postoperatively. This study is further evidence that hypotony is detrimental to the eye. Although further investigation is required, it may be that even though hypotony resolves in the short term, there is a risk of longer term filter failure. Avoidance of hypotony with measures such as meticulous closure of the scleral flap (but still allowing aqueous flow) and of the conjunctiva remain essential steps for short and long term bleb functioning. Competing interests The author(s) declare that they have no competing interests. Authors' contributions Data was collected by Sarah Benson and Kaveri Mandal. Statistical analysis was performed by Catey Bunce. Scott Fraser conceived of the study, and participated in its design and coordination and helped to draft the manuscript, along with Sarah Benson and Kaveri Mandal. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079873

Risk-based early prevention in comparison with routine prevention of dental caries: a 7-year follow-up of a controlled clinical trial; clinical and economic aspects

Background The results in an earlier study with 2–5-year-old children indicated that, in comparison with conventional prevention, a risk-based prevention programme was effective in reducing dental caries in a low-caries community. The aim of the present study was to examine the clinical and economic findings seven years after the cessation of the targeted programme, from the perspective of public health care. Methods The present material was collected from the dental records of the public health care centres, and included all dental visits after the 5-year examination until the 12-year examination. The groups were compared in relation to clinically detected caries at the age of 12 years, the number of dental visits needed from 5 to 12 years of age, and the estimation of running costs during these years. Statistical analyses included univariate analysis of variance, and calculation of absolute risk reduction and number needed to treat (NNT) values. Results At the age of 12 years, DMF was significantly related to the risk category determined ten years earlier, in both study groups. In the risk-based group, the absolute risk reduction for caries in permanent dentition was 0.13 (95% confidence interval 0.06 – 0.21), and the associated NNT value was 8 (95% confidence interval 5 – 17). The total number of preventive, as well as restorative visits was lower in the risk-based than in the routine prevention group. The findings indicate that early risk-based prevention can be correctly targeted, clinically effective, and economically profitable also from the long-term point of view. Conclusion Early prevention of dental caries also has long-term benefits in a 7-year follow-up perspective. This seems to hold true as regards targeting, as well as clinical and economic effectiveness. Success in risk-based prevention enables successful work division, and consequently, economic effectiveness.

Background The clinical and economic effectiveness of caries prevention cannot be correctly calculated without lengthy follow-ups. A prevention programme leading to postponement of the cavitation process can be favourable if the restorative treatment can be carried out more easily later when the children are older. Some observations and studies, however, suggest that if the timing of the prevention coincides with the eruption of the teeth, the preventive result may remain the same or be even better for several years after the cessation of the preventive programme. Those age cohorts, whose permanent teeth had erupted during the years when sugar consumption was strongly reduced because of the Second World War, still more often had intact teeth than younger cohorts about 40 years later [ 1 ]. The same observation was made by Honkala et al. in Karelia [ 2 ]. Köhler et al. have reported a long-term preventive effect in children after chlorhexidine treatment of the mother's dentition [ 3 ]. Analogously, several field trials with xylitol have indicated that the differences in caries increment figures between the xylitol and control groups increased after the cessation of the use of xylitol, especially in teeth which erupted in the mouth after the implementation of the xylitol programme [ 4 - 7 ]. The explanation for these results may be that the absence of sugar from the diet or the application of prevention before or around the time of eruption of the teeth can give an opportunity for the teeth to erupt and mature in a favourable environment. Therefore, the timing may play a crucial role in the success of caries prevention and, consequently, also in the economic results. Because there is a lack of long-lasting caries prevention trials we are also short of economic studies on the long-term effectiveness of these measures. In addition, a methodological problem is obvious: if the preventive measures are implemented in early childhood with success, the learned habits such as maintenance of good oral hygiene and use of fluorides may remain even after cessation of the intensive prevention programme. In the same way, if the dental professionals are happy with their results, they will pay special attention to the prevention also afterwards. For such reasons, it is quite impossible to reveal the exact role of the applied preventive measures or programmes afterwards or to measure their share in the long-term clinical and economic outcomes. The results in an earlier study with 2–5-year-old children indicated that, in comparison with conventional prevention, a risk-based prevention programme was effective in reducing dental caries in a low-caries community [ 8 , 9 ]. The screening was based on the presence of mutans streptococci (MS) and incipient carious lesions at the age of two years. MS-negative and caries-free children were considered to be at "low risk", MS-positive but caries-free children at "intermediate risk", and children with any caries lesions at "high risk" of caries. The intensity of the prevention increased with the increasing estimated risk. Basic prevention provided annually to all subjects included dental health education to parents on oral hygiene, fluoride toothpaste, and sweets. In the intermediate-risk category, the children additionally received fluoride varnish treatments twice a year. In the high-risk category, the treatment also included fluoride and/or chlorhexidine varnish treatments four times a year. The reduction in total costs was mainly based on the role of the preventive dental assistants carrying out the programme. The time spent on prevention at an early age reduced the need for restorative treatment, and, thus, the clinical time of a dentist/assistant team [ 8 , 9 ]. Examinations and the examination intervals are of great interest to the public health care. It is a routine system in Finland that the public health care centre determines the time and frequency of dental examinations of all under 18-year-olds. The aim of the present study was to examine the results from the early targeted prevention programme in relation to clinical and economic findings from the perspective of public health care seven years after the cessation of the targeted programme which had been followed by generally applied preventive measures in Finnish public health care centres. The article compares clinical and economic outcomes of a risk-based caries prevention programme with conventional prevention given in a neighbouring health centre, seven years after the 3-year programme was discontinued. Methods Subjects The first phase of the study was carried out as a field study in the Public Health Care Centres of Vanha Korpilahti and Saarijärvi, in Central Finland. The risk-based prevention group in Vanha Korpilahti and the routine prevention group in Saarijärvi included all two-year-old children born in 1987 or 1988. These children were followed up until the 5-year examination [ 8 ]. At the first examination, written informed consent was obtained from the parents of the children in the risk-based prevention group. The Ethical Committees of the Medical Faculty of Turku University and the Public Health Care Centre in Vanha Korpilahti approved the original study protocol. After the 5-year examination, the risk-based programme was discontinued. For the present study, the additional follow-up time was 7 years. The present material was collected from the dental records of the health care centres, and included all dental visits after the 5-year examination until the 12-year examination. The children had been examined regularly at individual intervals. The recommendations of the health care centre had included examination of all 12-year-olds. For the present study, the health care centres gave their permission to use the dental records. Drop-outs In the first phase of the study, the parents of five subjects refused to allow their children to participate in the programme. During the 3-year follow-up, the percentage of drop-outs was 13% in the risk-based and 16% in the routine prevention group of the children examined at baseline, the main reason being moving away from the district. During the additional 7-year follow-up, the percentages of drop-outs were 18% and 11%, respectively, of the children examined at the age of 5 years. Clinical examinations A mirror with 1.6 -fold magnification, a blunt periodontal probe and fibre-optic transillumination were routinely used. The examinations were carried out in a dental unit with good light and compressed air. X-rays were taken on an individual basis. Tooth decay, fillings and sealants were registered at surface level in the dental records of the public health care centre. A tooth surface was considered sound when no sign of demineralisation was observed. Incipient caries lesions in enamel and early dentinal lesions when no cavity was present clinically, were coded "I". When a defect was found clinically on the surface and restorative treatment was considered necessary, the lesion was coded "D". At the age of 12 years, the total caries experience was expressed at tooth level using the DMF index, and at surface level using the I+DMFS index. Treatment given After the subjects were 5 years of age no study-related recommendations were given to the dentists or the hygienists responsible for the dental care. The subjects received preventive and restorative treatment when the examining dentist considered it necessary. The prevention included dental health education, fluoride varnish treatments, sealants, and chlorhexidine treatments. The prevention was mainly delegated to dental hygienists or preventive dental assistants, but the dentists also gave preventive treatment in connection with other visits. Thirty minutes was the time reserved for each visit, regardless of the treatment or who carried it out. At the age of 12 years, the number of dental visits after the 5-year examination was collected and classified: first, the visits to preventive dental assistants or hygienists, consisting only of examinations and/or prevention, and, second, the visits to a dentist/assistant team, separating those consisting mainly of preventive non-operative treatment and those consisting mainly of restorative treatment. Economic evaluation The economic analysis was done from the perspective of a public health care centre. We used the actual running costs of the health care centre for the analyses. According to the annual report of Vanha Korpilahti Public Health Care Centre, in 1999, salary costs represented 73% of total running costs. In each personnel group, the hourly costs of clinical work were calculated by dividing the yearly salary of the group by their clinical working hours. The costs included the social security costs (31% of the salary), and they were calculated at 1999 rates. The total running costs (including materials, etc.) of the dentist/assistant team were estimated at 110 €/h. The equivalent costs of a preventive dental assistant and a hygienist were estimated at 43 €/h. The costs per child for the 7-year follow-up period were calculated by multiplying the time spent by the costs per hour of the required personnel. This was carried out for preventive dental assistants or hygienists, and for a dentist/assistant team (separately for preventive and restorative treatment). The sum of these three costs represented the total running costs. The time and costs related to treatment of traumas or orthodontic treatment were not included in the present analyses. The salary costs of the routine prevention group were calculated using the same figures as above. Analysis of data At the age of 12 years, the groups were compared in relation to clinical outcomes by using the DMF and I+DMFS indices, and the proportion of caries-free permanent dentitions (DMF = 0). In terms of use of clinical resources, the groups were compared using the number of sealants, the number of dental visits needed from 5 to 12 years of age, and the estimation of running costs during these years. Statistical analyses Univariate analysis of variance was used to test the effect of group, risk category, and their interaction effect on DMF, I+DMFS, the number of sealants, and the cumulative number of visits at the age of 12 years, as well as on the estimated running costs from 5 to 12 years of age. If the interaction effect was significant (p < 0.05), the significance of group differences was tested using the t-test for independent samples, separately, in the three risk categories. The analyses were carried out using SPSS statistical software (SPSS, release 10.0.5 for Windows). The absolute risk reduction and NNT values were calculated according to Sackett et al. [ 10 ]. Results Clinical outcomes DMF at the age of 12 years was significantly related to the risk category determined ten years earlier (the presence of MS in plaque and/or incipient caries lesions at the age of 2 years as caries risk indicators). The level of caries was higher with higher estimated risk in both groups, although the level of caries was highly significantly lower in the earlier risk-based group than in the routine prevention group (Table 1 ). Table 1 Number of DMF teeth, I+DMF surfaces, and sealants at the age of 12 years. Analysis of variance: DMF: Group effect, p < 0.001, Risk category effect, p < 0.001;I+DMFS: Group effect, not significant, Risk category effect, p < 0.001; Sealants: Group effect, p < 0.001, Risk category effect, p = 0.05 Prevention group DMF I+DMFS Sealants Risk category at 2 yrs n Mean S.D. Mean S.D. Mean S.D. Risk-based N = 245 0.2 0.6 2.8 4.4 2.6 2.5 Low-risk 166 0.2 0.6 2.4 3.8 2.4 2.5 Intermediate 52 0.2 0.7 3.1 4.7 2.8 2.2 High-risk 27 0.4 0.8 4.7 6.4 3.4 2.9 Routine N = 202 0.4 0.9 2.9 4.0 1.6 1.8 Low-risk 134 0.3 0.7 2.3 3.8 1.5 1.6 Intermediate 50 0.6 0.9 4.1 3.8 1.5 1.8 High-risk 18 0.9 1.4 4.6 4.7 2.2 2.9 When incipient caries lesions were included in the analyses the association between caries at the age of 12 years (I+DMFS) and risk category at the age of 2 was highly significant (Table 1 ). The group effect was not significant. At the age of 12 years, the proportion of permanent dentitions with caries was 0.13 in the risk-based and 0.26 in the routine prevention group. The absolute risk reduction for caries was thus 0.13 (95% confidence interval 0.06 – 0.21), and the associated NNT value was 8 (95% confidence interval 5 – 17). At the age of 12 years, the frequency of sealants was higher with higher estimated risk in both groups. The number of sealants was significantly higher in the earlier risk-based group than the routine prevention group (Table 1 ). Dental visits The total number of visits to a dental surgery from 5 to 12 years of age was associated with the risk category determined at the age of 2 years; the number of visits being higher with higher risk (Table 2 ). In the earlier risk-based group, the dentists gave less, and the preventive dental assistants or hygienists clearly more preventive treatment than in the routine prevention group. Altogether, the number of preventive visits was lower in the earlier risk-based than in the routine prevention group. Table 2 Number of visits for dental treatment from 5 to 12 years of age. Analysis of variance: Preventive/Dental hygienist: Group effect, p = 0.002, Risk category effect, p < 0.001; Preventive/ Dentist – dental assistant team: Group effect, p < 0.001; Restorative/ Dentist – dental assistant team: interaction term Group*Risk category, p = 0.001, Group difference: low-risk p < 0.09, intermediate p = 0.003, and high-risk category p < 0.03 Prevention group Dental hygienist Dentist-dental assistant team Preventive Preventive Restorative Risk category at 2 yrs n Mean S.D. Mean S.D. Mean S.D. Risk-based N = 245 8.2 4.0 3.9 2.3 2.0 2.9 Low-risk 166 7.5 3.6 3.9 2.4 1.8 2.6 Intermediate 52 8.8 3.9 4.0 2.1 2.4 3.4 High-risk 27 11.6 4.2 3.9 2.6 2.7 3.6 Routine N = 202 7.2 2.6 5.7 1.5 3.4 4.4 Low-risk 134 6.5 2.3 5.5 1.5 2.4 3.4 Intermediate 50 8.6 2.3 5.8 1.6 4.8 4.5 High-risk 18 8.9 3.3 6.3 1.1 7.1 7.4 The number of visits because of restorative treatment during the seven-year follow-up was associated with the risk category determined at the age of 2 years (Table 2 ). There were significant differences in the number of restorative visits between the groups in the intermediate and high-risk categories (Table 2 ). Running costs On average, the estimated running costs were lower in the earlier risk-based group (mean 505 €; S.D. 230) than in the routine prevention group (mean 656 €; S.D. 304). The difference was significant in all risk categories (Figure 1 ). The components of the costs are shown in Table 3 . Figure 1 Total running costs (mean and standard deviation) from 5 to 12 years of age. Risk categories were formed on the basis of information on MS and incipient caries at the age of 2. The figure within the column refers to the number of subjects within the category. Analysis of variance: Interaction term Group*Risk category, p = 0.009 Group difference: Low-risk category, p < 0.001; Intermediate category, p < 0.001; High-risk category, p = 0.02 Table 3 Mean running costs (in €) related to dental treatment from 5 to 12 years of age. Prevention group Dental hygienist Dentist- dental assistant team Preventive Preventive Restorative Risk category at 2 yrs n Mean S.D. Mean S.D. Mean S.D. Risk-based N = 245 177 86 216 129 111 160 Low-risk 166 161 79 215 132 99 142 Intermediate 52 190 84 220 114 130 188 High-risk 27 251 90 214 143 149 200 Routine N = 202 156 56 312 84 188 244 Low-risk 134 140 49 305 85 133 188 Intermediate 50 186 50 319 88 263 249 High-risk 18 192 72 346 59 388 406 Discussion The findings indicate that early risk-based prevention can be correctly targeted, clinically effective, and of economic benefit, also from the long-term perspective. This is in line with earlier studies among young children [ 11 , 12 ]. It seems to be possible to predict the first caries attack with acceptable accuracy, and also to succeed in the prevention itself with reduced costs. This is in contrast with several studies, which have without success tried to find an accurate method to identify risk subjects among schoolchildren or adolescents [ 13 , 14 ]. Even with relatively successful identification, the applied prevention does not suggest good results in these age groups [ 15 ]. Clinical outcomes indicated a clear association between caries level at the age of 12 years and the risk category determined at the age of 2 years. This association was found both in the risk-based study group and in the control group with conventional prevention. At the age of 12 years, in the risk-based group, a lower level of caries experience in permanent teeth was observed than in the routine prevention group, which indicated that the risk-based prevention had been correctly targeted throughout the years. The clinical effectiveness of early dental health education has been demonstrated in immigrant-background families in low socio-economic/high caries suburbs of Leeds in the United Kingdom [ 16 ]. The present study showed that even in subjects in communities with very low average caries occurrence figures, the early preventive measures led to increased prevention. In terms of population and economic structure, Saarijärvi and the three communities within the Vanha-Korpilahti public heath care centre catchment area were comparable. The soil in the area contains a low level of fluoride. Almost the whole population in both communities was included in the dental recall system within the public health care centres. After the subjects in the present study reached the age of 5 years, there was no strict programme concerning the dental preventive non-operative or restorative treatment in either health care centre. Thus, the first difference between the risk-based and the routine prevention groups was the screening and prevention programme introduced from 2 to 5 years. However, it is possible that the differences in the prevention programmes during 2 to 5 years of age are associated with the later differences in the measures used, as can be seen in the high number of sealants used in the risk-based group (Table 1 ). Therefore, a part of the result may be based on these differences between 5 and 12 years of age. Work division in dentistry is currently being hotly debated in Finland in an effort to find optimal cost-benefit ratios. The total clinical time used during the years 2–5 was the same for both the study and the control health care centres, but it was distributed unevenly. In Vanha-Korpilahti, more time was spent on prevention carried out by the hygienists, but less time on treatment calling for the dentist – dental assistant teams. Therefore, the economic results were mainly based on the work division. The second clear difference between the groups was in the structure of the personnel: in Vanha-Korpilahti, there were five hygienists and one preventive dental assistant for five dentist/assistant pairs, while in Saarijärvi, there was only one hygienist for every six dentist /assistant pairs. The salary costs represent the major proportion of the total costs. We did not discount the costs. Because the costs were lower in Vanha-Korpilahti than in Saarijärvi when the subjects were 2–5 years, the discounting had slightly increased the difference between the health care centres in favour of the risk-based targeted prevention. The costs related to parents and their children were not included. The time the parents and children spent on the programme is closely related to the number of dental visits, but not as much to the length of the visits. The present results should not be interpreted in such a way that all work division in prevention is effective if applied mechanically. If a child is already sitting in the dental chair for diagnosis, treatment etc., it is usually more practical for the dentist-dental assistant team to carry out the preventive clinical measures during the same visit. This saves a lot of time in the change-over of patients, and also in instrument maintenance. Conclusion Early prevention of dental caries has long-term benefits. This seems to hold true as regards targeting, clinical and economic effectiveness also from a follow-up perspective. Success in risk-based prevention allows successful work division, and consequently, economic effectiveness. List of abbreviations DMF the number of decayed, missed or filled teeth I the number of incipient caries lesions MS mutans streptococci NNT number needed to treat Competing interests The author(s) declare that they have no competing interests. Authors' contributions KP participated in the design of the study, performed the statistical analyses, and drafted the manuscript. JJ participated in the design of the study, and collected the data at the health care centre. PA participated in the design of the study and in writing the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079874

Women's health groups to improve perinatal care in rural Nepal

Background Neonatal mortality rates are high in rural Nepal where more than 90% of deliveries are in the home. Evidence suggests that death rates can be reduced by interventions at community level. We describe an intervention which aimed to harness the power of community planning and decision making to improve maternal and newborn care in rural Nepal. Methods The development of 111 women's groups in a population of 86 704 in Makwanpur district, Nepal is described. The groups, facilitated by local women, were the intervention component of a randomized controlled trial to reduce perinatal and neonatal mortality rates. Through participant observation and analysis of reports, we describe the implementation of this intervention: the community entry process, the facilitation of monthly meetings through a participatory action cycle of problem identification, community planning, and implementation and evaluation of strategies to tackle the identified problems. Results In response to the needs of the group, participatory health education was added to the intervention and the women's groups developed varied strategies to tackle problems of maternal and newborn care: establishing mother and child health funds, producing clean home delivery kits and operating stretcher schemes. Close linkages with community leaders and community health workers improved strategy implementation. There were also indications of positive effects on group members and health services, and most groups remained active after 30 months. Conclusion A large scale and potentially sustainable participatory intervention with women's groups, which focused on pregnancy, childbirth and the newborn period, resulted in innovative strategies identified by local communities to tackle perinatal care problems.

Background Participatory approaches to health have been advocated since the 1978 Alma Ata declaration in which the World Health Organisation emphasised the need for citizen participation in primary health care [ 1 ]. This paper details the development and implementation of a participatory project to improve perinatal care at the community level in rural Nepal. Community participation in health care The vision of Alma Ata was that increasing community participation in planning and implementation would lead to more cost-effective delivery of health care and increases in service utilisation. As communities took greater ownership of services they would become more culturally acceptable and responsive to local needs. Community participation also aimed to increase self-reliance and social awareness, which would lead to better health outcomes [ 2 - 4 ]. Opinions differ about the extent to which participation can achieve these results, and to what degree governments and agencies have facilitated participation, but the appeal of participatory approaches remains strong. Participation may be considered as a continuum [ 5 ]. In fully participatory approaches, needs are identified by the community themselves, who then may seek external support. At the other end of the continuum, superficial participation of community representatives is sought to validate the aims of programme planners, usually already decided. Harnessing the strengths of participation in community based interventions Reproductive health is an area where participatory approaches have been attempted. A structured literature search for community-based interventions focusing on perinatal health revealed no randomized, controlled trials, but two studies in developing countries which had evaluated impact on Perinatal health outcomes. The Warmi project in Bolivia, initiated by a collaboration of Save the Children Federation, USA and USAID MotherCare project [ 6 ], worked with women's groups to reduce maternal and neonatal mortality and morbidity. They used a participatory approach involving community diagnosis, planning together, implementation of plans, and participatory evaluation. The Warmi project, though neither randomized nor controlled, and based on a before-and-after analysis of 639 and 708 births, did report a reduction in the perinatal mortality rate from 117 to 44 per thousand births. The activities initiated by women's groups included literacy programmes, savings and credit schemes, and programmes to increase access to family planning. Studies based in the community that are towards the low end of the participation continuum also appear to have been successful in enabling improvements in pregnancy outcomes. A study in Maharashtra state, India, tested the effectiveness of early detection of warning signs of illness and village level management of neonatal sepsis (a cause of many neonatal deaths in developing countries)[ 7 ]. Village health workers were trained to visit newborn infants in their homes and identify and treat neonatal sepsis. This intervention appeared highly successful as a drop in neonatal mortality of 62% occurred. Village health workers were intensively managed and supported by the research team, and therefore large-scale implementation may be difficult. The study did, however, provide evidence that community level interventions to prevent or treat problems of the perinatal period in developing countries could be cost-effective. The Nepal MIRA Makwanpur trial The MIRA Makwanpur trial was designed to test the impact on neonatal mortality of a participatory intervention with women's groups, based on the Warmi Bolivia model, but on a much larger scale and using a randomized and controlled design. In south Asia infant mortality rates fell steadily from 1970 to 1990, but the decline has subsequently plateaued. In order to reduce infant mortality rates further, a focus on the neonatal period, in which most infant deaths occur, is necessary [ 8 , 9 ]. Primary and secondary care are deficient in rural areas of Nepal and where services exist, the reasons for their underuse are complex. The topographical barriers combined with limited expenditure on public health, poor quality of care, a high turnover of service providers, a lack of drugs and supplies and a lack of ownership of health programmes by communities all contribute to issues of demand and supply. The trial was implemented by a Nepali non-governmental organization, MIRA (Mother & Infant Research Activities). MIRA has been working in Nepal since 1992, conducting research specifically about newborn care, and is headed by a senior pediatrician (DM). The trial involved 24 Village Development Committees in rural Makwanpur district. Ethical approval was sought from the Nepal Health Research Council, and local meetings were held with the District Development Office and Chief District Officer to discuss the aims and objectives of the study. The chairpersons for each Village Development Committee agreed to take part in the study and provided signed consent, and links were made with community leaders, district health services and non governmental organisations. Each Village Development Committee has an average population of 7000 (range 1576 to 23 429) divided between nine wards. In twelve of the Village Development Committees a trained, locally based facilitator was employed to mobilize women's groups. All pregnancies and births to married women of reproductive age were monitored in the community. Details of the monitoring and the design of the trial have been described elsewhere [ 10 ] and the effect of this intervention on birth outcomes was reported in a recent publication [ 11 ]. Astonishingly, there was a reduction in neonatal mortality by 30% in intervention clusters, and an even larger and statistically significant effect on maternal mortality rates (78% reduction), although caution is required in interpretation given the relatively few maternal deaths. This paper describes and analyses the implementation of the first stages of the participatory intervention over a 30 month period. Methods Setting of intervention Nepal has a population of 23 million and a per capita gross national product of 240 US dollars. Literacy rates have improved steadily, particularly for females (currently 43%), but there remain gender disparities in literacy, school enrolment, and school dropout rates [ 12 ]. Life expectancy is now 61 years[ 13 ]. The total fertility rate is 4.1, the under-five mortality rate 91, the infant mortality rate 64, the neonatal mortality rate 39 per thousand live births, and the perinatal mortality rate 47 per thousand births [ 9 ]. The maternal mortality ratio is estimated at 539 per 100 000 live births [ 14 ]. Access to health care is limited as a result of geography, limited expenditure on public health, variable quality of care, high turnover of service providers, a lack of drugs and supplies, and lack of ownership of health programmes by communities. Makwanpur district, south west of Kathmandu, has a population of 376 000 [ 15 ] and a Human Development Index of 0.31, close to the national median. Makwanpur comprises hill and plain areas, with 15 different ethnic groups, the largest being Tamangs, a Tibeto-Burman group. Data from our baseline survey showed that more than 90% births take place at home and only five percent are attended by a trained birth attendant. The first health care provider in times of maternal or neonatal illness is the shaman ( dhami jhankri ) or traditional healer [ 16 ]. The intervention process The first ten meetings of the women's group participatory intervention were based on the design of the Warmi project in Bolivia [ 17 ]. In order to enter the communities successfully we gathered detailed information on local social networks and organizations, as well as attitudes and practice around the time of pregnancy and birth. Social mapping and qualitative research were conducted and served as a training exercise in facilitating focus group discussions and building rapport in the community [ 18 ] Establishing facilitated women's groups Meetings were facilitated by a paid, locally based woman, who was selected on merit and trained in facilitation techniques. The position of facilitator was locally advertised and suitable candidates were interviewed by senior MIRA employees. Each facilitator is paid a salary slightly higher than the government equivalent (5330 Nepalese rupees, or 71 US dollars). Her full-time responsibility was to plan and facilitate monthly women's group meetings, each facilitator leading nine groups per month, covering an average population of 7000. Meetings were organized in co-ordination with the local Female Community Health Volunteer, an unpaid community based health worker. In profiling our study area, we found that nongovernmental organisations or community based organisations did not routinely work in all 24 of our study Village Development Committees and had different agendas. The female community health volunteer works at ward level, and as part of her job description she runs women's groups to conduct health promotion activities. The facilitator used a meeting manual, adapted from the Warmi project, to guide the women's groups through problem identification and community planning using participatory iterative methods (see Figure 1 and Table 1 ). Facilitators were trained in the use of this manual and were allowed scope for their own input. Facilitation supervisors were also appointed after national advertisement and formal interview, and two men and three women were selected. One supervisor was provided for every three facilitators, providing support through community visits and regular meetings. Figure 1 The community action cycle Table 1 Content of first ten women's group meetings Stage in the cycle Meeting Content Introduction 1 To introduce the group to MIRA Makwanpur's work 2 To discuss why mothers and newborn infants die To introduce how MIRA will work in the community Problem 3 To find out how women understand maternal and neonatal problems Identification 4 To find out what kind of maternal and neonatal problems are in the community 5 To discuss whether the maternal and neonatal problems are common To identify strategies to collect information from the community Problem Prioritisation 6 To share the information collected from other women in the community To decide what are the three most important maternal and neonatal health problems Planning together 7 To discuss possible strategies for addressing the prioritised problems 8 To discuss which other community members should be involved in developing strategies 9 To discuss how to prepare for the community members meeting 10 The community members will learn about what the women have been doing The community members will learn about the three problems identified by the women The community members will learn about the possible strategies To reach a consensus of the strategies First meetings and problem identification Facilitators and supervisors were responsible for creating awareness and interest in their communities about the meetings, and in most wards at least 20 women attended the first few meetings (see Figure 2 ). Time was taken to introduce the study agenda to the groups, especially important in areas where many non governmental organisations work and expectations were often high. The first three meetings facilitated discussion of the reasons why mothers and newborn infants die in the community. The reasons for death were discussed in terms of social as well as medical factors with the aid of a story [ 19 ]. Women were introduced to the concept of ' learning together ' through another story, and were encouraged to discuss perinatal problems within the group and with their neighbours and friends. In this way the facilitator and the women learned which perinatal problems affected their community. Each group prioritised three problems of newborn infants and/or pregnancy which were recorded with justification for their inclusion. Most group members were illiterate and therefore facilitators used pictures and voting with stones to prioritise problems. Figure 2 Women's group and facilitation manager Planning together The objective of these meetings was to encourage women to identify local and low cost ways of tackling the prioritised problems using local resources. Many examples were listed in the manual and the supervisor was encouraged to support the facilitator during these meetings. The idea behind these meetings was to enable the women to prepare a plan to tackle the problems they had found, which would then be presented to their community. The community meeting A community meeting was planned and organized by the groups to enable increased community participation and to legitimize the work of the group. The community was invited to hear what the women's group had been doing, and to participate in planning together strategies. Most groups decided that community leaders would be invited by letter, and other households would be verbally invited. The groups also discussed the way they would present their findings and practised to develop their confidence. The supervisor supported the local facilitator and the group, and played a key role in facilitating the meeting. After introducing MIRA and its role in the community, the women's group presented their prioritised problems and suggested strategies to tackle them. Methods of data collection and analysis Data were collected through a variety of qualitative techniques. Participant observation was carried out by the technical advisors to MIRA (NM and JM). They helped design and implement the intervention, and lived in the vicinity of the head office in Hetauda, Nepal, throughout the study period (JM succeeded NM). They visited the field many times and attended facilitation team meetings regularly. The advisors are of British nationality but have an excellent spoken command of Nepali language and have a background in anthropology and sociology. Although they did not keep a diary as is usual in participant observation, their reflections and observations were noted in monthly reports and topic reports which have been used in this analysis. These reports were also contributed to and discussed with the facilitation team (facilitators, supervisors, and senior facilitation manager) who also added their reflections and observations. The technical advisor (JM) and senior facilitation manager (ST) analysed monthly reports and meeting minutes and reached consensus on themes emerging from the data, and issues of interest. Although there are limitations to this method of data analysis (the cultural background of the technical advisors and the fact that a diary was not kept), we wished to present the results of operational research that makes use of less formally recognized qualitative data collection techniques. Analysis of the data by more than one person strengthens the analysis and using different methods of data collection enables triangulation of the data. Results Out of 111 women's groups, 77 moved on to develop and implement strategies and 100 groups continue to meet to discuss perinatal health. Particular reasons for which the remaining groups did not meet include the unstable security situation, lack of support from local leaders, husbands or health workers, and general lack of interest. What makes an active women's group? The continuing activity of most groups suggests that usually group members found the experience useful and enjoyable. Not surprisingly, the activity of the groups varied. We found no specific formula for an active women's group. Previous studies suggested that homogeneity of members was conducive to a successful group [ 20 , 21 ], but our groups showed much ethnic and social diversity. Issues of ethnicity, geography and distance from a market area did not uniformly affect the activity of groups. For example, there were two particularly active groups near market areas, but in other areas factors concurrent with living near a market – such as higher socioeconomic status and less cohesion between households – did not facilitate enthusiastic women's groups. Some groups were dominated by women from higher castes, but in others these higher castes served as a stimulant to more traditionally subservient or timid ethnic groups. Issues of local support from political groups, local health staff and men also seemed important. Other studies have also found that supportive husbands make it easier for women to participate in groups [ 22 ]. In most communities supervisors and facilitators had been successful in establishing good community rapport, and strategies were agreed to help maintain community support, such as facilitators attending antenatal clinics and supervisors presenting reports to Village Development Committee chairpersons. Problem prioritization Women actively participated in learning together and gathered much information from their communities. The prioritized problems reflected local perceptions of the seriousness and frequency of specific problems and hence were different in each community (See Table 2 ). Table 2 Problems prioritised by women's groups Neonatal Problems Number of groups Pneumonia in the newborn infant 31 Low birth weight 16 Jaundice in the newborn infant 12 Neonatal death 7 Breathing problem in the newborn infant 6 Infant not feeding 4 Green stool in the newborn infant 3 Wounds in the newborn infant 2 Tetanus in the newborn infant 1 Eye and ear infection in the newborn infant 1 Maternal problems Retained placenta 58 Vaginal Discharge 42 Malpresentation 30 Headache in the mother 22 Post partum haemorrhage 20 Fever (unspecified if in mother or baby) 15 Breast problems 13 Ante partum haemorrhage 11 Miscarriage 11 Abdominal pain in the mother 6 Oedema of hands and legs in the mother 6 Prolonged labour 5 Maternal death (delivery complications) 2 Anaemia in the mother 2 Vomiting in the mother 1 Missing data 3 Total 330 Planning together During the community planning meeting groups nominated members to present their findings and eight groups performed small socio-dramas. When local health personnel and area chairmen attended, discussions were livelier and planning more productive. Issues of health care underutilization by the community or issues of poor service delivery were often raised. In nine places, communities appeared apathetic towards the group and were not prepared to commit or participate in planning. In these instances, they were happy for the group to plan and implement strategies and little discussion took place. In four places the group met with hostility from community leaders or health personnel or exceptionally low attendance from the community, usually due to local grievances with staff selection procedures or to the unstable security situation. Strategy development and implementation The strategies that were discussed during planning together and have been most successfully implemented were the mother and child health fund, locally produced clean home delivery kits, management and production of stretchers, and awareness raising through video shows. Mother and child health fund schemes 69 groups favoured mother and child health funds as a way of overcoming the cost barriers to seeking and obtaining care. The cost of consultation, medicine and transport is a real reason that families do not gain access to services in Nepal [ 23 ]. MIRA provided training to fund management committee members elected from each group. These committees sometimes included literate community members not attending the group. Each group developed their own policy with regard to how money would be collected, who would be able to access it, how often it would be collected, and who would be responsible for managing it. 23 months after the first mother and child health fund was established, groups had generated between 731 rupees (10.5 US dollars) and 9635 rupees (133.8 US dollars). Clean home delivery kits The clean home delivery kit is advocated by the World Health Organisation as an effective way to promote cleanliness during home delivery and to reduce the risk of maternal and neonatal infection [ 24 , 25 ]. In Nepal, a local private company (MCH Products Pvt Ltd) has produced a clean home delivery kit, approved by the Ministry of Health, which contains a blade, a bar of soap, three cord ties, a plastic coin for cord cutting, a plastic sheet, and a set of pictorial instructions. There are problems with distribution to remote rural areas and other difficulties regarding local acceptability and price [ 26 ]. A few groups were keen to develop their own locally produced clean home delivery kit, and facilitators disseminated this idea to motivate other groups by example. 19 groups have made clean home delivery kits and four groups have reproduced subsequent batches. Groups have decided the price, but all groups sell at a lower price than the MCH products kit, with profits going into the mother and child health fund. The pictorial instruction leaflet was developed with a local artist and was piloted in the community. The groups have also explored different selling points: local shops, Female Community Health Volunteers and Traditional Birth Attendants, and group members sold kits to their friends and neighbours. Recently, groups from one Village Development Committee have used free distribution of clean home delivery kits as an incentive to attend for antenatal care; the kits are free for women who attend at least four times. Stretchers In the study area, most births take place in the home [ 27 ] and transportation of women who encounter problems is difficult. Women's groups therefore identified the need for stretchers. 19 groups decided to raise money for stretchers themselves and the other 23 groups utilized local resources such as forest user groups or Village Development Committee offices. Women's groups investigated if there were any existing stretchers in their area, or if these needed repair. One group felt that the modern style of stretcher was not suitable for carrying women across difficult terrain, and therefore made a bamboo basket ( dhoko ) which is traditionally used to carry fodder or crops using a head strap ( tump line ). Some women's groups assumed management of unused stretchers, ensuring their accessibility and promoting their use, with 35 groups levying a fee for usage Awareness raising through video shows During the community meetings, many groups felt that there was a lack of awareness about perinatal health problems and how to deal with them. MIRA had previously researched and produced a 20 minute film about newborn care in Makwanpur and the groups were keen to use it in their communities. Group members approached those households in the community with electricity and a television, and the video was shown in homes or public buildings. Although not all of the study area has electricity, the video was shown in 10 out of 12 Village Development Committee areas, attracting an audience of more than 2100. Participatory health education During the identification of strategies to address problems, there was a tendency to mismatch prioritized problems and strategies. For example, one group suggested tackling the problem of post-partum hemorrhage by attending antenatal care. Another group considered that the problem of vaginal discharge during pregnancy could be addressed by training new Traditional Birth Attendants. During the first ten meetings, and from previous data analyses, the team found an overwhelming preference for care within the community, in terms of place of birth and seeking solutions to health problems [ 16 , 27 ]. Home practices with unequivocal allopathic clinical benefit were rarely mentioned. There was also little knowledge about what kind of problems could be managed at different health service institutions, and it appeared that communities define the "seriousness" of a problem in a different manner to the allopathic model. Therefore, the team concluded that perinatal health education would be useful during the development of the strategies. It was felt important to avoid turning the facilitators into educators, and therefore a participatory form of health education was developed, based around a picture card game. The picture card game A packet of small hand held cards of different shapes was developed in order to address the mismatch between problems and strategies and to promote participatory learning. Each shape of card represents either a problem (circle shape), a prevention activity (triangle shape), a home-care activity (house shape), or a health institution (square shape). The cards are pictorial and were developed with the MIRA health team and a local artist (see Figures 3 and 4 ). They were extensively piloted with women's groups and adapted accordingly. A manual for facilitators was also developed to accompany the cards. The card game is played in the group by passing round the cards and discussing the pictures. The group members match problem cards to their corresponding prevention activities, home care activities or type of health institution that could treat that problem. The card game worked well in facilitating discussion, and women and facilitators both enjoyed the learning experience. The team completed a participatory evaluation of the game with a sample of groups which indicated that the game also facilitated learning about danger signs, home care and prevention activities. Group members are presently taking the picture cards on visits to pregnant women in the community who are not group members. Figure 3 Women's group using picture cards Figure 4 Picture card game and manual Service quality spin-offs Community health volunteers The facilitator has worked to involve and support female community health volunteers with their work in the community. 70 group meetings have regular attendance and active involvement of the local female community health volunteer and traditional birth attendant. The female community health volunteer is the lowest cadre of government appointed health staff and is responsible for one ward. She is unpaid and has a broad job description mainly focused around health education. In theory, she should run monthly women's group meetings to facilitate health education and discuss issues of maternal and newborn health. Although she receives initial and refresher training, she is often left to work unsupervised and unsupported, and in practice female community health volunteers find it difficult to run women's groups. By seeking the participation of the female community health volunteer in the groups, we gave her a forum to conduct her work and increase her contact with her user group. In twelve wards the women's group was invited by the local health institution to play an active role in selection of new female community health volunteers and traditional birth attendants. The group had created good links with the health institution, which could be exploited for future service quality improvements. Clearly, the health institution and the community consider many groups as legitimate entities with a role to play in the health of women and their children. It also appears that members of women's groups have become more involved with their local health services. In one area women's group members responded to the needs of women visiting an outreach clinic for antenatal care and family planning. Women were complaining of a lack of privacy and there was no furniture in the clinic. The group contacted the local forest user group to supply materials for rudimentary furniture and collected money for the purchase of cloth for curtains. In several cases, the women's group was a medium for brokered links between health service providers and users. Proxies for empowerment effects? One group put a sign on the door of their meeting place indicating a sense of ownership. Ten out of 12 facilitators have been invited to participate in other meetings and community activities as their role as key actors in the community is being recognized and developed. Women's groups have sung the song from the video film at the annual women's festival, and a supervisor initiated discussion about newborn health in a local bus using a cassette of the song from the film. Another women's group organised a perinatal care quiz that was carried out with nearby women's groups and community members. Discussion We have described the development and implementation of a large scale participatory intervention with women's groups. This was adapted from a smaller scale project developed in Bolivia, and implemented in a poor rural population of 86 000 in Nepal. The effects on health outcomes, reported elsewhere, were dramatic. During the process of developing and implementing the intervention we had to be flexible and respond to the needs of the group. Group members and the wider community clearly faced difficulties when thinking about ways to tackle perinatal problems. These difficulties raised issues around culture and our facilitation role. Striking a balance between support and directiveness It is highly likely that the facilitation team's attempts to adequately support the facilitators may have led to less participatory processes taking place, especially in the case of strategy development. The facilitation manual was considered by the facilitation team as an essential resource. Examples were often given to enable facilitators to grasp key concepts before conducting a meeting. The manual was designed as a reference guide but evidently became more like an instruction booklet, as the strategies most commonly adopted during community meetings were those given as examples in the manual. The reasons behind this usage of the manual illustrate some of the key issues in implementing a participatory project. To truly facilitate, and not be directive, is a difficult technique to learn, especially in a hierarchical society where the facilitator's education has emphasised rote learning rather than independence of thought [ 28 ]. Our facilitators were also keen to educate and provide the answers, and it may have appeared easier (in the short term) to suggest things for groups to do than to facilitate open discussion. The self-confidence or ability of the facilitator to manage the chaos and unpredictability resulting from a truly participatory process was often lacking, although their facilitation skills developed with time. Power and culture Difficulties in linking problems to strategies may also be explained in part by the cultural phenomenon of fatalism, ' ke garne' ( what to do ?). Bista described ' ke garne ' as a belief in fatalism which leads to the feeling that " one has no personal control over one's life circumstances, which are determined through a divine or powerful external agency " [ 29 ]. He argued that this fatalism and dependency affects the work ethic and achievement motivation in Nepal. Concepts of planning, orientation to the future, and sense of causality, are all affected. Our study experience was that fatalism affected both the way people viewed themselves in relation to a problem, and also the power and capacity they believed themselves to have in overcoming it. What has been the impact of the women's groups and their strategies? In cases where there was a mismatch between problem and strategy, or when groups developed strategies suggested by MIRA, we hope that these groups will benefit from the implementation process alone. The strategies in the manual are not necessarily evidence-based, and it may be that the process of implementation is more beneficial than the strategy itself. Through implementation, interaction between the wider community and the group may be increased, knowledge about the group may spread, and more people may become interested and involved in issues of perinatal health. To enable a better understanding of the intervention process, evaluations using both qualitative and quantitative methodologies are underway. The impact of the women's group intervention was evaluated in a cluster randomized controlled trial which showed a 30% reduction in neonatal mortality rates and a reduction in maternal mortality rates in the first 30 months of the trial[ 11 ]. Qualitative analysis will explore perceptions and process indicators to assess how the intervention affected the study area and community stakeholders. Cost analysis of the intervention will enable estimates of cost-effectiveness and sustainability to be made. A comparison of the socio-economic status of women's group members with non-group members will allow an estimate of the equitability of the intervention. The Millennium Development Goals for reductions in maternal and neonatal mortality in developing countries are unlikely to be met by 2015. In populations where maternal and newborn mortality rates are highest, most deliveries occur at home. It is essential that Safer Motherhood and Newborn Care Programmes design interventions which reach out to the poorest groups in order to change care practices at home, and care seeking for illness or complications of childbirth. Our participatory work with women's groups provides a model for an intervention that can be scaled rapidly in even the poorest and most remote communities. Conclusion A large scale participatory intervention to improve pregnancy outcomes in rural Nepal through 111 women's groups has been described. Although we have faced contextual, cultural and security problems, we believe that the participatory approach can be a powerful tool in unleashing the creative potential to solve perinatal health problems in communities. Such an approach may have lasting benefits, affecting behaviour in subsequent pregnancies. Competing interests The author(s) declare that they have no competing interests. Contribution by authors JM wrote the first draft of the paper and contributed to the study design and collection of field data. ST, and BS contributed to the study design, collection of field data and analysis, and criticised later drafts of the paper. NM and DO contributed to the study design and analysis, and criticised later drafts of the paper. MM and HS contributed to the design of the study and criticised drafts of the paper. DM and AC contributed to the design of the study and supervision of the field programme, and criticised drafts of the paper. JM and AC will act as guarantors for the paper. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079875

Further investigation of confirmed urinary tract infection (UTI) in children under five years: a systematic review

Background Further investigation of confirmed UTI in children aims to prevent renal scarring and future complications. Methods We conducted a systematic review to determine the most effective approach to the further investigation of confirmed urinary tract infection (UTI) in children under five years of age. Results 73 studies were included. Many studies had methodological limitations or were poorly reported. Effectiveness of further investigations: One study found that routine imaging did not lead to a reduction in recurrent UTIs or renal scarring. Diagnostic accuracy: The studies do not support the use of less invasive tests such as ultrasound as an alternative to renal scintigraphy, either to rule out infection of the upper urinary tract (LR- = 0.57, 95%CI: 0.47, 0.68) and thus to exclude patients from further investigation or to detect renal scarring (LR+ = 3.5, 95% CI: 2.5, 4.8). None of the tests investigated can accurately predict the development of renal scarring. The available evidence supports the consideration of contrast-enhanced ultrasound techniques for detecting vesico-ureteric reflux (VUR), as an alternative to micturating cystourethrography (MCUG) (LR+ = 14.1, 95% CI: 9.5, 20.8; LR- = 0.20, 95%CI: 0.13, 0.29); these techniques have the advantage of not requiring exposure to ionising radiation. Conclusion There is no evidence to support the clinical effectiveness of routine investigation of children with confirmed UTI. Primary research on the effectiveness, in terms of improved patient outcome, of testing at all stages in the investigation of confirmed urinary tract infection is urgently required.

Background UTI in children is an important clinical problem. Renal scarring, which occurs in a small proportion of children (approximately 6%[ 1 ]), is the most important outcome of infection as it is associated with significant future complications[ 2 ], and ultimately with end stage renal disease[ 3 ]. Young children are considered particularly vulnerable to renal scarring and its consequences[ 4 ]. However, a recently completed 20-year follow-up study suggested that compensatory mechanisms mean no significant changes in overall GFR occur in patients with unilateral scaring[ 1 ], and the risk of hypertension is low in all patients (regardless of the degree of scarring)[ 5 ]. Current UK recommendations state that all children under 5 years of age should be investigated after their first confirmed UTI[ 6 , 7 ], although the benefit from this strategy has been questioned[ 8 ]. Further investigation of children with confirmed UTI has a number of different clinical aims: the localisation of infection, the prediction and detection of renal scarring and the detection of VUR. The current reference standards for these investigations are Tc-99 m-DMSA renal scintigraphy (DMSA scan) for the localisation of infection and for the detection and prediction of renal scarring, and micturating cystourethrography (MCUG) for the detection of VUR. These investigations have the disadvantages of being invasive and involving exposure to ionising radiation. It is desirable to minimise the number of invasive examinations and radiation load to which children are exposed. Alternative tests that offer a potential advantage over the current reference standards, such as ultrasound or laboratory-based tests, are therefore required. An additional aim of the investigation of children with UTI is the detection of anatomical abnormalities that may be amenable to surgery, and a role has been suggested for ultrasound in this context. We did not identify any studies evaluating tests with this objective; therefore it could not be assessed in this review. However, a recently published observational study has suggested that routine ultrasound post-UTI, in children under five years, does not change management[ 9 ]. The role of pre-natal ultrasound is unclear[ 9 , 10 ] and was outside the scope of this review. We reviewed the diagnostic accuracy of tests evaluated for the further investigation of UTI together with evidence of their long-term effectiveness, with a view to determining the optimum diagnostic pathway. A previous systematic review has evaluated ultrasound for the detection of scarring[ 11 ]. This review was published in 1999 with searches undertaken in 1997 and only included 10 studies. We are unaware of any other systematic reviews in this area. This review therefore represents the most complete review of the area. Methods We assembled a database of published and unpublished literature from systematic searches of 16 electronic databases (inception to between October 2002 and February 2003), hand searching of 12 journals, consultation with experts in the field, and screening bibliographies of included studies. Update searches were conducted in May 2004. There were no language restrictions. Full details of the search strategy will be reported elsewhere[ 12 ]. We included controlled trials that compared different imaging strategies and reported patient based outcomes. We also included diagnostic cohort studies that included at least 20 children, some of whom were aged five years or under, and that reported sufficient information to construct 2 × 2 tables. Table 1 presents a summary of the tests evaluated in this review and details the potential advantages that these tests have over the current reference standards. Although other tests have been evaluated, this article will focus on those offering potential advantages over the current reference standard. Studies had to compare one of the index tests listed in this table to the listed reference standard, for each of the different aims. Table 1 Summary of tests used for different clinical applications Aim Current Reference standard Tests Advantage over the reference standard Localisation of infection Tc-99 m-DMSA renal scintigraphy Clinical features Laboratory-based tests Ultrasound All less invasive, no exposure to ionising radiation Detection of reflux Micturating cystourethrography (MCUG) Ultrasound Less invasive, no exposure to ionising radiation Indirect radionuclide cystography Single procedure test for reflux and scarring Prediction of renal scarring Follow-up Tc-99 m-DMSA renal scintigraphy Clinical features Would allow earlier prediction of children at risk of renal scarring Ultrasound MCUG Acute DMSA renal scintigraphy Detection of renal scarring Tc-99 m-DMSA renal scintigraphy Ultrasound Less invasive, no exposure to ionising radiation Intravenous pyelography (IVP) Provides a detailed anatomic map, considered essential where surgery is planned. Radionuclide cystography Advocated as a single procedure test for reflux and scarring Two reviewers independently screened titles and abstracts for relevance, any disagreements were resolved by consensus. One reviewer performed inclusion assessment, data extraction and quality assessment; a second reviewer checked this. We extracted 2 × 2 data from each of the studies and used this to calculate estimates of test performance. Where insufficient details were reported, we contacted authors to request further information. We assessed the methodological quality of diagnostic accuracy studies using QUADAS[ 13 ]. Individual QUADAS items were used to investigate heterogeneity and to present a detailed assessment of quality to the reader. We analysed results grouped by clinical aim. Where studies presented more than one estimate of test performance for the same test, we only included one estimate in the pooled analysis. We aimed to select the most appropriate data set or the one most similar to that used by other studies in terms of population, technique or unit of analysis. For example, data for tests used to localise infection were analysed by patient in preference to kidney or renal unit (kidney and ureter), whereas data for tests used to detect VUR were analysed by renal unit. For each test, or test combination, we calculated the range in sensitivity, specificity, positive (LR+) and negative (LR-) likelihood ratios, and diagnostic odds ratios (DOR). Where sufficient studies were available, pooled likelihood ratios were calculated for each test[ 14 ]. Heterogeneity of likelihood ratios was investigated using the Q statistic[ 15 ] and through visual examination of forest plots of study results[ 16 ]. We presented individual studies results graphically by plotting estimates of sensitivity and specificity in receiver operating characteristic (ROC) space. Where sufficient data were available, we used regression analysis to investigate heterogeneity. We extended the summary ROC (sROC) model[ 17 ], estimated by regressing D (log DOR) against S (logit true positive rate – logit false positive rate), weighted according to sample size, to include the variables for patient age (<2 years, <5 years, <12 years and <18 years), geographic region, and QUADAS items[ 18 ]. For ultrasound for the detection of VUR a variable for ultrasound technique (contrast-enhanced or standard) was also included. Results We identified more than 10,000 studies. Of these, 73 studies met our inclusion criteria: 72 diagnostic accuracy studies and one RCT of the clinical effectiveness of investigation. Figure 1 shows the flow of studies through the review process. The results of individual included studies are presented [see Additional file 1 ]. Table 2 presents summary results for each included test. Figure 1 Flow of studies through the review Table 2 Summary of the results of imaging studies Test Number studies Range LR+ Pooled LR+ (95% CI) Range LR- Pooled LR- (95% CI) Localisation of infection Ultrasound 20 1.6 to 55.0 3.5 (2.5, 4.8) 0.10 to 0.91 0.57 (0.47, 0.68) Clinical features 5 1.1 to 26.6 - 0.09 to 0.89 - Infection markers 10 1.0 to 8.8 - 0.09 to 1.00 - Renal function markers 4 0.7 to 36.7 - 0.02 to 1.51 - Immunofluorescence detection of bacteria 1 1.8 - 0.55 Detection of reflux Ultrasound: standard 12 1.0 to 8.7 1.9 (1.2, 2.9) 0.05 to 0.98 0.76 (0.63, 0.93) Ultrasound: contrast enhanced 16 3.8 to 71.2 14.1 (9.5, 20.8) 0.04 to 0.51 0.20 (0.13, 0.29) Indirect radionuclide cystography 3 11.2 and 25.0 - 0.41 and 0.68 - Prediction of renal scarring Ultrasound 2 1.3 to 3.0 - 0.60 to 0.86 - Micturation cystourethrography 2 2.6 and 2.7 - 0.71 and 0.64 - Temperature 1 1.1 - 0.44 - CRP 1 1.1 - 0.44 - Intravenous pyelography 1 12.9 - 0.88 - Acute renal scintigraphy 1 3.1 - 0.54 - Detection of renal scarring Ultrasound 7 1.3 to 35.9 - 0.14 to 0.99 Intravenous pyelography 4 10 to 171.3 - 0.15 to 0.80 - Indirect radionuclide cystography 2 2.1 to 12.6 - 0.15 to 0.75 - Clinical effectiveness One RCT evaluated the effectiveness of routine follow-up investigation for children with confirmed UTI. This study was published as an abstract and we are unable to obtain further data[ 19 ]. The objective was to determine whether routine imaging, using ultrasound and MCUG, of children with their first UTI significantly reduced renal scarring or recurrent UTI. Children aged 2–10 years (n = 172), with confirmed UTI, were allocated to routine (all received Ultrasound and MCUG) or selected imaging (Ultrasound and MCUG for recurrent UTI or persistent problems). Routine investigation lead to higher rates of imaging (100% vs 21%), identification of VUR, and antibiotic prophylaxis compared to the selective investigation group. However, there was no difference in the proportion of children with recurrent UTI or in the rate of renal scarring between the two groups after two years of follow-up. The authors concluded that routine imaging of toilet trained pre-school and school aged children with their first uncomplicated UTI is not worthwhile. Diagnostic accuracy None of the studies fulfilled all QUADAS criteria. Inadequate reporting was a problem in many studies; only two studies reported sufficient information to determine whether each criterion had been met. Less than half of studies included an appropriate spectrum of patients, and reported selection criteria. Incorporation bias, verification bias, and disease progression bias were also inadequately addressed by around half of all studies. Results of the quality assessment are presented [see Additional file 2 ]. Localisation of infection A total of 31 studies (61 evaluations) evaluated the diagnostic accuracy of one or more tests to localise infection. Ultrasound was evaluated in 20 studies [ 20 - 39 ]. Performance was poor both in terms of ruling in and ruling out renal involvement: the pooled positive likelihood ratio was 3.5 (95% CI: 2.5, 4.8), and the pooled negative likelihood ratio was 0.57 (95% CI: 0.47, 0.68). Figure 2 shows estimates of sensitivity and specificity for these studies plotted in ROC space. There was significant between study heterogeneity in the ultrasound evaluations (p < 0.0001). None of the items investigated in the regression analysis showed a significant association with the DOR. Thirteen studies investigated clinical or laboratory-based tests[ 22 , 25 , 40 - 50 ]. The tests investigated varied greatly, and in general, showed poor performance. Figure 2 Sensitivity and specificity plotted in ROC space for ultrasound for the localisation of infection Detection of VUR We identified 30 studies (40 evaluations) evaluating the diagnostic accuracy of tests to detect VUR. Ultrasound was assessed in 28 studies, 12 using standard ultrasound techniques[ 35 , 51 - 61 ], and 16 using cystosonography or other contrast-enhanced techniques [ 62 - 77 ]. Standard ultrasound techniques performed poorly: the pooled positive LR was 1.9 (95% CI: 1.2, 2.9), and the pooled negative LR was 0.76 (95% CI: 0.63, 0.93) Contrast-enhanced ultrasound techniques showed much better performance, with higher pooled positive likelihood ratios (14.1, 95% CI: 9.5, 20.8) and lower pooled negative likelihood ratios (0.20, 95% CI: 0.13, 0.29). Figure 3 shows estimates of sensitivity and specificity for all ultrasound studies plotted in ROC space. Both techniques showed between study heterogeneity (p < 0.001). Regression analysis found that ultrasound technique, disease progression bias and use of an appropriate reference standard showed a significant association with the DOR. The DOR was 16.87 times greater (95% CI: 7.03, 40.48) in studies that used contrast enhanced ultrasound rather than standard ultrasound; 2.65 (95% CI: 1.02, 6.90) times higher in studies in which there was no clinically significant delay between the index test and reference standard; and 7.14 (95% CI: 1.13, 50) times higher in studies that used an inappropriate reference standard. Figure 3 Sensitivity and specificity plotted in ROC space for ultrasound for the detection of reflux Two studies evaluated indirect radionuclide voiding cystography[ 78 , 79 ]. These reported good positive likelihood ratios (11.2 and 25.0), but negative likelihood ratios were poor (0.41 and 0.68). Prediction of renal scarring Five studies (nine evaluations) investigated the ability of a variety of tests (clinical, laboratory-based, and imaging techniques) to predict renal scarring[ 29 , 32 , 80 , 81 ]. The diagnostic accuracies reported in these studies were poor. Positive LRs were in the range of 1.1–3.1 for four of the studies, with the fifth (the only study of IVP) reporting a positive LR of 12.9. Negative likelihood ratios ranged from 0.44 and 0.88. Detection of renal scarring Thirteen studies evaluated the diagnostic accuracy of tests to detect renal scarring. Four studies evaluated the diagnostic accuracy of IVP: positive likelihood ratios were high (10.0 to 171.3), but corresponding negative likelihood ratios were poor (0.15 to 0.80)[ 80 , 82 - 84 ]. Only one study included an appropriate patient spectrum[ 80 ], and this reported a much lower positive likelihood ratio (10.0 compared to next lowest of 58.8), and higher negative likelihood ratio (0.80 compared to next highest of 0.42) than the others. Seven studies evaluated standard ultrasound techniques[ 60 , 85 - 90 ]. Figure 4 shows estimates of sensitivity and specificity for these studies plotted in ROC space. Performance characteristics varied greatly, although positive likelihood ratios were generally moderate to high (1.3–35.9). Negative likelihood ratios showed poorer performance for ruling out scarring (0.14–0.99). Three studies assessed the diagnostic accuracy of indirect radionuclide cystography[ 78 , 84 , 91 ]; positive likelihood ratios were moderate and ranged from 3.3 to 12.6, and negative likelihood ratios ranged from 0.15 to 0.63. Figure 4 Sensitivity and specificity plotted in ROC space for ultrasound for the detection of scarring Discussion When considering further testing following confirmation of UTI, it is important to bear in mind clinical aim. If the information derived cannot be used to prevent renal disease there is little benefit in testing. Tests should only be conducted if (a) the results will lead to a change in management and (b) this change is likely to lead to an improved outcome. For this reason, the ideal study would be a randomised controlled trial of different testing strategies, or no testing. We identified only one such study, and this was only available as an abstract reporting limited information, and we were unsuccessful in obtaining further details[ 19 ]. Our results are therefore primarily derived from diagnostic accuracy studies, which assume the validity of the clinical aims of current testing. Localisation of infection can be considered a first step in the investigation of UTI. Lower UTI does not involve the kidneys and so cannot lead to renal scarring. Children with lower UTI are therefore unlikely to benefit from immediate investigation. Given that therapeutic delay is thought to be associated with renal damage[ 92 ], the possibility that they may benefit from monitoring for recurrence remains open to question. The ideal test to localise infection would be non-invasive, inexpensive, and quick to perform. Further investigation of children with infections of the lower urinary tract could thus be avoided. Our results do not support the use of any of the minimally invasive tests evaluated as alternatives to renal scintigraphy for the localisation of infection. However, the available evidence was limited, and further primary research in this area would be useful. Testing with the specific aim of localisation of infection is not common in current practice. Baseline renal scintigraphy in all children with confirmed UTI, in whom further investigation is planned, may be beneficial. This approach would eliminate a substantial proportion of children from further invasive investigations. The detection of VUR has historically been considered an important element in the investigation of UTI, as it has been thought to indicate an increased risk of scarring. This idea is currently the subject of considerable debate. The only study of the effectiveness of imaging identified by our review compared routine and selective imaging, using US and MCUG for the detection of VUR[ 19 ]. This study found increased rates of VUR detection and prophylaxis with routine imaging, but no reduction in scarring or recurrent UTIs. Other studies have shown that the presence of VUR, as determined by MCUG, correlates poorly with the presence of renal scarring[ 78 , 93 - 95 ]. A recent systematic review also found that VUR is a weak predictor for renal damage in children hospitalised with UTI[ 96 ]. The management of VUR and how this impacts on the risk of future renal disease is also the subject of debate. A clinical trial comparing surgical and medical management found no difference in outcome[ 97 ], and a systematic review evaluating antimicrobial prophylaxis for the prevention of UTI in children found a lack of data for children with VUR[ 98 ]. Given the considerable doubts surrounding both the link between VUR and renal scarring, and the benefits to be derived from treating VUR, it appears difficult to justify the routine use of MCUG. This is an invasive, and costly test, involving considerable exposure to ionising radiation; its use should therefore be minimised where possible. Should the evaluation of VUR be considered clinically necessary, the available evidence supports the use of contrast-enhanced ultrasound techniques as an accurate alternative. Although not currently in widespread use in the UK, these techniques have the advantage of not involving exposure to ionising radiation. In addition, standard ultrasound forms part of the examination, allowing simultaneous screening for anatomical abnormalities and some types of calculi. A test predicting risk of renal scarring would be useful were a treatment available to prevent its development. Anti-microbial therapy is usually initiated in children with confirmed UTI prior to investigation, as there is some evidence that treatment delay may affect scarring[ 3 , 7 ]. Predicting risk of renal scarring as a result of a current infection would, therefore, appear to be of academic interest alone. We identified no acute tests that were able to accurately predict the development of renal scarring. The prediction of risk of future infection is of potential interest in guiding the initiation of prophylactic antimicrobial therapy, but is outside the scope of this paper. Although the presence of renal scarring represents the initial stages of renal disease, there is little that can be done to treat patients with scarring in order to prevent complications. If progressive scarring is assumed to be the consequence of repeat infections then anti-microbial prophylaxis may be initiated, although the effectiveness of this strategy remains open to debate. Imaging for the detection of renal scarring may be seen as a means of monitoring disease progression. If repeat examination is required then a less invasive alternative to the reference standard (renal scintigraphy), and one which avoids the use of ionising radiation, would seem particularly desirable. We found standard ultrasound examination to be a potentially useful test for ruling in scarring, but poor for ruling it out. This fits with anecdotal opinion that ultrasound is good at identifying gross scarring, but poor at detecting minor lesions. It may be that ultrasound images are insufficiently subtle to enable their use in monitoring disease progression. Further research on the accuracy of ultrasound in grading scarring is therefore required. Indirect radionuclide cystography is sometimes advocated as an alternative test for renal scarring, on the grounds that it may combine detection of VUR and scarring in a single test. We found it to be an accurate test for scarring, but poor for ruling out VUR. Conclusion There is no evidence to support the clinical effectiveness of routine investigation of children with confirmed UTI. There is limited evidence that routine imaging to detect VUR, following first UTI in older children, has no effect on recurrence or renal scarring. High quality primary research on the effectiveness, in terms of improved patient outcome, of testing at all stages in the investigation of confirmed UTI is urgently required. Competing interests The author(s) declare that they have no competing interests. Authors' contributions All authors contributed towards the conception and design of the study and the interpretation of the data. They also read and approved the final manuscript. MEW and PFW participated in data extraction, the analysis of data, and drafted the article. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Additional file 1 is a Microsoft Word file containing a table of the results of individual studies included in the review. Click here for file Additional File 2 Additional file 2 is a Microsoft Word file containing a table of the results of the quality assessment of included studies. Click here for file

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1079876

Anticipatory nausea in cyclical vomiting

Background Cyclical Vomiting Syndrome (CVS) is characterised by discrete, unexplained episodes of intense nausea and vomiting, and mainly affects children and adolescents. Comprehending Cyclical Vomiting Syndrome requires awareness of the severity of nausea experienced by patients. As a subjective symptom, nausea is easily overlooked, yet is the most distressing symptom for patients and causes many behavioural changes during attacks. Case presentation This first-hand account of one patient's experience of Cyclical Vomiting Syndrome shows how severe nausea contributed to the development of anticipatory nausea and vomiting (ANV), a conditioned response frequently observed in chemotherapy patients. This conditioning apparently worsened the course of the patient's disease. Anticipatory nausea and vomiting has not previously been recognised in Cyclical Vomiting Syndrome, however predictors of its occurrence in oncology patients indicate that it could complicate many cases. Conclusion We suggest a model whereby untreated severe and prolonged nausea provokes anxiety about further cyclical vomiting attacks. This anxiety facilitates conditioning, thus increasing the range of triggers in a self-perpetuating manner. Effective management of the nausea-anxiety feedback loop can reduce the likelihood of anticipatory nausea and vomiting developing in other patients.

Background Cyclical Vomiting Syndrome (CVS) has been recognised for over 100 years [ 1 ]. It is probably a manifestation of migraine diathesis [ 2 ], and is characterised by recurrent episodes of intense [ 3 , 4 ] nausea and vomiting, with normal health between emetic crises. Attacks are self-limiting, lasting several hours to over a week, and their duration is often stereotypical for each patient [ 5 ]. Episodes of CVS are most commonly precipitated by infections, physical or emotional stress, excitement, or specific foods [ 6 ], and often commence during the night or early morning [ 3 , 6 ]. The following case report is that of the first author. It is derived from patient memory and family diaries, and describes a previously unrecognised complication of CVS that is invisible to the clinician due to its subjectivity. It suggests a triggering mechanism based upon classical conditioning and exacerbated by anxiety about the prospect of severe nausea, similar to the anticipatory nausea and vomiting (ANV) seen in chemotherapy patients. Recognition and effective management of nausea can reduce the risk of this complication from developing in other CVS patients. Case report A 28-year-old postdoctoral researcher experienced multiple episodes of vomiting from the age of 3 years. The episodes occurred with upper respiratory tract infections and asthma, and generally lasted 24–48 hours. The patient's mother had nearly identical episodes during childhood. The maternal grandmother experienced hyperemesis gravidarum. The patient's sister and paternal grandfather had classical migraine headaches. The patient first presented to the hospital at age 10 1/2 years. A typical vomiting attack had been triggered by a cold, but had not resolved after four days. Despite treatment with intravenous fluids, the vomiting continued for a further two days before resolving. Cyclical vomiting was diagnosed. She was subsequently hospitalised repeatedly with episodes of about four days duration, with vomiting occurring every 5 to 10 minutes at peak. She continued to have similar attacks during every infection, but never without prior infection. The symptoms were refractory to anti-emetics available at the time (eg. metoclopramide, prochlorperazine). Shortly before Christmas of that year (1986), a 6 1/2-day vomiting episode was triggered by pneumonia, and the patient spent the Christmas period in hospital. The following year (1987), a cold triggered a CVS attack two days before Christmas. Having spent two successive Christmases in hospital, the patient hoped to be well the next year. However, she was woken by nausea in the early hours of Christmas Day 1988 and began vomiting. Uniquely, this episode had not been triggered by an infection, and the patient (then aged thirteen) recognised this. Subsequently, episodes rapidly increased in frequency: compared with an average of 7.3 episodes per year in the three preceding years, the patient averaged 22.3 episodes per year over the three years following Christmas 1988. Many further episodes now occurred without infection, but coincided with visits to certain places (e.g. friend's house), or commenced on specific days of the week (e.g. Saturday). Most episodes resulted in 3–8 day hospitalisations. At one point, a repetitive cycle commenced, where each episode occurred exactly two days after discharge from hospital following the previous attack. The patient was aware of this pattern and consciously afraid of becoming ill at the expected time. Her attacks always commenced nocturnally and she became reluctant to go to sleep, fearful of another episode beginning. The patient experienced exhaustion and severe, intractable nausea for the entire length of her emetic episodes (up to 6 1/2 days), and the nausea caused marked behavioural changes. Sleep was the only state that allowed her to be insensible to nausea, but she was often unable to lie down, as this intensified her nausea, causing retching and heartburn. Instead, she spent hours sitting motionless in bed with her hips and knees flexed, her arms and head resting on her knees and her eyes closed. She was unable to lie supine for abdominal palpation. She would repeatedly expectorate, as she experienced hypersalivation [ 7 ] and was too nauseated to swallow. Accumulated oral secretions, and the profound fatigue and confusion that accompany nausea, contributed to her verbal unresponsiveness. Attempting to reduce intolerable nausea, she frequently induced vomiting by drinking large volumes of water. This also diluted bile and gastric acid, thus lessening oesophageal and oropharyngeal discomfort during vomiting [ 7 ]. Consistent with the natural history of the condition, the attacks gradually became less frequent as the patient grew older. Availability of newer anti-emetic agents (5-HT 3 antagonists) also contributed to the improvement: prompt treatment of episodes with intravenous ondansetron, ranitidine and analgesics reduced the duration and severity of attacks, and the same medications were partially successful as prophylactics. Thus, having achieved partial symptom control, and an understanding of how her anticipation of episodes was exacerbating her condition (and therefore when to use prophylactic medication), the patient felt more in control of her illness, feared it less, and conditioned responses to situations previously associated with CVS attacks gradually diminished. To date, the patient has been hospitalised nearly 100 times, however now experiences vomiting attacks only rarely (fifteen episodes over the past decade, including only one episode over the past five years). Discussion The emetic reflex has evolved to expel ingested toxins [ 4 ]. Nausea is a vital component of this protective reflex: as an intensely unpleasant sensation it subsequently causes potent aversion to the offending food through association. Thus a key feature of nausea is its rapid conditioning, and non-vomiting species (e.g. rodents) rely solely upon this 'conditioned taste-aversion' for avoiding toxins. Unfortunately, this evolutionary legacy is problematic in clinical situations. Oncology patients whose vomiting is caused by cytotoxic drugs may also develop emesis prior to subsequent treatments [ 8 ]. This anticipatory nausea and vomiting (ANV) occurs through classical (Pavlovian) conditioning [ 8 , 9 ]. Chemotherapy (the unconditioned stimulus) is administered in hospital (the conditioned stimulus). Chemotherapy causes emesis (the unconditioned response). Patients subsequently associate the hospital with nausea and vomiting: smells, sights, or thoughts of the hospital can then elicit emesis (the conditioned response) without the emetogenic agent. ANV is also seen in animal models [ 10 ], and is possibly involved in pregnancy-sickness [ 11 ]. However, its occurrence in CVS has remained unrecognised. In this patient's case, ANV increasingly precipitated CVS episodes. Before Christmas 1988, attacks only occurred during infections. Christmas (associated with infection and vomiting in two successive years) was the first conditioned stimulus. Christmas subsequently elicited the conditioned response (vomiting) in the absence of the unconditioned stimulus (infection). Occurrence of attacks without prior infection caused the patient to fear the illness and search for alternative triggers. A concurrent increase in the frequency of episodes occurred, and increasingly insignificant events associated with past episodes became sufficient to trigger vomiting. This 'stimulus generalisation', whereby conditioned stimuli become progressively less specific, is a feature of conditioned responses [ 9 ]: in some oncology patients the sight of any nurse can eventually induce emesis [ 9 ]. In our CVS patient, fear or expectation of an attack in itself became a trigger, and she became conscious of this. Consequently, many attacks occurred before important occasions (e.g. holidays, family celebrations, school examinations, university interviews), when she particularly wished to remain well: this constantly reinforced conditioning. As her episodes always commenced during sleep, she felt powerless to control her ANV. One study [ 12 ] found ANV in 59% of paediatric cancer patients. Development of ANV is positively correlated with severity of vomiting (intensity, frequency, duration) and number of chemotherapy cycles ('conditioning trials'); and inversely with patient age [ 9 , 13 ]. In CVS, the severity of emesis [ 4 ] and number of conditioning trials can exceed that in chemotherapy patients, and CVS mainly affects children. Based on these predictors, and the intrinsic evolutionary links between conditioning and nausea, other CVS patients might develop ANV, as illustrated by another young woman's description: " [For] about two months...I would get sick every Saturday morning. I would be sick until Wednesday, feel good on Thursday and Friday and then the cycle would start all over. Looking back at it now, I know I worried when next Saturday would roll around. I think I worried myself so much that in a way I helped my body into the cycle." [ 14 ]. We believe that ANV could complicate many CVS cases in addition to these two, but is extremely difficult for clinicians to recognise. Whilst triggers for chemotherapy-associated ANV are specific, controlled, and can be objectively observed by doctors within the hospital; the conditioned stimuli for CVS-associated ANV are subtle (e.g. certain day of the week), subjective (e.g. fear of a CVS attack), and occur outside of the hospital, thus are invisible to doctors. As a first-hand account, this case report therefore provides a unique, qualitative perspective on triggering factors as viewed through the eye of the patient, and reveals the hidden role of conditioning. ANV progressed in this patient because of her extremely severe nausea, and her consequent fear of future episodes. Conditioning is more potent when the patient is anxious [ 13 , 15 , 16 ]; and in patients who expect and experience much distress from nausea [ 12 ]. Based on these observations, therapy to prevent ANV in CVS should have two aims: reduction of nausea, and reduction of anxiety. Consistent with this, it is noted that CVS patients who are given prompt anti-nausea treatment during an attack experience recurrence of attacks less frequently [ 7 ]. Unfortunately, most CVS patients are treated by non-specialists, who may overlook nausea as it is subjective and unquantifiable [ 17 , 18 ]. Standard management therefore focuses on rehydration, which, without controlling nausea, cannot prevent dread of future attacks and the development of ANV. Iatrogenic anxiety may exacerbate this: nausea-induced behavioural changes seen during attacks [ 2 , 7 ], which may appear 'psychotic' [ 19 ] and 'regressive' [ 20 ], can prompt the misdiagnosis of factitious vomiting [ 2 ], hence raising patient anxiety through stigmatisation. For example, the patient reported here feared being ill before school examinations not only because of the physical unpleasantness of the illness and the educational consequences of missing important exams, but also because she believed that her doctors would interpret a CVS attack at such a time as evidence of psychosomatic illness. Management of nausea should comprise an individualised plan devised between patient and physician. Where attacks are sufficiently frequent to justify daily medication, anti-migraine prophylaxis (e.g. cyproheptadine, pizotifen, amitriptyline or propranolol) might help prevent episodes. Prophylaxis also requires amelioration of known triggering factors, e.g. acute or chronic infection. In patients with prodromal symptoms, oral ondansetron and/or lorazepam sometimes abort the episode. If vomiting commences, an intravenous infusion containing glucose, sodium, potassium and ranitidine should be started immediately. Intravenous ondansetron and lorazepam may terminate vomiting; otherwise the patient should be sedated to reduce sensations of nausea. This can be achieved using intravenous chlorpromazine plus diphenhydramine every 3 to 4 hours until the episode abates. The second goal of therapy, reduction of anxiety about the illness, will result from successful treatment of nausea, but is also facilitated by a patient-centred, holistic approach to care. Established ANV can be treated by relaxation-based behavioural approaches such as 'counterconditioning' (systematic desensitisation) and hypnosis [ 11 ]. However, as ANV is mediated through the normal psychological process of classical conditioning, it should be regarded as a normal response to severe nausea, and not as a primary anxiety disorder (although pre-existing anxiety could exacerbate ANV). Oncology patients often assume that ANV is indicative of something psychologically wrong with them (which causes further anxiety), and are therefore reluctant to report it to hospital staff [ 21 ], so reassurance of its normality is important. Conclusion This report illustrates the potential importance of ANV as a self-perpetuating triggering mechanism in CVS, and describes the positive feedback loop between nausea and anxiety. Conditioning might contribute to regular timing of episodes in some patients [ 2 ], account for seemingly implausible triggers, and explain attacks that superficially appear to be triggered by excitement or stress (e.g. Christmas, exams). Its prevalence in other CVS patients should therefore be investigated. Correct management of CVS should help to prevent ANV, and to reverse it where it has already arisen. Competing interests The author(s) declare that they have no competing interests. Authors' contributions FEM developed the hypothesis and wrote most of the manuscript. DRF critically revised the article and wrote guidelines for treatment of CVS patients. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079877

An exceptional Albanian family with seven children presenting with dysmorphic features and mental retardation: maternal phenylketonuria

Background Phenylketonuria is an inborn error of amino acid metabolism which can cause severe damage to the patient or, in the case of maternal phenylketonuria, to the foetus. The maternal phenylketonuria syndrome is caused by high blood phenylalanine concentrations during pregnancy and presents with serious foetal anomalies, especially congenital heart disease, microcephaly and mental retardation. Case presentation We report on an affected Albanian woman and her seven children. The mother is affected by phenylketonuria and is a compound heterozygote for two pathogenetic mutations, L48S and P281L. The diagnosis was only made in the context of her children, all of whom have at least one severe organic malformation. The first child, 17 years old, has a double-chambered right ventricle, vertebral malformations and epilepsy. She is also mentally retarded, microcephalic, exhibits facial dysmorphies and small stature. The second child, a girl 15 years of age, has severe mental retardation with microcephaly, small stature and various dysmorphic features. The next sibling, a boy, died of tetralogy of Fallot at the age of three months. He also had multiple vertebral and rib malformations. The subsequent girl, now eleven years old, has mental retardation, microcephaly and epilepsy along with facial dysmorphy, partial deafness and short stature. The eight-year-old child is slightly mentally retarded and microcephalic. A five-year-old boy was a premature, dystrophic baby and exhibits mental retardation, dysmorphic facial features, brachydactyly and clinodactyly of the fifth finger on both hands. Following a miscarriage, our index case, the youngest child at two years of age, is microcephalic and mentally retarded and shows minor facial anomalies. All children exhibit features of phenylalanine embryopathy caused by maternal phenylketonuria because the mother had not been diagnosed earlier and, therefore, never received any diet. Conclusion This is the largest family suffering from maternal phenylketonuria reported in the literature. Maternal phenylketonuria remains a challenge, especially in woman from countries without a neonatal screening program. Therefore, it is mandatory to be alert for the possibility of maternal phenylketonuria syndrome in case of a child with the clinical features described here to prevent foetal damage in subsequent siblings.

Background Phenylketonuria (PKU; OMIM *261600) is an autosomal recessive disorder of phe metabolism which can cause severe damage to the patient or, in the case of maternal PKU, to the offspring. The teratogenic effects of elevated maternal phe levels was first recognised in the mid nineteen sixties, at a time when routine newborn screening and diet treatment of PKU was being established in most developed countries [Mabry et al., 1966]. Typical features in offspring with phe embryopathy include microcephaly, mental retardation and heart malformation. The severity of maternal PKU syndrome is proportional to maternal blood phe concentrations, and a strict dietary control prior to conception and throughout pregnancy is mandatory to prevent congenital foetal anomalies [Rouse et al., 2000]. With our case report on an exceptional Albanian family, we wish to highlight the problem of untreated or undiagnosed PKU in adult women, resulting in the risk of severe maternal PKU syndrome in children. Case presentation Our index case, a 2-year-old girl, is the 7 th living child born to her mother (Figure 1 ). She was admitted to our hospital for further diagnostic work-up. Her birth weight was below 2500 g, as was the case for all her siblings. Additionally, she showed microcephaly, mental retardation and facial anomalies (long underdeveloped philtrum, high palate, anteverted nostrils). She also exhibited a large diastase of the abdominal rectus muscle. Figure 1 Portrait of the index patient. Case 1, the oldest child of the non-consanguine family, a 17-year-old girl, has a double-chambered right ventricle, multiple vertebral malformations of the thoracic and lumbal spine and epileptic seizures. She also shows severe mental retardation, microcephaly, facial dysmorphology (long underdeveloped philtrum, broad nasal bridge, micrognathism, high palate, divergent strabism) and stunted growth. Case 2, the 15 year-old sister, presented with severe mental retardation, microcephaly, facial dysmorphology (long underdeveloped philtrum, micrognathism, high palate, divergent strabism) and stunted growth. In addition, he has brachydactyly and clinodactyly of the fifth fingers of both hands. Case 3, a dystrophic boy, died at the age of 3 months of Fallot's tetralogy. He also had vertebral malformations such as hemivertebrae, rib fusions and thoracic scoliosis (Figure 2 ). Figure 2 Chest x-ray of case 3 who died of Fallot's tetralogy. The picture exhibits vertebral and rib malformations and fusions along with thoracic scoliosis. Case 4, an 11-year-old girl, presented with microcephaly and facial dysmorphology (long underdeveloped philtrum, broad nasal bridge, micrognathism, dysplastic ear helices, strabism), partial deafness, mental retardation, absence epilepsy and stunted growth. Case 5, an 8-year-old girl, was slightly mentally retarded and microcephalic, with minor facial anomalies and short stature. Case 6, a 5-year-old boy, was a preterm baby and small for gestational age. He also presented with microcephaly, facial dysmorphology and micrognathism. Brachydactyly and clinodactyly of the fifth fingers of both hands were noted. The mother had one miscarriage between her 6 th child and our index patient, case 7. The stillbirth occurred in the 7 th month of pregnancy. Further diagnostic work-up in case 1 to 7 was unremarkable. Because of the characteristic features of all her children, maternal serum phe concentration was analysed. The blood phe level was greatly elevated with 1560 μmol/L (normal < 90 μmol/L). Urinary organic acid analysis showed high excretion of phenylpyruvic, phenylacetic, hydroxyphenylacetic and phenyllactic acids as well as mandelic acid. Mutation analysis revealed that the mother was compound hetereozygous for the mutations L48S and P281L in the PAH gene. This disease was previously unknown to the mother. A newborn screening program had not yet been established in 1963 in Albania when the mother was born. The mother did not wish to maintain a phe-restrictive or protein-restrictive diet, and the children were unavailable to us for a long-term follow-up. Conclusion The case histories of the patients reported here provide further information on the maternal PKU syndrome, which causes heart malformation, microcephaly, mental retardation and stunted growth, respectively [Koch et al., 2003;Lee et al., 2003;Levy et al., 2001;Rouse et al., 1997], since it is the largest family suffering from maternal PKU reported in the literature. There are several reports of undiagnosed maternal PKU in the English language literature [Starostecka et al., 2001;Hanley et al., 1999], however, none with as many as 7 offspring. The disease was previously unknown to the mother who had visited a primary school and was not noted to have subnormal intelligence. The mother did not speak German but was socially integrated. Interestingly, the couple reported restricted food, especially of protein-rich meals, in Albania due to their low social status. However, seven of the children were born in Germany. All offspring had normal phe serum concentrations. The genotype identified in the mother of our patients, compound heterozygosity for the common mutations L48S and P281L, is associated with complete loss of enzyme activity of PAH and a classical PKU phenotype [Zschocke, 2003]. Since untreated PKU does not, in all patients, lead to severe neurological symptoms and profound mental handicap, as in the mother described here, there is a continuing need to be alert for the possibility of maternal PKU syndrome to prevent phe embryopathy in subsequent siblings. Although the condition is well known, it remains a problem, particularly in women born in countries without neonatal screening programs. However, the incidence of undiagnosed maternal PKU is currently estimated at 1 case per 100,000 births or even higher in Europe and the US [Hanley et al., 1999]. In conclusion, maternal blood phe concentrations should be investigated in the case of an otherwise unexplained neonatal syndrome presenting with microcephaly, facial dysmorphy and heart malformation, particularly in mothers born in countries without neonatal screening programs even if there is no obvious maternal mental retardation. Our case report clearly demonstrates that maternal PKU is still a challenge to be faced. Competing interests The author(s) declare that they have no competing interests. Authors' contributions Knerr, principal investigator; Zschocke, molecular studies, laboratory investigations; Schellmoser, clinical studies; Topf, Weigel, manuscript discussion; Dötsch, Rascher, scientific discussion. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079878

Smoking and tooth discolouration: findings from a national cross-sectional study

Background Smoking is a risk factor of a number of oral diseases; the extent to which tobacco products influence dental aesthetics has not been widely investigated. The aim of this study was to determine satisfaction with own tooth colour of smokers and non-smokers and to investigate whether smokers have higher levels of self-assessed tooth discolouration compared to non-smokers Methods A cross sectional national study was conducted on sample of 6,000 UK adults. A total of 3,384 adults was interviewed. Smoking behaviour was recorded together with satisfaction with own tooth colour. Prevalence of perceived discolouration was measured by asking respondents to match their own tooth colour to one of a set of seven photographs of differing severities of discolouration. Results Twenty eight percent of smokers reported having moderate and severe levels of tooth discolouration compared to 15% in non-smokers. As well as more often perceiving discolouration smokers were also more likely to be dissatisfied with their own tooth colour compared to non-smokers. Conclusion The study provides further evidence of the negative impact of tobacco smoking on dental aesthetics in the general public. The evidence provided by the study may be of value in short interventions for smoking cessation in the dental setting.

Background In the last few decades there has been a substantial growth in strategies and measures to reduce smoking, particularly among young people. These have resulted in a decline in smoking prevalence in UK between the 1950's and the1990's [ 1 ]. Nevertheless, in the most recent survey of smoking Britain, more than a quarter of the UK adult and 10% of children said they were regular smokers [ 1 , 2 ]. Smoking is a major risk factor for general health. In the oral cavity it can lead to oral mucosal lesions, oral cancer, periodontal disease and consequent tooth loss [ 3 - 6 ]. However, perhaps the most visible and immediate dental manifestation seen by the public is tooth discolouration. Smokers' teeth tend to develop tobacco stains; these may be yellow, brown, dark brown or even black stains, the severity depending partly on duration and frequency of the habit. Tooth discolouration may therefore have a deleterious effect on individual's appearance, which in turn may result in social disadvantage for smokers. In terms of efforts to encourage and support smoking cessation, it has been shown that when smokers are shown the adverse effect of smoking then they are more likely to quit than if other measures are used to motivate the same change [ 7 ]. In Finland, short intervention involving an exercise where adolescents identified stains on their teeth as a result of their smoking behaviour, helped to reduce the habit [ 8 ]. Labelling of cigarettes and tobacco carries mandatory health warnings. These are usually in the form of words, often against a white background and in marked contrast to the often seductive, design of the remainder of the packaging. The majority of warning labels on tobacco products are aimed at the more serious health problems of tobacco use; they are more often focused on lung cancer, impact of smoking on pregnancy, fertility or on general health. However, in the UK it was reported that smokers were not greatly influenced by the warring labels [ 9 ]. Very few used aesthetics or pictures in their health messages. In some tobacco products manufactured in Canada, actual pictures of tobacco staining are shown clearly on tobacco packs. The effectiveness of using images particularly those showing teeth, gums or lung diseases was tested in Canada. Results showed that three quarters of smokers recalled these images on tobacco products and reported that they believe that using these images is more effective than the commonly used worded warnings [ 10 ]. Aesthetics may be a significant supporting measure in the anti-smoking campaigns or interventions; this can be particularly important when targeting adolescents or females in general as appearance is more important to these two groups [ 11 ] who are often the desired target. Perhaps there is need of selecting the most effective warning message for the desired target. In the United Kingdom there has been no national recording of the prevalence of tobacco staining. The aims of this study were firstly to investigate the prevalence of self-assessed discolouration in smokers and non-smokers, secondly to compare satisfaction with own tooth colour in the same two groups. Methods The data collected here is part of a larger study investigating perception of dental fluorosis in the UK. Details have been published elsewhere (12). The office of national statistics omnibus survey was utilised to collect the data. A random stratified probability sample of UK adults was selected using the Postcode Address File (PAF). The study population included 6,000 addresses of adults aged 16 and above. The survey was carried out by 16 trained interviewers. The questionnaire used in the study included a question about smoking behaviour (whether the respondent was a regular smoker or not) as part of socio-demographic and behavioural information gathered from the respondents. A set of six photographs showing varying severities of discolouration (a pair for each level) and one showing normal tooth colour was shown to respondents, who were asked to match their own tooth colour to the closest photograph in the set. Satisfaction with own tooth colour was measured on a five point scale which was then grouped into three point scale for analytical purposes. Classification of discolouration used in the photographs is described in detail in a separate paper (12). The questionnaire was piloted and tested for reliability and validity on samples of the public and professionals. Logistic regression was undertaken to calculate odds ratios and to adjust for potential confounding factors and descriptive analysis was carried out to provide frequency distribution. Data was analysed using the SPSS statistical package (version 11). Results Three thousand nine hundred and fifty five of the 6,000 selected addresses were eligible and contactable. 3,384 adults agreed to take part in the study (representing a 69% response rate) and 3,215 provided information about tooth discolouration. Profile of the study sample is summarised in table 1 . Almost three quarters 74% (2,387) of these were non-smokers; the remaining 26% (828) were regular smokers. Prevalence of tooth discolouration is presented in table 2 . More than half of non-smokers (52%) reported themselves to have normal tooth colour whilst less than half (46%) of smokers reported the same. Moderate and severe discolourations were more prevalent in smokers. Table 1 Profile of the study sample Sex Male 1500 (44.3%) Female 1884 (55.7%) Age 16–34 957 (28.3%) 35–54 1214 (35.9%) 55+ 1213 (35.8%) Education High education 748 (22.1%) Middle education 1304 (38.5%) Lower education 1332 (39.4) Income Average or higher than national average 1046 (30.9%) Lower than average 2338 (69.1%) Smoking Smoker 817 (24.1%) Non smoker 2567 (75.9%) Table 2 Prevalence of tooth discolouration in smokers and non-smokers Tooth discolouration Non-smokers N (%) Smokers N (%) Normal 1236 (51.8%) 379 (45.8%) Mild 774 (32.4%) 224 (27.1%) Moderate 275 (11.5%) 149 (18.0%) Severe 102 (4.3%) 76 (9.2%) Results of the logistic regression of smoking and tooth discolouration are shown in table 3 . The difference in the prevalence of mild tooth discolouration in smokers and non-smokers was not statistically significant ( P > 0.05). However, in the case of moderate discolouration, smokers were more likely to report themselves to have discolouration of this severity [OR = 1.7 (1.4–2.2)] ( P < 0.01), likelihood of having severe discolouration was even greater amongst smokers [OR = 2.4 (1.7–3.3)] ( P < 0.01). Table 3 Logistic regression of smoking and tooth discolouration P Value OR 95% CI** Smokers Mild 0.54 0.94 0.78 1.13 Moderate 0.00* 1.76 1.40 2.22 Severe 0.00* 2.43 1.76 3.34 * Statistically significant at 0.05 level ** Confidence Interval Age, sex and income were adjusted for in the regression. Non-smokers is the reference Satisfaction with own tooth colour decreased with increasing severity of reported discolouration. Results are shown in table 4 . Thirty percent of smokers were dissatisfied with their tooth colour compared to 15% of non-smokers. This difference was confirmed in the logistic regression where smokers were more likely to be dissatisfied compared to non-smokers [OR = 2.4 (2.0–-3.0)] ( P < 0.01) (Table 5 ). Table 4 Satisfaction with own tooth colour in smokers and non-smokers Satisfied N (%) Dissatisfied N (%) Neither Satisfied nor dissatisfied N (%) Smokers 464 (56.1%) 250 (30.2%) 113 (13.7%) Non-smoker 1678 (70.7%) 366 (15.3%) 334 (14.0%) Table 5 Logistic regression of smoking and satisfaction with tooth colour P Value OR 95% CI** Smokers Satisfied 0.00* 0.43 0.35 0.52 Dissatisfied 0.00* 2.48 2.05 3.00 Neither 0.08 1.23 0.97 1.55 Discussion Prevalence of self-assessed tooth discolouration in smokers was almost twice of that reported by non-smokers. However, this was true only for moderate and severe levels. Not all tooth discolourations may be attributed to smoking; excessive intake of fluoride in early childhood is another risk factor for tooth discolouration as a result of dental fluorosis. However, in the case of fluorosis, the likelihood of developing moderate or severe discolouration in a low fluoridated area such as UK is low so that where mild discolouration may be attributed to fluoride intake, more severe forms of discolouration may be attributed to smoking. Thus the pattern of perception may be logical. Not only respondents who smoked were more likely to perceive discolouration they were also more likely to be dissatisfied with their appearance. Findings therefore suggest that smoking does have a negative impact on tooth colour and on perceived dental aesthetics. The potential for dental aesthetics to have more general effects such negative personal and social impacts has been shown by other researchers [ 13 ] Conclusion The study has shown that smokers have higher prevalence of tooth discolouration than non-smokers as anticipated. Variations in the prevalence tooth discolouration were clearer in the case of more severe levels. It has been reported that short interventions for smoking cessation are applicable by the dental team [ 8 , 14 ]. Information from this study highlighting the adverse cosmetic effect of smoking may provide a strong evidence base for strategies used for such interventions in primary care settings or in the dental practices. Use of pictures of teeth with tobacco staining as warning messages on tobacco products may be also worth consideration as it was very clear that smokers recognised the deleterious effect of smoking on their dental appearance. Further research is needed to explore the impact of different types of tobacco products and other related factors such as duration and frequency of use on tooth colour. Authors' contributions MNK and RB designed the study and produced the results, RH organised the analysis. MNK and RH have written the paper. All authors read and approved the final manuscript. Competing interests The author(s) declare that they have no competing interests. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079879

A qualitative study to identify community structures for management of severe malaria: a basis for introducing rectal artesunate in the under five years children in Nakonde District of Zambia

Background Malaria is a serious illness among children aged 5 years and below in Zambia, which carries with it many adverse effects including anemia and high parasites exposure that lead to infant and childhood mortality. Due to poor accessibility to modern health facilities, malaria is normally managed at home using indigenous and cosmopolitan medicines. In view of problems and implications associated with management of severe malaria at home, rectal artesunate is being proposed as a first aid drug to slow down multiplication of parasites in children before accessing appropriate treatment. Methods A qualitative study using standardised in-depth and Focuss Group Discussions (FGDs) guides to collect information from four (4) villages in Nakonde district, was conducted between February and March 2004. The guides were administered on 29 key informants living in the community and those whose children were admitted in the health facility. Participants in the 12 FGDs came from the 4 participating villages. Participants and key informants were fathers, younger and older mothers including grandmothers and other influential people at household level. Others were traditional healers, headmen, village secretaries, tradtional birth attendants, church leaders and black smiths. FGDs and interview transcriptions were coded to identify common themes that were related to recognition, classification and naming of malaria illness, care-seeking behaviour and community treatment practices for severe malaria. Results Parental prior knowledge of the disease was important as the majority of informants (23 out of 29) and participants (69 out of 97) mentioned four combined symptoms that were used to recognise severe malaria. The symptoms were excessive body hotness, convulsions, vomiting yellow things and bulging of the fontanelle. On the other hand, all informants mentioned two or more of symptoms associated with severe malaria. In all 12 FGDs, participants reported that treatment of severe malaria commenced with the family and moved into the community as the illness progressed. Although treatment of severe diarrheal effects, were common among the winamwanga, no rectal medicines to treat severe malaria were identified. Apart from the anti-malarial fansidar, which was mentioned by 23 in IDIs and 40 in FGDs, participants and informants also frequently mentioned indigenous medicines provided by healers and other respectable herbalists for repelling evil spirits, once a child had severe malaria. Mothers were the important arms for administration of ant-malarial drugs in the villages. Referrals began with healers to CHWs, where no CHWs existed healers directly referred sick children to the health facility. Conclusion Our findings showed that there is a precedent for rectal application of traditional medicine for childhood illness. Therefore rectal artesunate may be a well-received intervention in Nakonde District, provided effective sensitisation, to mothers and CHWs is given which will strengthen the health care delivery system at community level.

Background The problem of malaria infection in the under five years children has been widely described [ 1 - 5 ]. It is responsible for up to three million deaths annually throughout the world, with Africa having more than 90% of this burden. Almost 3% of disability-adjusted life years are due to malaria mortality globally, of these 10% are in Africa [ 6 ]. One of the most significant complications of the disease is Malaria-induced anemia causing more deaths than any of the other conditions of this disease [ 7 ]. In Zambia malaria continues to be the leading cause of deaths and illnesses, responsible for 47% of all deaths [ 8 ]. The incidence of the disease, and related mortality, in children under the age of five years is even higher [ 9 ]. Contributing factors to high child case fatality are the sudden progression from the simple form of the disease to severe malaria and malaria-induced anaemia, exacerbated by a lack of timely or inappropriate treatment. Simoes and colleagues in a review of the management of severely ill children in sub Saharan Africa found that over half of these children die before reaching the health facility for specialised care [ 10 ]. Where new drugs such as rectal artesunate have been developed and used for the emergency management of acute malaria in patients who cannot take drugs by mouth and for whom parenteral treatment is indicated but not available, positive results have been recorded [ 11 , 12 ]. The treatment should be followed by effective oral or parenteral therapy for malaria as soon as possible. In providing emergency therapeutic coverage, the drug is intended to give significant therapeutic advantage in situations where, at present, treatment does not exist. There is a potential for this approach to save lives of patients [ 13 ]. A lack of knowledge at family level on transmission of malaria hampers prevention of the disease. In the same study Simoes and others have shown that malaria case fatality rates were related to environmental and socio-economic status rather than poor knowledge of the disease 'malaria' [ 10 ]. In rural Zambia prompt access to treatment is hampered by the distances between families and the health facilities [ 9 , 14 ]. As a consequence most malaria-related morbidity is treated at home and within the community by informal health providers and networks [ 15 , 16 ]. In a clinical trial the rectal administration of artesunate has resulted in adequate drug absorbtion in all subjects and 92% children had achieved a parasite density lower than 60% of baseline after 12 hours compared with 14% of those administerd parenteral Quinine. Results were shown to be highly significant [ 12 ]. Its use in the emergency management of acute malaria in patients who cannot take drugs by mouth but require urgent treatment has therefore been established. However, for a significant reduction in case fatality in children to be achieved, prompt administration is essential. In Zambian rural households this implies that the responsibility for administration of rectal artesunate must lie with the caretakers of young children or those providing health care services at village level. The treatment should then be followed by effective oral therapy for malaria at a recognized health facility as soon as possible. In preparation for the introduction of rectally administered artesunate at population level it is necessary to gain an understanding in three areas; firstly whether caretakers can identify malaria in their children under 5 years and distinguish between severe manifestations of the disease; secondly whether they would administer a drug, per rectum, if this was made available to them and thirdly and of great importance, whether the caretakers will then proceed to take the sick child to the health facility for follow up oral treatment. This paper describes the finding of a study designed to address these gaps in knowledge to inform the design of a study introducing rectal administered artesunate into the community. More specifically the paper covers the following areas: how communities defined symptoms of severe malaria, treatment seeking behaviour; decision making processes at a household level and the current use of rectally administered preparations to treat malaria. Health services in Nakonde Nakonde District is situated in Northern Province of Zambia, it was chosen to conduct this study here because it is usually hard to reach communities. Average distances to health facilities range between ten and sixty kilometers. The District Health Management Board [DHMB], at the helm, controls all government activities in health care provision including one District hospital for referred patients. Of twelve administrative Wards only seven boast a rural health centre servicing all 154 villages in the district. Communication between families and the health centres is through the Neighbourhood Health Committees, established per 'zone' covering a catchment of 500 populations or 5 to 10 km radius. Community Health Worker (CHWs), trained Traditional Birth Attendants (TBAs) and Village Health Motivators (VHMs), who are trained by the District Health Management Board, also report directly to the Rural Health Centres. Community Health Workers cover between three and five villages in their catchment areas. The presence of local healers and untrained Traditional Birth Attendants (TBAs) providing indigenous health care has a direct effect on the health seeking behaviour of the families in the villages. The villages in Nakonde are spread over a vast area and are sparsely populated, making it difficult for the government health structures to be easily accessible and effective. Methods The study was conducted in Nakonde District between February and March 2004. Data collected was qualitative in nature, primarily using key informant interviews. Focus group discussions provided a means of triangulation. Sample population Primarily interviews were conducted with caretakers and influential relatives at household level. Caretakers comprised of fathers of the under five years children, younger and older mothers and grandmothers. In view of the important role of traditional healers and other community members, village headmen, village secretaries, Traditional Birth Attendants, church leaders and blacksmiths were interviewed. The Focus Group Discussions (FGDs) were conducted to provide more responses and accommodated the emergence of hidden issues from the respondents. Finally, interviews with health care providers working at the closest Rural Health Centre (at Mwenzo) and the next referral level, the District Hospital (at Nakonde) were conducted. Focus Group discussions and key informant interviews To provide a means of triangulating the information, key informant interviews and focus group discussions were conducted. Key informants consisted of 12 community informants, 11 mothers of children admitted at either Mwenzo Rural Health Centre or Nakonde District Hospital for severe malaria and 6 health care providers working either at Mwenzo Rural Health Centre or Nakonde Rural Health Centre. A total of 12 Focus Group Discussions (FGD), distributed equally in the four villages, were conducted. They lasted for between forty minutes and one hour. Separate groups of younger mothers, fathers and older mothers were organised and lastly a discussion with opinion leaders (all genders) was conducted (Table 1 ). Table 1 distribution of participants per village Young Mothers Fathers of <5 years Older Mothers Opinion Leaders Total Village 1 1 1 0 1 3 Village 2 0 1 1 1 3 Village 3 1 1 1 0 3 Village 4 1 0 1 1 3 Total 3 3 3 3 12 The number of people participating in each FGD varied between six and ten. The major criteria for participating in the FGD was availability of an under five child in the care of the mother or caretaker. Only younger mothers were categorized according to their age groups (15–24 years). However, no age restrictions were imposed on older mothers, fathers of under five or opinion leaders who participated in the FGDs. Procedures The scene was set by the research team who visited the study site prior to the interviews, explaining the purpose of the study. All those who participated in the study were identified by the research team. The village headmen mobilised participants for FGDs through the criteria provided by the investigators. The interviews were conducted by local residents following a training session by the research team at Mwengu Social and Health Research Center. Despite the permission that was granted by the community leadership, individual participants were requested to consent participating in the study. This arrangement ensured that the quality of data was not compromised, as enough time was given to allow for respondents to gain confidence in the research assistants. The interviews with caretakers lasted about one hour and about two hours with traditional healers and other opinion leaders. At every stage, interviewees were encouraged to talk freely. Transcription was completed following each interview before moving on to the next one. The authors closely supervised both data collection and transcriptions of materials. All the interviews were tape recorded, transcribed and translated from Chinamwanga to English. The authors listened to recordings and checked the accuracy of the transcripts. During the course of the research, investigators took turns to witness an in-depth being conducted by the research assistants and, in some cases, assisted in manual recording of responses. At every interview, the presence of the investigator was critical. During focus group discussions the investigators actively participated as facilitators. Research tools A field guide was developed for use by interviewers and piloted in a community with the same socio-economic characteristics as the study site. The guide provided an opportunity to explore various community treatment behaviours of severe malaria, questions were related to serious illnesses in the community; information about health care; local terminologies of malaria; symptoms suggestive of simple and severe malaria; causes and treatment of different symptoms for simple and severe malaria; access to health facilities; dispensing practices at community and health facility levels; health information and communication channels regarding malaria and finally knowledge and practices regarding administration of rectal medicines. Research assistants Criteria for selection of research assistants were completion of 12 years of schooling, previous experience as a research assistant, ability to speak the local language, Chinamwanga, fluently, resident in Nakonde, mature and respected by the people in the community. The four research assistants selected were all males aged between 27–35 years. The research assistants had not had any medical training; this assisted in minimizing the likely interviewer bias. Training of research assistants The research team conducted an eight-hour training session for ten days familiarizing research assistants with research tools and practicing interviews. They were also taught basic techniques of recording responses and probing, identifying informants and note-taking for easy transcription. The fundamentals of organizing and conducting focus groups discussions were also included in the training. Investigators demonstrated in-depth interviewing. Analysis The authors read and re-read the transcripts and developed codes to identify important themes. All transcriptions were typed in MS Word and converted into an Atlas.ti readable text file. Each interview and focus group discussion was entered as a single file. There were 41 single files for analysis. The files were then coded and independently prepared matrices for interpretation of substantive themes were developed. The purpose of the texture analyses was to interpret the data and findings by interrelating codes and data, in order to create common concepts. To achieve this, data was segmented into variables, coded and then frequencies of codes were electronically assigned. Focus group discussion and in-depth interview transcripts were coded to identify common themes related to recognition, classification and naming of malaria illness, care seeking behaviour and traditional treatment practices for severe malaria. Codes were used to find specific occurrences of common responses in the data, which could not be searched by simply applying text based search techniques. They were also used to classify different levels of summaries that fell under different variables, for purposes of comparison between FGDs and in-depth interviews, as well as between categories of informants and participants. The process was followed to ensure a systematic basis for frequencies of the views expressed by participants during discussions and interviews. Codes were standardized in all files according to variable under analysis, in order to ensure inter-code reliability. Assessment and validity of information was made by re-reading the transcripts. Results General description of malaria The results are presented in themes previously selected to meet the objectives of the study; identification of malaria (all types), causes of malaria, use of rectally administerd medicines, health seeking behaviour and decision-making processes. Information from twenty-nine (29) interviews, the point at which no new information was forthcoming, and 12 focus group discussions are presented. Identification of malaria Malaria was recognized as being a common problem in the area. At least 24 out of 29 key informants and 78 among the 97 FGD participants continuously mentioned this. An in-depth interview with a grandmother caring for under five child reported this: There is this disease number one, malaria. The children have finished [meaning many children have died], when diarrhea starts, at the same time with vomiting, you haven't arrived in time and the child.. we have buried many children, and the child is dead. Eee, this is the disease, which has defeated us very, very much . In one of the focus group discussions with opinion leaders, an old man aged over 60 years expressing himself on the prevalence of malaria in the district said: The biggest problem, which we face in this area, especially for young children, is malaria [said it in English], inzekema is common here . The Winamwanga at times considered fever as an illness on its own in the under five children. They used the term ' Inzekema ' to describe the syndrome of fever, chills, body pains and headache. The term fever was used synonymously with the English word malaria. The categories of variables varied as shown in table 2 by the variable criteria for 'fever' [malaria, simple and severe]. Table 2 Types of malaria and terms used in the local language Indigenous word English word 1. Inzekema Malaria 2. Impepo Simple malaria 3. Ichinzekema Severe malaria Two types of malaria were recognized, simple and severe, the latter being associated with convulsions. The Winamwanga use 'Umuwili ukulungua' as a general term to describe the symptom of fever. 'Impepo' which is translated as cold, includes body hotness, shivering, headache and general body pains. This is considered as less serious, as the 46-year-old traditional healer put it: There are two types of malaria. There is one where a child is shivering and the body is hot, that is one type of malaria, we call it impepo . The two symptoms associated with an episode of simple malaria, that were commonly mentioned by both informants and participants included shivering and body hotness. The local term ' sinsa ' [convulsion], was one of the key symptoms used by FGD participants to describe the progression of an illness, 'it is big malaria' they could say. Making a statement on severe malaria, the 46 year old traditional healer continued and said: The other type of malaria comes with convulsions [sinsa], this is the other type. There are 2 different types of convulsions [sinsa] . Describing severe malaria a 40 year old key informant said: Vomiting [ukuluka] yellow things, when he starts vomiting yellow things, shivering too much [ukuzakaza], coldness/hotness [impepo], this is how we know that this is big malaria . In focus group discussions 78 out of 97 participants recognized that vomiting yellow things, convulsions, bulging of the fontanelle and excessive body hotness were some of the symptoms associated with severe malaria. These participants included those who had at one time taken their children to either Nakonde district hospital or Mwenzo rural health Centre, Convulsions were associated with severe malaria. At this point the reference to fever changes and they call ' E malelya mukulu' , meaning (big malaria). A 54-year-old opinion leader in one of the FGDs said: we see when a child starts to say things we cannot understand; in the end you see the child prostate/stretching [wanyuluka, Informant stretches his hands upwards showing how the child stretches]. Among the Winamwanga, vomiting yellow things was a clear sign that the malaria was coming out and that the child was at least breathing [meaning was relieved]. During the FGDs, it was common for participants to interpret vomiting yellow things as a sign of relief and beginning of healing of the illness. One participant in the FGD with younger mothers reported: That is when he is breathing; it means the illness is coming out A mother aged 39 years old, whose under five child was admitted at Nakonde hospital, reported that though most children with severe malaria vomited, not all vomiting was malaria. To show how vomiting due to severe malaria could be identified, she went on to say: They look yellow and they are slippery, if it is severe malaria [Inzekema]. But if it is another disease, because illnesses come different, he/she may just be vomiting [ukuluka] yellow things only and we say maybe it is severe malaria [Inzekema] . [Meaning, they are unsure of the diagnosis] Caretakers were capable of identifying different causes of some symptoms, which included diarrhea [ Ukupolomya ]. A mother whose child was admitted at the hospital stressed on ' diarrhea' that was caused by witchcraft and said,' Cannot be cured until you find the African medicines, those one which when you cut tattoos, if there is no witch who is increasing it, but when he is there washing it [meaning diluting the medicine], it is not cured easily. Child will keep on crying, keep on crying. That is how it is . It was common to have mothers associating symptoms such as diarrhea with witchcraft, not with the malaria episodes. When analyses were made on symptoms of convulsion and bulging fontanelle, caretakers knew differences between convulsions that occurred because of epilepsy and convulsions due to severe malaria attacks. They were able to associate a bulging fontanelle due malaria and that resulting from other illnesses. A father of the under five, who was 52 years old, said, ' there are two. There is that one suffered by young children... an illness, which they say [akatuwi] sinking of fontanel, it causes the fontanel not closing quickly, this one is [ichapamutwe]. Now those... those illnesses are which differentiate them from malaria. And that one...that ... [akatuwi], when you use a malaria drug, you find a child still sick. It closes without working. We... oh, this person needs African medicines which the people have been using long ago for cure. That is when we go to those who know the medicines . Reported malaria infection ranged from 50 to 80% in all villages. The prevalent conditions were convulsions, vomiting, high fever and loss of appetite. Rectal medicines Rectally administered medications are already being used in this community. During an FGD with older women, one participant described how the medicine for Ilonda was administered, she reported: Yes, turning the child upside down Sikaona [referring to the interviewer], sprinkling him/her just there where the dirt comes out [anus], that is where I sprinkle... there since it is Ilonda . It was common for most informants and participants, to explain in detail how medicines given through the rectum were administered. In an interview with a male key informant, regarding administration of medicine for Ilonda, he said: We just apply it on the sores on the anus, bath in medicine in the water and put the powder inside the anus, and then some you drink it. [ Meaning medicine is applied on the rectum]. But...but some they buy those things from Tanzania, which they put medicine and inject people (those things, referring to syringes). When asked whether they would be willing to let their children have the drug inserted into the rectum, almost all the participants said it was alright as long as it made their children reach a health facility where they would continue seeking further treatment. As one member of the FGD said, ' me... I would not care even if the medicine was inserted in the ears or nose, for as long as the child survived that is alright Decision making The results from this study showed that the decision to use a facility when a child had severe malaria lay with the mother. Most informants and participants stressed on this point. During a Focus Group Discussion with opinion leaders, when asked who takes the decision for the child to go to the health facility for treatment, all participants shouted : We mama [the mother]. In another FGD with older mothers, a participant aged 25 years, with seven years of schooling and having three children reported; Just myself the brains come to decide that here it is not looking well. Me... when I tell the father [Father here meaning husband], you father this child is sick, aah.... It is just malaria you are just troubling let us just buy 'to' panadol that's how sometimes he will go and buy panadol, but we who are taking care of a child [meaning one sleeping closer to the child]. I am the one who is knowing how the child is becoming, so when I see that the child has not slept, I tell him my dear this child has not slept its better we take him/her where...? To the hospital . During discussions with opinion leaders, the responsibility for the mother to take the first decision was strongly stressed, as was evident in the following discussion: Inf.6: It's the mother of the child . Inf.3, 5 and 7 almost at the same time: Its the mother Inf.4: The very mother of a child, is she not the very one who can tell the father? That lets take the child to the hospital . Inf.5: She has the right Inf.4: Because she is the first one to know the disease, so it means she has the right to decide . According to this discussion, a father was only consulted in order to provide financial and material support, which enabled a mother to use services from specialised health facilities or service. Health seeking behaviour The majority of the participants believed that once a child is sick, unless treated, it was inevitable that he/she would die. Households started using structures and medicines that they were familiar with, before they could start looking for help outside what they knew. The chart below demonstrates caretakers' movements. Indigenous Healer CHWs Admitting Health Centre During an in-depth interview with a mother admitted at the hospital, she reported: It is just like when you meet the disease... some days you can waste even three days. Now if you have not met the illness, some days they fail like that they come and change and say go and buy this such you change again it is just like when you meet the illness like when it meets [referring to drugs bought and administered at home] with the illness it can even take two days [says it in English], and the child is better . While attempting to treat symptoms associated with severe malaria, local individuals, such as vendors and other people who knew some indigenous medicines were consulted. During an FGD, one informant aged 34 years stressed on this point when she reported: and those which you find here [respondent touching on her top of the head showing where it sinks] it is getting in side, even those they treat in the village, some people even here [referring to the group], they treat it . The informants emphasizing treatment-seeking patterns reported thus: We get ourmedicine from wa sing'anga, here (Pointing at a healer). If things do not work, then we go to the hospital where doctors try . (FGDs). One informant reported: we just use traditional medicines here. If we all fail, then we send them to the hospital. [ Q: Do you have someone in this village that gives drugs from the hospital?] A: Yes, we have one, Mr. Sikaona, if he has medicine, that is why I am saying, when we fail we send to Mr. Sikaonga [referring to a CHW], then he knows what to do next . Normally, a healer would take between two and four days before a referral was made to a community health worker. It was common for both informants and participants to report this number of days. One key informant in the village said: It takes 2 days...3 days to 4 days and when you give a child treatment, you now see a child starts playing, now you know he/she is okay . In an interview with mothers whose children were admitted in hospital, it was common for them to mention this pattern. One informant aged 37 years old reported: We take him/her to the village doctor [referring to the CHW]... the reason why it takes days because we first go to the village doctor, that is why it takes days because he gives us a dose and then we go back to him, or go to the hospital, when there is no change... If it is Fansidar, we wait for four days, if she/he has not changed, we go back to the village doctor and the village doctor says, to go to the hospital. Then we lift the child to the hospital, that's all . Discussion The study showed that severe malaria was well described by the Nakonde residents and was consistent across all the age groups of participants and key informants. They were able to correctly define and differentiate simple from severe malaria (simple malaria known as 'inzekema intichi' and severe malaria 'ichinzekema).' Both participants and informants expressed high level of knowledge of symptoms of severe malaria 'ichinzekema', which was directly reflected in their definition of 'big' malaria and were able to report symptoms in pairs or in groups, which might appear one at a time, accompanied with high temperature. The studies among the Tumbuka of Lundazi in the Eastern part of Zambia [ 17 , 18 ] presented similar findings and were consistent with studies conducted in other African Countries [ 19 - 21 ]. There was however, in this study, a confusion with the bulging of the fontanelle, which people regarded as a condition for severe malaria, while the medical field associates bulging of fontanelle with meningitis. The study showed that caretakers recognise fever as being associated with malaria, they can determine severe and simple malaria and can differentiate between convulsions due to malaria and those due to epilepsy. In respect of rectal administration of drugs, it was found that local practices included rectal administration of local medications already exist. To determine whether caretakers then proceed to take the sick child to the health facility for follow up oral treatment, several factors were apparent. Firstly it is the mothers who would make this decision, secondly that health seeking behaviour follows a pattern of using the local traditional healers/CHWs and then, if no success is found then taking the child to the health facility. Mothers and other caretakers in Nakonde could also identify severe malaria with many other complications and distinguish between convulsions due to other conditions. For example, only convulsions that were accompanied by high fever were regarded as 'big malaria' or severe malaria. Tchinda and others [ 22 ] in their study showed that anaemia, hypoglycemia and jaundice were recognized by caretakers as complications of malaria, this was not identified in the Nakonde study [ 23 - 25 ]. These parameters are critical foundations for an intervention strategy into home-based management of malaria. While the majority of participants and informants perceived malaria to be an inevitable part of life in Nakonde others believed the causes for symptoms included evil spirits. This has implications for the introduction of home-based management of malaria since the casting out of evil spirits will delay seeking of biomedical treatment. Rectal medicines In this study, investigations were made on whether there were any local medicines that were administered rectally to treat acute illness. During the in-depth interviews, it was shown that, although rectally administered medicines were not used for treatment of severe malaria in the Nakonde community; it was common for rectal medicines to be used for treating sores/wounds resulting from episode/s of severe diarrhea. The rectal medicines were (Inkula), local herbs and some backs of trees, like (Umulombwa and Umulala ntemelale)]. Inkula was widely used in the Northern and most of the Eastern part of Zambia. Inkula is sometimes called cassia in English. Treating 'Ilonda', sometimes a child was made to sit in cold or warm water in which the medicine had been soaking. It was believed medicines soaked in water would go in the wound and treat it. The other interesting finding from this study was that healers were able to administer rectal medicines using syringes that were bought from chemist shops in Tanzania. Medicines that were in powdered form could be injected in the child's rectum. Healers, who could not afford syringes, used local materials such as straws to inject the rectal medicines into the children's rectum. With this background, introducing rectal artesunate in the Nakonde community might not face any resistance, as people were already administering some of their medicines in the same way as the modern rectal artesunate. We believe that the most important element in the intervention would be to provide accurate and correct information to caretakers. Other studies [ 26 , 27 ], had recommended that there was need for effective system of providing information, in order to influence treatment seeking behaviour for severe malaria. CHWs have been identified as the first port of call, along with the indigenous healers, once home treatment had been given. However the lack of essential drugs available to these workers contributes to delay in providing appropriate treatment. This study suggests that the provision of artesunate rectal suppositories to these health workers for the initial management of malaria patients who were not able to take medication by mouth would also reduce case fatality. Women decision-making In this study, results revealed that a woman had a right to decide when to take a sick child to the health facility. According to these findings, a father was only consulted in order to provide financial and material support, which enabled a mother to use services from specialised health facilities. This finding is consistent with a study by Kaona et al [ 4 ], which indicated that health care in homes in rural Zambia and rural Africa as a whole, was predominantly the responsibility of women. Young women in particular, felt very strongly that a severely sick child should never be kept at home or would certainly die. They appreciated the fact that early treatment would help the family avoid expending the family's' meager resources. Moreover, participants and informants categorically stated that children affected by severe malaria should be quickly referred to the health centres. Residents were deeply concerned that even the health facilities run without the qualified staff. If emergency treatment has been given prior to arrival at the health facility then unqualified staff can provide the oral treatment required to complete the cure. Community treatment seeking behaviour The people of Nakonde commonly manage malaria episodes at household level initially. This is consistent with similar studies in other parts of Africa [ 16 , 26 , 25 ]. Breman for example showed that over 80% of malaria cases in the under five children, were managed at home [ 28 ]. Home treatment of malaria, has both advantages and disadvantages, one major disadvantage is that it may delay appropriate treatment during the acute stage of the disease. It was established in this study, that the family was able to use various methods including sponging which was most common. Applications of these medicines varied though most of the respondents mentioned preparations through mixing with the child's porridge and drinks. Situations like this, made mothers and other caretakers to rely heavily on the indigenous medicines from healers, which were not anti-malarial drugs. There is no doubt, therefore, that caretakers can be trusted to identify when they would need to give rectal artesunate. However, there will be need for some extra sensitisation for mothers. The study by Savingry and colleagues [ 29 ], found that the traditional healers attended over 28% of all deaths that had occurred. This is true for most of the societies in Eastern and Southern part of Africa. The use of indigenous plants in the treatment of malaria in Africa was reported by Waako, Fob and Smith [ 30 ]. They demonstrated that communities in Uganda could use plants such as Aspilia africana and Momordica foetida, for malaria treatment. The Winamwanga people revealed an extensive knowledge of both modern and traditional medicines for treating malaria. The Nakonde people used various types of medicines, for instance, there were medicines that could be used to repel evil spirits as perceived cause of convulsions, however, the main local remedies for treating malaria were, 'namayoka', referred to as cinchona in English, which was taken orally by the children. They use medicines which included ' nankololwe, utunvumbe, imbozyo, isyoyo, inkowamatete and umupene wambuzi , as repellants for evil spirits. Sensitization messages must therefore address the need for the rectal artesunate to be administerd before evil spirits are expelled. Limitations 1. Lack of knowledge of the local language by the PI, could have made the research assistants fail to stress important issues during interviews. However, the Co-PI who comes from the area, managed to complement this. 2. The study recruited younger men as research assistants, to interview elderly men and women. This could have affected the responses from some of the participants. 3. The sampled individuals were purposively selected; these have led the study to generalize the findings. 4. The nature of the qualitative study, does not allow a researcher to measure the significance of the occurrence of an event. Conclusion Our findings showed that there is a precedent for rectal application of traditional medicine for childhood illness. Therefore rectal artesunate may be a well-received intervention in Nakonde District, provided effective sensitisation, to mothers and CHWs is given which will strengthen the health care delivery system at community level. Competing interests The author(s) delclare that they have no competing interests. Authors' contributions FADK the Principal author of this paper, assisted in the design of the project to be adapted to the country's situation. He was instrumental in data collection, supervision during data collection, analysis and the production of this manuscript. MT was PI to the project. She contributed to the design of the project, supervised all field activities including data collection, transcription and editing of typed manuscripts. She was instrumental in data analysis. She has worked closely with the Principal author in the production of the manuscript. Authors have read and approved the final manuscript. Table 3 Local terminologies for describing symptoms of severe malaria and their interpretations Local terminology English word 1. Sinsa Convulsions 2. Ukuzanzauchila/usawawiya Confusion/mental disorder 3. Wakapanga ama picture ngati akulola a film Hallucination 4. Akakalila Prostration 5. Insingo ikakalila Stiff neck 6. Ukusanta Fitting 7. Ukukana ukulya Loss of appetite 8. Ukuchuluchila Twitching 9. Ukutwinka Sweating 10. Ukupolomia Diarrhea 11. Icapamutwe Fontanelle 12. Ukuluka inyonga Vomiting yellow stuff 13. Ukuzakaza Shivering 14. Ukulungula nkaninye Very hot body 15. Ukunyonga Stomach pains Pre-publication history The pre-publication history for this paper can be accessed here:

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1079880

Application of population pharmacokinetics to cladribine

Background The nucleoside analog cladribine is used for the treatment of a variety of indolent B- and T-cell lymphoid malignancies. The primary aim of the study was to evaluate the population distribution of pharmacokinetic parameters in patients undergoing treatment with cladribine and to detect the influence of different covariates on the pharmacokinetic parameters. Methods This pharmacokinetic study presents the results of a retrospective population pharmacokinetic analysis based on pooled data from 161 patients, who were given cladribine in different administration routes in various dosing regimens. The plasma concentrations of cladribine were determined by reversed-phase high-performance liquid chromatography using a solid phase extraction with a limit of quantitation of 1 nM using 1 mL of plasma. Results A three compartment structural model best described the disposition of cladribine. Clearance was found to be 39.3 L/hour, with a large interindividual variability. The half-life for the terminal phase was 16 hours. Bioavailability was 100% and 35% for subcutaneous and oral administration, respectively, with low interindividual variability. None of the investigated covariates were found to be correlated with the pharmacokinetic parameters. Conclusion As interindividual variability in apparent clearance after oral administration was not significantly higher compared to that following infusion, cladribine could be administered orally instead of intravenously if compensated for its lower bioavailability. Individualized dosing on basis of body surface area or weight does not represent an improvement in this study as compared to administering a fixed dose to all patients.

Background Cladribine [Leustatin ® ] is a purine analogue that entered clinical testing fifteen years ago, with major activity in the treatment of B- and T-cell lymphoid malignancies. Cladribine has an outstanding therapeutic activity against hairy cell leukemia, a disease in which the drug induces long-lasting complete remissions in the vast majority of patients treated. The activity of cladribine has also been demonstrated in chronic lymphocytic leukaemia (CLL) non-Hodgkin's lymphoma, cutaneous T-cell lymphoma and myeloid leukemia. Cladribine and the other newer purine analogues are unique, when compared to traditional antimetabolites, in that they are equally cytotoxic to both dividing and resting cells [ 1 ]. Cladribine is usually administered at 0.09 mg/kg daily as a continuous intravenous infusion over 7 days. However, pharmacokinetic studies supporting the use of intermittent intravenous (iv) infusions have shown a long terminal half-life of cladribine after a 2-hour infusion with the same anti tumour activity seen with continuous iv infusion [ 2 ]. The pharmacokinetic profile of oral administration of cladribine resembles that of a 2-hour iv infusion, with a bioavailability of 37–51% [ 3 , 4 ]. Subcutaneous administration gives a high peak concentration of short duration with an area under the curve (AUC) identical to that of the iv infusion and a bioavailability of 100%. The primary aim of the study was to evaluate the population distribution of pharmacokinetic parameters in patients undergoing treatment with cladribine and to detect the influence of different covariates on the pharmacokinetic parameters. This analysis was performed in order to evaluate the plasma concentration-time profiles in relation to previously presented pharmacokinetic data and to elucidate the possibilities to create a better tailoring of cladribine dosing. Results The population pharmacokinetic analysis of cladribine was based on 1102 plasma concentrations obtained from 161 individuals. The observed plasma concentrations of cladribine versus time are presented in Figure 1 and 2 . The initial runs carried out were aimed at finding a base model (pharmacokinetic and statistical submodels). A three compartment structural model best described the time course of plasma concentrations of cladribine for all patients and was therefore chosen for the present analysis. Figure 1 Observed plasma concentration versus time profile for cladribine after a 2- hour iv infusion, oral and subcutaneous administration once daily. Concentrations are given on a logarithmic scale. Figure 2 Observed plasma concentrations after repeated intravenous administration (steady -state observations). Concentrations are given on a logarithmic scale. The final disposition model is described as follows: a three compartment model with interindividual variability on clearance ( CL ), central and peripheral volumes of distribution ( V1 , V2 , V3 ), intercompartmental clearances ( Q2, Q3 ), and with a proportional residual error model for residual variability. The final population parameter estimates based on the model are given in Table 3 . The clearance in the typical patient was calculated to be 39.3 L/h, with relative standard error (RSE) of 4.9 % while the interindividual variability, as expressed by the coefficient of variation, was 54%. Diagnostic plots of the observed and population model predicted concentrations after iv infusion, oral and subcutaneous administrations are shown in Figure 3A–C . Table 3 Population pharmacokinetic parameter estimates for the typical individual after administration of cladribine as; an infusion, orally or subcutaneously. The relative standards errors are given in parentheses. The estimates of intersubject variability are given as coefficients of variation (%). Parameter Population average Interindividual variability Estimate (RSE%) 1 Estimate % (RSE%) Clearance (L/h) 39.3 (4.9) 54 (17) V1 (L) 71.7 (13) 34 (62) Q2 (L/h) 51.1 (6.8) 61 (17) V2 (L) 475 (1.8) 70 (31) Q3 (L/h) 105 (21) 61 (17) V3 (L) 73.6 (13) 61 (17) Oral Ka (h -1 ) 1.31 (14) 75 (50) Oral F 0.353 (7.9) 4.1 (63) Subcutaneous Ka (h -1 ) 2.48 (9,6) N.E. Parameter Infusion (RSE %) Oral (RSE %) Subcutaneous (RSE %) IIV 3 (RSE %) Residual error 2 Proportional 0.191 (8.2) 0.232(9.8) 0.162 (8.8) 24 (28) 1 Rellative standard error. given as %. 2 Residual error presented as population average of estimate and relative standard error in parentheses. 3 Interindividual variability in residual variability expressed as coefficient of variation Abbreviations: CL, clearance from central compartment; V1 , V2 and V3 , central and peripheral volumes of distribution, Q2 and Q3, intercompartmental clearances; Ka, absorption rate constant (hour -1 ); F, bioavailability of drug; N.E., not estimated Figure 3 Model predicted versus observed cladribine concentrations after intravenous infusion (A), oral (B) and subcutaneous (C) administration. In the left panel predictions are based on the parameters of the typical individual whereas in the right panel predictions are based on individual parameter estimates. ID numbers has been used as plotting symbols and all observations for an individual are connected by a broken line. Concentrations are given on a logarithmic scale to facilitate model inspection. The oral data were best described with a first order absorption without lag time. The bioavailability was found to be 0.353 with RSE 7.9% and the interindividual variability was estimated to be 4 % (expressed as coefficients of variation). The estimated value for the absorption rate constant was 1.31 hour -1 with RSE 14%. As bioavailability and interindividual variability in F were of particular interest, some additional investigation was performed. First, the 95% confidence interval for interindividual variability in F was obtained using likelihood profiling. This resulted in a 95 % confidence interval ranging from 0% to 10%. Second, in order to get a broader insight into the first pass metabolism, bioavailability and variability in CL following different administration routes, F was omitted from the model and instead apparent CL and its interindividual variability was estimated separately for each route of administration (other disposition parameters were still estimated jointly). The estimated CL for the intravenous route was 39.7 L/h (RSE 8%) while the estimates of the oral CL (or rather CL/F) was 105 L/h (RSE 13%). The estimated interindividual variability in CL for the intravenous and oral routes were 59% (RSE 22%) and 55 (50%) respectively. Third, a model allowing correlation between F and CL was tested, but it did not result in any improvements and variability in F was still not appreciable. For the subcutaneous data the bioavailability was calculated to be close to 1 and therefore was fixed to this value, as estimation of this parameter did not result with a significant model improvement. The corresponding result for ka was 2.48 hour -1 with RSE 9.6%. The terminal half-life was relatively long, 16 hours. The half-life for the first distribution phase was calculated to be 0.2 hour and the corresponding half-life for the second distribution phase was 1.3 hours. The highest and the lowest values for terminal half-life were 58 hours and 5 hours respectively. The corresponding accumulation index, the amount at steady state compared to the corresponding value after the first dose at the same time, was for the highest 3.05 and for the lowest 1.02. None of the covariate relations, included in the generalised additive model, were found to be significant when included in the NONMEM model. Discussion In this study we have evaluated pooled data from 161 patients at different centres, receiving different doses of cladribine, administered by three different routes, using different treatment schedules and leaving different numbers of blood samples at different times. To perform this evaluation we used nonlinear mixed effect modelling for the pharmacokinetic analysis of the data. A three compartment structural model best described the time course of plasma concentrations of cladribine, which is also in agreement with previously published individual pharmacokinetic modelling [ 4 , 5 ]. Cladribine, administered as an oral solution, was rapidly absorbed and the absorption was best described using a model with first order absorption without lagtime. We calculated that the bioavailability after oral administration of cladribine was 35.3% and this value is slightly lower compared to the previous reports of 37–51% [ 3 , 4 , 6 , 7 ]. The estimate of interindividual variability (IIV) in oral bioavailability was surprisingly low, 4%. Only seven patients had data following both intravenous and oral administration and even if in theory, bioavailability and variability in bioavailability can be estimated from a parallel group study, the sparsity of crossover data might be one explanation for the negligible value of variability in bioavailability and a lower value for bioavailability. Further, as all variability estimates are based on the nominal dose being exact, deviations from this would inflate variability in pharmacokinetic parameters. Therefore, another possible explanation is that the apparent lack of variability in F is due to that the variability between the nominal and actual dose is larger for intravenous administration than for oral administration. Variability between nominal and exact dose will arise in the manufacturing process, but may also occur due to deviations between nominal and actual volume infused and number of tablets administered. Last, estimates of variability in bioavailability obtained by traditional non-compartmental methods are upwards biased as any error in determining the AUC following intravenous or oral administration will be translated into an individual bioavailability estimate differing from the true one. Thus, even a drug without variability in bioavailability will appear to have variability when this is estimated by classical two-stage methods. Nonlinear mixed effects methods are in general showing less bias in variability estimates than the classical two-stage methods [ 8 ]. Many factors contribute to the bioavailability of cladribine, but the low variability in bioavailability was confirmed with classic pharmacokinetics (data not shown), which is also in agreement with previous analyses [ 3 , 4 ]. Consequently, the variability in AUC after oral administration is of the same magnitude as after intravenous administration, as the variability in CL is more pronounced than that for the bioavailability Table 3 . To reach the general circulation, a drug given orally must pass through the liver via the portal system. A shorter onset and a more intense response may occur when giving a compound orally, rather than as a 2-hour intravenous infusion, if the compound is rapidly absorbed and undergoes extensive first-pass conversion to an active metabolite more potent than the parent. The metabolite of cladribine, 2-chloroadenine, has 8 times lower cytotoxic effect than cladribine, while five times more metabolite is formed after an oral administration compared with after an iv infusion [ 9 ]. After subcutaneous administration the bioavailability was found to be 100% with a low interindividual variability less than 1%. These results are in agreement with the previous analysis [ 3 , 7 ]. For the typical patient, 65 % of the elimination is associated with the terminal slope, 11% with the first and 24% with the second slope. Although distribution kinetics cannot be ignored, the majority of cladribine elimination is clearly associated with events defined by the terminal phase, which has a mean half-life of 16 hours. It is standard practice in oncology to individualise chemotherapy dosing, and to dose according to BSA or weight, with the aim of reducing the interpatient variability of drug effect and toxicity. However, neither weight nor BSA explains more than a minor part of the variability seen. In this study, the modelling indicates that dosing according to body weight or BSA does not represent any significant improvement as compared to administration a fixed dose to all patients. For a convincing evaluation of dosing cladribine, more patients need to be studied, but the lack of reduction in interindividual variability when entering relationships with body size make it unlikely that body size can explain more than a small portion of the interindividual variability. Likewise, it has been shown that BSA fails to standardize the marked interpatient variation in pharmacokinetic variables for most cytotoxic drugs [ 10 ]. There is no simple satisfactory method for calculating drug dose, and other non-BSA-based dose calculation methods have been proposed and discussed [ 10 - 14 ]. Patients with a dose corrected for BSA or weight, but with a low clearance, tend to have an excessive accumulation of the drug and a high risk of toxicity. The amount at any time within the dosing interval at plateau was maximum 3.05 times and minimum 1.02 times the values at the corresponding times after a single dose. It seems therefore valuable to adjust the dose according to pharmacodynamic events such as observed toxicity, which is one parameter used in non-BSA-based dose calculation methods to individualise treatment [ 10 ]. This is possible when patients are treated with repeated courses, e.g. in low-grade lymphomas, but not in the treatment of hairy cell leukemia where only one course of treatment is given. The reasons why people differ in their responsiveness to drugs are manifold. Age, weight, height, disease stage etc. are important because they are sources of variability that can be taken into account. None of the covariates included in this study was found to have a significant influence on the pharmacokinetics of cladribine. In this study no patient with severe reduction of liver- or kidney function was participating. Separate studies with such patient groups are warranted. Conclusion As interindividual variability in apparent clearance after oral administration was not significantly higher compared to that following infusion, cladribine could be administered orally instead of intravenously if compensated for its lower bioavailability. Individualized dosing on basis of body surface area or weight does not represent an improvement in this study as compared to administering a fixed dose to all patients. Methods Patients This population pharmacokinetic study is based on drug concentration data from previously published pharmacokinetic studies [ 2 , 3 , 5 - 7 ]. Patients from five centres in Sweden, Department of Oncology and Department of Hematology at Karolinska Hospital in Stockholm, Department of Medicine at Huddinge Hospital in Stockholm, Department of Hematology at Linköpings Hospital in Linköping and one centre in the United Kingdom, Taunton Hospital, Somerset, participated in these studies after giving their informed consent. The study period, from February 1990 to March 1996, included 215 patients and 227 courses. Fifty-three courses in 52 patients were excluded due to lack of information. Thus, 173 courses in 163 patients, (129 male, 34 female) with a mean age of 60 years (range 22–89) were initially included in population analysis. However, during the analyses, one subject was omitted as concentration time profile showed seven-fold increase after ending the infusion and one subject was excluded as an outlier in that the absorption was considerably slower than for other individuals, possibly indicating another site of administration. Two courses were excluded as drug was given in other administration route (rectal) than the ones that are of interest for the current analysis. Thus the final population analysis included 168 courses in 161 patients. More details regarding demographics and distribution of the different diagnoses among patients are presented in Table 1 . The intravenous infusion results were based on 93 doses during 63 courses, the subcutaneous results on 83 doses during 83 courses and the oral results on 24 doses during 22 courses. For seven patients observed concentrations following both intravenous and oral administration was available. There was no difference in demographic data between patients included and not included in the analysis. Table 1 Descriptive statistics and distribution of diagnoses for patients initially included in the population pharmacokinetic analysis. Gender m/f Age (years) 2 Weight (kg) 2 Height (cm) 2 BSA 1 (m 2 ) 2 Total 129/34 60 (13) [22–89] 76 (14) [48–118] 174 (9) [152–198] 1.9 (0.2) [1.5–2.4] Diagnosis CLL HCL AML NHL CML LCH Total 63 84 3 7 4 2 1 body surface area 2 data is presented with mean (SD) Abbreviations: CLL, chronic lymphocytic leukemia; HCH, hairy cell leukemia; AML, acute myeloic leukemia; NHL, non-Hodgkin's lymphoma; CML, chronic lymphocytic leukaemia; LCH, Langerhan's cell histiocytosis. Drug administration The trials, on which this study was based, had previously been approved by the local Ethics Committee at the Karolinska Institute (Dnr 91:4, 91:190, 92:41, 93:62) and by the Swedish Medical Product Agency. The dose for the iv administration (2-hour infusion) was 5 mg/m 2 or 0.12 mg/kg. The corresponding oral dose was 10 mg/m 2 or 0.24 mg/kg administered in saline after overnight fasting. No food was allowed until two hours after dosage. The subcutaneous dose, 5 mg/m 2 (2 mg/mL) was given in the adipose tissue in the abdominal wall as an injection. Six patients received a continuous iv infusion for four to seven days, with a dose between 4.0–5.6 mg/m 2 /24 hours. Three patients had a 2-hour iv infusion the 1st day and a continuous iv infusion the 2nd day with a dose of 4.8–5.7 mg/m 2 . Dosing and sampling history was collected including the dosing date and time, dose, and treatment period. The covariate data collected were gender, age at treatment, body weight, body height, diagnosis and are summarized in Table 1 . Also laboratory data was included in the analysis, creatinine clearance according to Cockcroft-Gault formula [ 15 ] presented kidney function, liver function tests and lymphocyte count. There was no difference in the laboratory data between the different routes of administration. The laboratory data for the mean patient presented with standard deviation and range are presented in Table 2 . Table 2 Distribution of laboratory data for patients included in the population pharmacokinetic analysis Mean SD Range Outliers Creatinine clearance (mL/min) 81 27 5–162 5 i , 16 i , 27 i , 161 sc , 162 i, o S-aspartate aminotransferase (μkat/L) 0.52 0.44 0.07–4.4 1.9 i , 2.1 sc , 2.2 i , 4.4 i S-alanine amintransferase (μkat/L) 0.49 0.5 0.08–5.3 1.6 i , 2.0 i , 2.1 sc , 5.3 i S-alkaline phosphatase (μkat/L) 1.3 6.7 1.0–64 12 i , 12 i , 14 sc , 54 i , 64 i S-bilirubin (μmol/L) 14 25 3.0–284 41 sc , 88 i , 284 i Outliers were defined as: Creatinine clearance <30->150 (mL/min), aspartate aminotransferase >1.5 (μkat/L), alanine amintransferase >1.5 (μkat/L), alkaline phosphatase >10 (μkat/L), bilirubin >30 (μmol/L) Abbreviations:i, infusion; o, oral; sc, subcutaneous The lymphocyte count was for the mean patient with CLL 124(123) [3.2-472] × 10 9 /L. CLL patient's response to treatment was collected and was distributed as 32% complete remission, 30% partial remission and 38% no remission in 73 patients. Staging according to Rai was 23% stage 1, 27% stage 2, 19% stage 3 and 30% stage 4 and staging according to Binet was 19% A, 33% B and 48% C. Response, staging according to Rai and Binet were included in the analysis of the 73 CLL patients. These data were collected at the beginning of the treatment. Blood sampling Venous samples (10 mL) were collected from an indwelling catheter into heparinized Venoject ® tubes. The blood was stored in ice water. The plasma was isolated within 6 hours after sampling and frozen immediately at -20°C. The average patient had 8 (range 1–27) samples taken up to 36 (range 3–303) hours. Determination of cladribine in plasma The plasma concentrations of cladribine were determined by reversed-phase high-performance liquid chromatography using a solid phase extraction [ 2 , 16 ]. The limit of quantitation was 1 nM for cladribine using 1 mL of plasma, when determined cladribine at 265 nm. Population pharmacokinetic analysis Nonlinear mixed effect modelling was applied for the pharmacokinetic analysis of the data using the software NONMEM[ 17 ]. The program Xpose [ 18 ] was used for data set checkout, exploration and visualisation, model diagnostics, candidate covariate identification and model comparison. Structural pharmacokinetic model The model building strategy involved a development of an integrated model that simultaneously fit the data from all administration routes. Thus, the estimates from this model of the population disposition parameters and interpatient variabilities are based on data from all patients receiving cladribine by all administration routes. Two- and three compartment disposition models were evaluated for cladribine. A first-order absorption model, characterized by the absorption rate constant (ka) with estimated bioavailability (F) was used for extravascular administration. The presence of a lag-time was investigated for oral administration. The analyses were made using the first order conditional estimation method with interaction in NONMEM. Statistical model The interpatient variability for the parameters was described using exponential models. Interpatient variability was included on clearance (CL), central volume of distribution (V1), peripheral volumes of distribution (V2 and V3), intercompartmental clearance (Q2 and Q3), absorption rate constant (ka) and bioavailability of drug (F). Covariances between interpatient variabilities in different pharmacokinetic parameters were investigated. Residual variability, which represents the composite influence of assay variability, patient compliance and model misspecification, were described as a proportional component with separate estimates for the different routes of administration. The residual errors were assumed to be symmetrically distributed. Further, it was assumed that the residual error may not be constant across the individuals since data were pooled from different centres and collected over a long period of time. In order to handle such a variation in the residual variability, the individual contribution to the residual error was accounted for by including an interpatient variability in the residual error model [ 19 ]. Covariate model The relationships between individual pharmacokinetic parameters and covariates were explored using the software Xpose and auxiliary program PsN [ 20 ]. The covariate model was built in a stepwise fashion within the population model, in which both linear and nonlinear relationships between the pharmacokinetic parameters and covariates were considered. The algorithm and assumptions have been described in detail elsewhere [ 21 ]. A generalised additive model was used to analyse the relationship between covariates and preliminary individual parameter estimates [ 22 ]. This procedure, based on the Akaike information criteria [ 23 ], will search for significant relationships between each of the parameters and the candidate covariates. These models guided the inclusion of covariates into the population model. The identified candidate covariates were evaluated in the base model by testing each covariate individually on each parameter and then by testing combinations of covariates on the parameters. The P level for inclusion of a covariate into the final population model was 0.01. This test was based on the objective function value produced by NONMEM, which is minus twice the Log Likelihood value. The difference in the objective function value between hierarchical models is approximately chi-squared distributed. Model diagnostics and validation Basic goodness of fit plots including population and individual predictions versus observed concentrations, as well as individual predictions versus individual weighted residuals, were checked for diagnostic purposes. The population predictions are based on the typical population parameters in the final population model, and the individual predictions on the empirical Bayes estimates of individual parameters. Other calculations The extent of accumulation following intravenous administration was obtained through model simulations and was based on AUC following the first and a steady state dosing interval. Authors' contributions All authors participated in designing the study and/or writing the paper. Physician in charge of cladribine chemotherapy were JL and GJ. SL, RS and MOK were responsible for pharmacokinetic analyses. SL had the primary responsibility for the data collection. We thank Mats Strömberg for his skilful and reliable technical assistance and the staff at the Department of Oncology and Department of Haematology, Karolinska Hospital, for performing the blood sampling. We also like to thank Birgitta Pettersson for performing HPLC analysis.

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1079881

Differential modulation of microglia superoxide anion and thromboxane B2 generation by the marine manzamines

Background Thromboxane B 2 (TXB 2 ) and superoxide anion (O 2 - ) are neuroinflammatory mediators that appear to be involved in the pathogenesis of several neurodegenerative diseases. Because activated-microglia are the main source of TXB 2 and O 2 - in these disorders, modulation of their synthesis has been hypothesized as a potential therapeutic approach for neuroinflammatory disorders. Marine natural products have become a source of novel agents that modulate eicosanoids and O 2 - generation from activated murine and human leukocytes. With the exception of manzamine C, all other manzamines tested are characterized by a complex pentacyclic diamine linked to C-1 of the β-carboline moiety. These marine-derived alkaloids have been reported to possess a diverse range of bioactivities including anticancer, immunostimulatory, insecticidal, antibacterial, antimalarial and antituberculosis activities. The purpose of this investigation was to conduct a structure-activity relationship study with manzamines (MZ) A, B, C, D, E and F on agonist-stimulated release of TXB 2 and O 2 - from E. coli LPS-activated rat neonatal microglia in vitro . Results The manzamines differentially attenuated PMA (phorbol 12-myristate 13-acetate)-stimulated TXB 2 generation in the following order of decreasing potency: MZA (IC 50 <0.016 μM) >MZD (IC 50 = 0.23 μM) >MZB (IC 50 = 1.6 μM) >MZC (IC 50 = 2.98 μM) >MZE and F (IC 50 >10 μM). In contrast, there was less effect on OPZ (opsonized zymosan)-stimulated TXB 2 generation: MZB (IC 50 = 1.44 μM) >MZA (IC 50 = 3.16 μM) >MZC (IC 50 = 3.34 μM) >MZD, MZE and MZF (IC 50 >10 μM). Similarly, PMA-stimulated O 2 - generation was affected differentially as follows: MZD (apparent IC 50 <0.1 μM) >MZA (IC 50 = 0.1 μM) >MZB (IC 50 = 3.16 μM) >MZC (IC 50 = 3.43 μM) >MZE and MZF (IC 50 >10 μM). In contrast, OPZ-stimulated O 2 - generation was minimally affected: MZB (IC 50 = 4.17 μM) >MZC (IC 50 = 9.3 μM) >MZA, MZD, MZE and MZF (IC 50 > 10 μM). From the structure-activity relationship perspective, contributing factors to the observed differential bioactivity on TXB 2 and O 2 - generation are the solubility or ionic forms of MZA and D as well as changes such as saturation or oxidation of the β carboline or 8-membered amine ring. In contrast, the fused 13-membered macrocyclic and isoquinoline ring system, and any substitutions in these rings would not appear to be factors contributing to bioactivity. Conclusion To our knowledge, this is the first experimental study that demonstrates that MZA, at in vitro concentrations that are non toxic to E. coli LPS-activated rat neonatal microglia, potently modulates PMA-stimulated TXB 2 and O 2 - generation. MZA may thus be a lead candidate for the development of novel therapeutic agents for the modulation of TXB 2 and O 2 - release in neuroinflammatory diseases. Marine natural products provide a novel and rich source of chemical diversity that can contribute to the design and development of new and potentially useful anti-inflammatory agents to treat neurodegenerative diseases.

Background The hallmark of brain inflammation is the activation of glia, particularly microglia, the resident immune cells of the brain [ 1 ]. Microglia activation in brain pathologies, as caused by infectious diseases, inflammation, trauma, brain tumors, ischemia and AIDS, may result in neuronal injury and ultimately neurodegeneration [ 1 ]. Similar to other tissue macrophages, when microglia become activated they release potentially neurotoxic mediators [ 2 ], followed by sublethal and lethal injury to the central nervous system. The two different phenotypic forms of microglia, namely the activated but nonphagocytic microglia found in inflammatory pathologies and the reactive or phagocytic microglia present in trauma, infection and neuronal degeneration, appear to have the capacity to express cell-surface receptors and release mediators of inflammation, such as cytokines, coagulation factors, complement factors, proteases, nitric oxide, eicosanoids and reactive oxygen species [ 2 ]. Over the last three decades, the marine environment has been demonstrated to be a source of novel therapeutic agents, many of which have anti-inflammatory properties [ 3 ]. We have previously shown that selected marine natural products modulate eicosanoids [ 4 , 5 ] and O 2 - generation from activated rat [ 6 ] and human neutrophils [ 7 ], as well as liver [ 8 ] and alveolar macrophages [ 9 ]. Based on these observations we hypothesized that selected marine natural products might potentially attenuate activated brain microglia [ 2 ]. Since the discovery by Sakai and Higa that the marine sponge-derived manzamine A (MZA) had potent antitumor activity [ 10 ], there has been a sustained interest in the chemistry [ 11 ] as well as the pharmacology of the manzamines, a class of β-carboline marine-derived alkaloids. More than 40 manzamine-type alkaloids have been isolated from 9 different genera of marine sponges from the Indian and Pacific Oceans, and in addition to the antitumor activity [ 10 ], manzamines have been shown to be immunostimulatory [ 12 ], insecticidal [ 13 ], antibacterial[ 13 ], antimalarial [ 14 ], antiparasitic [ 15 ], antiviral [ 16 ] and to possess antituberculosis activity [ 17 ]. In preliminary communications we have reported that MZA, isolated from the Okinawan marine sponge Haliclona sp., potently inhibited TXB 2 and O 2 - generation by activated rat neonatal microglia while showing very low concomitant toxicity [ 18 - 20 ]. We now extend these previous communications by reporting the results of a structure-activity relationship study with manzamines A, B, C, D, E and F on agonist-stimulated release of O 2 - and TXB 2 from LPS-activated rat neonatal microglia. Results Effect of manzamine A on LPS-activated neonatal brain microglia TXB 2 , O 2 - and LDH release As shown in Fig. 1 , MZA has a pentacyclic diamine group attached to the β-carboline moiety and it was tested as its hydrochloride salt. As is shown in Fig. 2A , MZA potently inhibited PMA-stimulated TXB 2 generation (IC 50 = 0.016 μM), with a maximum 95.5 % inhibition observed at 10 μM (MZA vs. vehicle, respectively 78.3 ± 45 vs. 1,413 ± 439 pg of TXB 2 per 200,000 microglia per 70 min, P < 0.01, n = 4). Furthermore, as depicted in Fig. 3A , MZA inhibited PMA-stimulated O 2 - generation with an apparent IC 50 = 0.1 μM (MZA vs. vehicle, respectively 7.5 ± 1.8 vs.13.8 ± 1.9 nmol of O 2 - per 200,000 microglia per 70 min, P < 0.01, n = 4). Significantly, increasing MZA concentrations to 10 μM resulted in O 2 - inhibition of 55.9 ± 6.1 %, P < 0.01, n = 4. Figure 1 The chemical structures of manzamines A, B, C, D, E and F. Manzamines are indole-derived alkaloids isolated from the marine sponges Haliclona sp . [10], Amphimedon sp . [66] and Xestospongia sp . [10,67]. Molecular weights are respectively = 585.2, 550.8, 347.5, 591.3, 564.7, 580.8. Figure 2 Differential effects of manzamines A, B, C, D, E and F on PMA and OPZ-stimulated TXB 2 generation by LPS-activated rat neonatal microglia . Rat neonatal microglia (200,000 cells/well) were activated with LPS (0.3 ng/mL) for 17 hours. Manzamines were added 15 min before stimulation with either PMA (1 μM) or OPZ (0.5 mg/mL). After 70 min, agonist-triggered TXB 2 was measured as described in Materials and Methods . LDH release, indicator of cytotoxicity, was determined as described in Materials and Methods . Data are expressed as percentage of control TXB 2 release triggered by either PMA (MZA: 1,423 ± 439 pg TXB 2 /70 min; MZB, C, D, E, F: 3,279 ± 281 pg TXB 2 /70 min), or OPZ (4,504 ± 308 pg TXB 2 /70 min). Data show mean ± SEM of indicated number (n) of experiments. * P < 0.05, ** P < 0.01. Figure 3 Differential effects of manzamines A, B, C, D, E and F on PMA and OPZ-stimulated O 2 - generation by LPS-activated rat neonatal microglia . Rat neonatal microglia (200,000 cells/well) were activated with LPS (0.3 ng/mL) for 17 hours. Manzamines were added 15 min before stimulation with either PMA (1 μM) or OPZ (0.5 mg/mL). After 70 min, agonist-triggered O 2 - release was determined as described in Materials and Methods . LDH release, indicator of cytotoxicity, was measured as described in Materials and Methods . Data are expressed as percentage of control O 2 - release triggered by PMA (MZA: 13.8 ± 1.9 nmoles O 2 - /70 min; MZB, C, D, E, F: 10.8 ± 0.6 nmoles O 2 - /70 min), or OPZ (9.4 ± 0.7 nmoles O 2 - /70 min). Data show mean ± SEM of indicated number (n) of experiments. * P < 0.05, ** P < 0.01. In contrast, as shown in Fig. 2A , the effect of MZA on OPZ-stimulated TXB 2 generation was weaker (apparent IC 50 = 3.16 μM), with a maximum 58 % inhibition at 10 μM (MZA vs. vehicle, respectively 1,927 ± 474 vs. 4,504 ± 308 pg of TXB 2 per 200,000 microglia per 70 min, P < 0.05, n = 2). Similarly, as depicted in Fig 3A , MZA did not appear to affect OPZ-stimulated O 2 - generation even at 10 μM (MZA vs. vehicle, respectively 9.5 ± 1 vs. 9.4 ± 0.7 O 2 - nmol per 200,000 microglia per 70 min, P > 0.05, n = 2). As shown in Fig. 2A and 3A , the cytotoxicity of MZA to neonatal brain microglia measured as LDH release was not significantly different from controls even at 10 μM (MZA vs. vehicle, respectively 21.3 ± 7 % vs. 13.9 ± 3.7 % of total LDH released by 0.1 % Triton X-100 treated-microglia, n = 6, P = 0.1735). This data suggests that the effect of MZA on both PMA-stimulated TXB 2 and O 2 - may be of a pharmacological nature. Effect of manzamine B on LPS-activated neonatal brain microglia TXB 2 , O 2 - and LDH release MZB differs from MZA in having a tetracyclic diamine complex and an epoxide ring system (Fig. 1 ). As shown in Fig. 2B , MZB which was tested as a free base, was less potent than MZA in affecting PMA-stimulated TXB 2 generation (IC 50 = 1.6 μM). MZB 10 μM reduced TXB 2 release to 15.5 % of control (MZB vs. vehicle, respectively 546.3 ± 281 vs. 3,279 ± 281 pg of TXB 2 per 200,000 microglia per 70 min, P < 0.01, n = 3). Similarly, as depicted in Fig. 3B , MZB was less potent than MZA in affecting PMA-stimulated O 2 - generation (IC 50 = 3.16 μM). MZB 10 μM reduced O 2 - release to 20 % of control (MZB vs. vehicle, respectively 2.14 ± 2.14 vs. 10.8 ± 0.6 nmol of O 2 - per 200,000 microglia per 70 min, P < 0.01, n = 3). As shown in Fig. 2B , MZB affected OPZ-stimulated TXB 2 (IC 50 = 1.44 μM) more than MZA. MZB 10 μM reduced TXB 2 generation to 25.6 % of control (MZB vs. vehicle, respectively 1,160 ± 207 vs. 4,504 ± 308 pg of TXB 2 per 200,000 microglia per 70 min, P < 0.05, n = 2). Furthermore, as shown in Fig. 3B , MZB reduced OPZ-stimulated O 2 - generation (IC 50 = 4.17 μM) more than MZA. MZB 10 μM reduced O 2 - generation to 16.5 % of control (MZB vs. vehicle, respectively 1.5 ± 0.7 vs. 9.4 ± 0.7 O 2 - nmol per 200,000 microglia per 70 min, P < 0.01, n = 2). As shown in Fig. 2B and 3B , in contrast to MZA, MZB was cytotoxic to neonatal brain microglia at concentrations above 1 μM. In fact, considerable LDH release was observed at 10 μM (88.3 ± 12 % of total LDH released by 0.1 % Triton X-100 treated-microglia, n = 5, P < 0.01). Taken together, these data suggest that the reduction of both O 2 - and TXB 2 generation resulted from both pharmacological and toxic effects of MZB on LPS-activated microglia cells. Effect of manzamine C on LPS-activated neonatal brain microglia TXB 2 , O 2 - and LDH release MZC differs from MZA in having a monocyclic amine ring attached to the β-carboline moiety (Fig. 1 ). As shown in Fig. 2C , MZC which was tested as a free base, was less potent than MZA in affecting PMA-stimulated TXB 2 generation (IC 50 = 2.98 μM). MZC 10 μM reduced TXB 2 release to 19.6 % of control (MZC vs. vehicle, respectively 677 ± 293 vs. 3,279 ± 281 pg of TXB 2 per 200,000 microglia per 70 min, P < 0.01, n = 3). Similarly, as depicted in Fig. 3C , MZC was less potent than MZA in affecting PMA-stimulated O 2 - generation (apparent IC 50 = 3.43 μM). MZC 10 μM reduced O 2 - release to 36.1 % of control (MZC vs. vehicle, respectively 3.7 ± 2.2 vs.10.8 ± 0.6 nmol of O 2 - per 200,000 microglia per 70 min, P < 0.05, n = 3). As shown in Fig. 2C , MZC affected OPZ-stimulated TXB 2 (IC 50 = 3.34 μM), similar to MZA. MZC 10 μM reduced TXB 2 generation to 29.8 % of control (MZC vs. vehicle, respectively 1,345 ± 160 vs. 4,504 ± 308 pg of TXB 2 per 200,000 microglia per 70 min, P < 0.05 n = 2). Furthermore, as depicted in Fig. 3C , MZC reduced OPZ-stimulated O 2 - generation (apparent IC 50 = 9.3 μM), higher than MZA. MZC 10 μM reduced O 2 - generation to 41.6 % of control (MZC vs. vehicle, respectively 3.9 ± 0.3 vs. 9.4 ± 0.7 O 2 - nmol per 200,000 microglia per 70 min, P < 0.05, n = 2). As shown in Fig. 2C and 3C , and in contrast to MZA, MZC was cytotoxic to neonatal brain microglia though not as much as MZB. Substantial LDH release was observed at 10 μM (59.8 ± 11 % of total LDH released by 0.1 % Triton X-100 treated-microglia, n = 5, P < 0.05). In summary, similar to MZB, the data suggests that the reduction of both O 2 - and TXB 2 generation resulted from both pharmacological and toxic effects of MZC on LPS-activated microglia cells. Effect of manzamine D on LPS-activated neonatal brain microglia TXB 2 , O 2 - and LDH release MZD differs from MZA in having a tetrahydrocarboline group in the molecule (Fig. 1 ). As shown in Fig. 2D , MZD that was tested as its hydrochloride salt, strongly affected PMA-stimulated TXB 2 generation (IC 50 = 0.23 μM). MZD 10 μM reduced TXB 2 release to 18.8 % of control (MZD vs. vehicle, respectively 611 ± 342 vs. 3,279 ± 281 pg of TXB 2 per 200,000 microglia per 70 min, P < 0.01, n = 3). Furthermore, as shown in Fig. 3D , MZD also strongly affected PMA-stimulated O 2 - generation. MZD 0.1 μM reduced O 2 - release to 14 % of control (MZD vs. vehicle, respectively 0.7 ± 0.7 vs. 10.8 ± 0.6 nmol of O 2 - per 200,000 microglia per 70 min, P < 0.01, n = 3). In contrast, as shown in Fig. 2D the effect of MZD on OPZ-stimulated TXB 2 generation was limited. MZD 10 μM reduced TXB 2 generation to 79.9% of control (MZD vs. vehicle, respectively 3,613 ± 469 vs. 4,504 ± 308 pg of TXB 2 per 200,000 microglia per 70 min, P > 0.05, n = 2). Furthermore, as shown in Fig. 3D , MZD did not affect OPZ-stimulated O 2 - generation even at 10 μM. As shown in Fig. 2D and 3D , and in contrast to MZB and C, MZD was very cytotoxic to microglia when PMA was used as an agonist to trigger O 2 - and TXB 2 release: MZD at 0.1 μM caused 61.5 ± 13 % of total LDH released by 0.1 % Triton X-100 treated-microglia (n = 5, P < 0.05). In contrast to the limited effect of MZD on OPZ-stimulated microglia, the data suggests that the reduction of PMA-stimulated O 2 - and TXB 2 generation resulted from both pharmacological and toxic effects of MZD on LPS-activated microglia cells. Effect of manzamine E on LPS-activated neonatal brain microglia TXB 2 , O 2 - and LDH release As shown in Fig. 1 , MZE differs from MZA in having a saturated ketone functionality in the eight-membered amine ring. As depicted in Fig. 2E , MZE inhibited PMA-stimulated TXB 2 generation with a maximum 49.4 % inhibition observed at 10 μM (MZE vs. vehicle, respectively 1,614 ± 628 vs. 3,279 ± 281 pg of TXB 2 per 200,000 microglia per 70 min, P > 0.05, n = 3). Furthermore, as shown in Fig. 3E , MZE inhibited PMA-stimulated O 2 - generation with a maximum 26.3 % inhibition observed at 10 μM (MZE vs. vehicle, respectively 8.5 ± 2.1 vs.10.8 ± 0.6 nmol of O 2 - per 200,000 microglia per 70 min, P > 0.05, n = 3). As shown in Fig. 2E , MZE had limited effect on OPZ-stimulated TXB 2 generation, with a maximum 43.1 % inhibition at 10 μM (MZE vs. vehicle, respectively 2,594 ± 646 vs. 4,504 ± 308 pg of TXB 2 per 200,000 microglia per 70 min, P > 0.05, n = 2). Similarly, as depicted in Fig. 3E , MZE did not affect OPZ-stimulated O 2 - generation even at 10 μM. As shown in Fig 2E and 3E , cytotoxicity of MZE to microglia measured as LDH release was low even at 10 μM (MZE vs. vehicle, respectively 20 ± 6.7 % vs. 19.8 ± 7.7 % of total LDH released by 0.1 % Triton X-100 treated-microglia, P > 0.05, n = 5). Effect of manzamine F on LPS-activated neonatal brain microglia TXB 2 , O 2 - and LDH release MZF differs from MZA in having a saturated ketone functionality in the eight-membered amine ring and hydroxylation at the C-8 position of the β-carboline ring system (Fig. 1 ). As shown in Fig. 2F , MZF did not inhibit PMA-stimulated TXB 2 generation. In the presence of 10 μM MZ, TXB 2 release was 104.1 ± 24.7 % of control TXB 2 generation (MZF vs. vehicle, respectively 3,202 ± 1,139 vs. 3,279 ± 281 pg of TXB 2 per 200,000 microglia per 70 min, P > 0.05, n = 3). Similarly, as shown in Fig. 3F , MZF did not inhibit PMA-stimulated O 2 - release. In the presence of 10 μM MZF, O 2 - release was 113 ± 14.5 % of control O 2 - generation (MZF vs. vehicle, respectively 12.8 ± 1.6 vs. 10.8 ± 0.6 nmol of O 2 - per 200,000 microglia per 70 min, P > 0.05, n = 3). As shown in Fig. 2F , MZF effect on OPZ-stimulated TXB 2 generation was weak, with a non-statistically significant 26.2 % inhibition at 10 μM (MZF vs. vehicle, respectively 3,317 ± 121 vs. 4,504 ± 308 pg of TXB 2 per 200,000 microglia per 70 min, P > 0.05, n = 2). Similarly, as depicted in Fig. 3F , MZF was minimally effective in inhibiting OPZ-stimulated O 2 - generation, only a non-statistically significant 15.7 % inhibition observed at 10 μM. As shown in Fig. 2F and 3F , cytotoxicity of MZF to neonatal brain microglia measured as LDH release was low even at 10 μM (MZF vs. vehicle, respectively 19 ± 7.2 % vs. 19.8 ± 7.7 % of total LDH released by 0.1 % Triton X-100 treated-microglia, n = 5). Effect of manzamine A, B, C, D, E and F on hypoxanthine-xanthine oxidase generated O 2 - In order to determine a potential scavenging effect of MZA, B, C, D, E and F on O 2 - , a standard hypoxanthine-xanthine oxidase system was used as a cell-free source of O 2 - [ 21 ]. As shown in Fig. 4 , O 2 - generation by incubation of purified xanthine oxidase with hypoxanthine was abolished by superoxide dismutase. Furthermore, DMSO, the vehicle used to prepare the manzamines, did not affect O 2 - formation (control vs. DMSO, respectively 12.3 ± 2.3 vs. 12 ± 1.1 nmoles/30 min, n = 2, P > 0.05). Similarly, MZA, B, and E did not significantly affect O 2 - generation (MZA, B, E vs. control, respectively, 15.3 ± 4.4, 15.3 ± 2.4, 16.3 ± 2.2 vs. 12 ± 1.1 nmoles/30 min, n = 2, P > 0.05). In contrast, MZC, D and F appeared to enhance O 2 - formation (MZC, D, F vs. control, respectively 20.7 ± 3.1, 22.2 ± 2, 20.1 ± 3.6 vs. 12 ± 1.1 nmoles/30 min, n = 2, P < 0.05 (MZC, F), P < 0.01 MZD). Thus we conclude that the inhibition of either PMA or OZ stimulated-O 2 - release from LPS-activated microglia by the manzamines was not the result of a direct O 2 - scavenging effect. Figure 4 Differential effect of manzamine A, B, C, D, E and F and superoxide dismutase on hypoxanthine-xanthine oxidase system-generated O 2 - . Cell-free source of O 2 - was generated by a standard hypoxanthine-oxidase system as described in Materials and Methods . Data are expressed as O 2 - released (nmoles O 2 - /30 min) and correspond to the mean ± SD of 2 representative experiments in duplicate. * P < 0.05, ** P < 0.01 versus control. Discussion The important role of neuroinflammation and glial activation in the pathogenesis of brain disorders has progressively been established [ 1 , 2 , 22 ]. Because in vitro LPS-activated microglia appear to mimic the functions of activated microglia found in neuroinflammatory conditions in vivo [ 2 , 23 ], we used LPS-activated rat microglia as a relevant in vitro paradigm to search for marine natural products that may modulate the enhanced release of TXB 2 and O 2 - from activated microglia [ 24 ]. Using this in vitro model we have previously communicated that MZA, a secondary metabolite isolated from the Okinawan marine sponge Haliclona sp[ 10 ], inhibited TXB 2 and O 2 - generation by microglia [ 18 ]. The current study extends our initial observations, and reports a structure-activity relationship study with manzamines A, B, C, D, E and F on both PMA and OPZ-stimulated release of TXB 2 and O 2 - from LPS-activated rat neonatal microglia. Members of the eicosanoid family (i.e. prostaglandins, leukotrienes and thromboxanes) are important mediators of inflammation that would appear to be play a causative role in the pathogenesis of several CNS disorders [ 25 - 27 ]. Increased levels of eicosanoids have been observed in neurodegenerative disorders such as amyotrophic lateral sclerosis [ 28 ], multiple sclerosis [ 29 ], ischemia and seizures [ 30 ], prion diseases [ 31 ], human immunodeficiency virus-associated dementia [ 32 ] and Alzheimer's disease [ 33 ]. Following the seminal observation that microglia release eicosanoids [ 34 ], numerous studies have increasingly supported the notion that activated brain macrophages may be the main source of both prostaglandins [ 33 , 35 - 39 ] and thromboxanes [ 23 , 37 , 39 , 40 ] in these neurodegenerative diseases. Thus modulation of microglia enhanced prostanoid synthesis has been investigated as a potential drug therapeutic approach for intervention in neuroinflammatory disorders of the CNS [ 36 , 41 , 42 ]. One possible approach to diminish enhanced eicosanoid production has been to search for inhibitors of signal transduction pathways involved in eicosanoid synthesis in activated microglia [ 42 ]. In our study, we used PMA and OPZ, agonists known to activate p44/42 and p38 mitogen-activated protein kinases in microglia [ 43 ], to target distinct signal transduction pathways that cause TXB 2 release in rat neonatal microglia activated by an in vitro exposure to 0.3 ng/mL of LPS for 17 hours [ 23 ]. As shown in Fig. 2 , the 6 marine-derived manzamine analogs attenuated PMA-stimulated TXB 2 generation differentially and in the following order of decreasing potency: MZA>MZD>MZB >MZC>MZE and F. In contrast, the manzamine analogs inhibited OPZ-stimulated TXB 2 generation with reduced potency: MZB>MZA>MZC>MZD, MZE and MZF. Thus, with the exception of MZB and MZC which modulated both PMA and OPZ-stimulated TXB 2 release with similar potency, MZA, MZD, MZE and MZF inhibited TXB 2 release with lower potency when OPZ was used as an agonist. It is interesting to compare our differential results with the MZA, B, C, D, E, and F with those reported for other agents that have been shown to modulate microglia eicosanoid release by targeting the cyclooxygenase I and II enzymes which are expressed in activated rat and human microglia [ 35 , 44 ]. PGE 2 and TXB 2 synthesis that occurs concomitantly with LPS-induced activation of rat [ 38 , 45 , 46 ] and human microglia [ 35 ], has been shown to be attenuated by nonsteroidal anti-inflammatory drugs (NSAIDs) with differing activities towards the two isoforms of COX. Thus, LPS-induced microglia PGE 2 synthesis was reduced by COX-1 inhibitors: acetylsalicylic acid (aspirin) (IC 50 = 3.12–10 μM) [ 38 , 46 ], flurbiprofen (apparent IC 50 = 100 nM) [ 45 ] and indomethacin (apparent IC 50 = 1 nM) [ 38 ], and the COX-2 inhibitor NS-398 (apparent IC 50 = 1–5 nM) [ 38 ]. Even though NSAIDs have been reported to attenuate neurotoxicity in vitro [ 47 ] and neuroinflammation in animal models [ 48 - 50 ], an important caveat is the fact that determining the best NSAIDs for clinical neurodegenerative disease management appears to remain a matter of considerable debate in view of their well known adverse effects [ 51 - 53 ]. Thus, although the molecular mechanism by which the manzamines inhibit TXB 2 release in LPS-activated cells remains currently undetermined, MZA inhibited PMA-stimulated eicosanoid generation in vitro with potency similar to that of the COX-1 inhibitor indomethacin [ 38 ], potency that was higher than that of other NSAIDs that have been reported to modulate enhanced eicosanoid release in both activated rat and human microglia [ 35 , 38 , 45 , 46 ]. The involvement of reactive oxygen species (ROS) has been documented in CNS pathologies, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, Down's syndrome, cerebral ischemia and reperfusion, amyotrophic lateral sclerosis, multiple sclerosis and meningitis [ 54 ]. Prolonged exposure to ROS may potentially damage neurons, particularly their synapses [ 55 ] as well as oligodendrocytes, the myelin producing cell of the CNS [ 56 ] by overriding normal CNS antioxidant defense mechanisms, e.g. superoxide dismutase, catalase, glutathione-S-transferase, glutathione peroxidase, permanently affecting cellular function [ 57 ]. Thus the mechanism of ROS generation by CNS leukocytes, i.e. infiltrating neutrophils and monocytes as well as resident microglia production of O 2 - , hydrogen peroxide and nitric oxide in CNS, has received considerable attention since the mid-1980s [ 2 ]. In fact, during the past 18 years, numerous research groups have shown that O 2 - may be generated by microglia isolated from rat, mice, hamsters, dogs, swine and humans, when stimulated with a variety of agonists such as phorbol ester, opsonized zymosan, calcium ionophore, antiviral antibodies, antibody-coated red blood cells and myelin (reviewed in [ 2 ]). We and others have hypothesized that rather than scavenge ROS with antioxidants, the modulation of the signal transduction mechanism leading to microglia ROS generation might be putatively a better therapeutic strategy to turn off or reduce ROS generation that could lead to neuronal injury [ 2 , 58 , 59 ]. As depicted in Fig. 3 , the manzamine analogs attenuated PMA-stimulated O 2 - generation in the following order of decreasing potency: MZD>MZA>MZB>MZC>MZE and MZF. In contrast, and similarly to their weaker effect on OPZ-stimulated TXB 2 generation, all the manzamine analogs modulated OPZ-stimulated O 2 - generation with lower potency: MZB>MZC>MZA, MZD, MZE and MZF. Interestingly, as shown in Fig. 4 , the effect of MZD, A, B and C on PMA-stimulated O 2 - formation was not the result of any detectable scavenging O 2 - , because none of the manzamines inhibited a standard hypoxanthine-xanthine oxidase system that was used as a cell-free source of O 2 - . Thus, although the exact mechanism by which the manzamines modulate O 2 - release by microglia remains currently undetermined, we have demonstrated that these compounds clearly modulate the signal transduction pathway that PMA triggers in microglia and ultimately leads to O 2 - generation. It is interesting to compare our differential results with the MZD, A, B and C with those reported for other agents that modulate PMA-stimulated O 2 - generation in microglia. Interestingly, MZD, A, B and C demonstrated higher potency than three clinically available agents shown to inhibit PMA-stimulated O 2 - generation: propentofylline, a selective phosphodiesterase inhibitor (IC 50 >100 μM) [ 60 ], cabergoline, a potent and selective agonist of D2-dopamine receptors (IC 50 >100 μM) [ 59 ], and nicergoline, an ergoline derivative used for cerebrovascular diseases (IC 50 = 10–15 μM) [ 58 ]. It is noteworthy that these agents have been proposed to confer protective effects against neurodegenerative diseases which may involve O 2 - release by activated rat microglia. In order to determine if the effect of the manzamines on PMA or OPZ-triggered TXB 2 and O 2 - generation was either pharmacological or toxic, we investigated LDH release from LPS-activated rat neonatal microglia. LDH has extensively been used as a marker for cell cytotoxicity [ 61 ]. The results from our investigation appear to demonstrate that the manzamine analogs clearly differed in their effect on LDH release from LPS-primed microglia: MZA, MZE and MZF generated less than 50% of maximal LDH release at 10 μM, and thus were the least toxic analogs; MZB and MZC induced greater than 50% of LDH release at 10 μM; while MZD showed greater than 50% of LDH release at 0.1 μM when PMA rather than OPZ was used as an agonist, thus its toxicity contrasted with the other analogs tested. Thus even though MZD inhibited PMA-stimulated O 2 - generation with slightly higher potency than MZA, because MZD caused concomitant LDH release at low concentrations (0.1 μM), the nature of the inhibitory effect on PMA-triggered O 2 - and TXB 2 release, either toxic or pharmacological, remains currently unresolved. In summary, the in vitro studies described herein suggest that MZA is the most potent manzamine analog of the series investigated because both PMA-stimulated O 2 - and TXB 2 were potently inhibited with the lowest concomitant release of LDH. It is of interest to consider the results of our structure-activity relationship (SAR) study with the manzamines, alkaloids characterized by a complex heterocyclic ring system attached to the C-1 of the β-carboline moiety. From the SAR perspective, the potent effect of MZA and D hydrochloride salts on PMA-stimulated O 2 - and TXB 2 suggests that the solubility or ionic forms are contributing factors to their bioactivity. Furthermore, the fused 13-membered macrocyclic and octahydroisoquinoline ring system, and any substitutions in these rings would appear to be less important for their in vitro activity. Finally, changes such as saturation or oxidation of the β carboline or the 8-membered amine ring tended to decrease bioactivity in both O 2 - and TXB 2 assays. Taken together, our current data demonstrates that the most potent and least toxic manzamine analog, namely MZA, was less effective in attenuating O 2 - and TXB 2 from LPS-activated microglia when the triggering agonist was OPZ rather than PMA. Similar differential effects between PMA and OPZ-triggered signaling have been observed with other natural products [ 62 ]. Furthermore, the current data suggest the following on the as yet undefined mechanism of action of MZA: Firstly, that the MZA molecular target plays a critical role in O 2 - and TXB 2 generation initiated by PMA upon binding to PKC [ 63 , 64 ] and activation of the p44/42 mitogen-activated protein kinase signaling pathway [ 43 ]; Secondly, that the MZA molecular target probably plays a less critical role in O 2 - and TXB 2 release elicited by OPZ, a ligand of the microglial cell surface complement receptor 3 shown to activate the p38 mitogen-activated protein kinase signaling pathway [ 43 ]. Studies to determine which element is targeted by MZA in the p38 and/or p44/42 mitogen-activated protein kinase pathways in LPS-activated rat microglia are currently underway in our laboratory. Conclusion Our present results provide the first experimental evidence to support the hypothesis that the marine-derived β-carboline alkaloid manzamines differentially modulate both O 2 - and TXB 2 generated by E. coli LPS-activated rat neonatal microglia. Additional conclusions are the following: Firstly, SAR studies demonstrated that at in vitro concentrations that were non-toxic to E. coli LPS-activated rat neonatal microglia, MZA was the most potent inhibitor of O 2 - and TXB 2. Secondly, although the mechanism by which MZA inhibited PMA-stimulated TXB 2 generation in vitro is as yet unclear, its potency was similar to the COX-1 inhibitor indomethacin [ 38 ], and thus higher than other NSAIDs reported to modulate enhanced eicosanoid release in both activated rat and human microglia [ 35 , 38 , 45 , 46 ]. Thirdly, although the mechanism by which MZA inhibited PMA-stimulated O 2 - generation in vitro remains undetermined, MZA was more potent than propentofylline, a selective phosphodiesterase inhibitor, cabergoline, a potent and selective agonist of D2-dopamine receptors and nicergoline, an ergoline derivative used for cerebrovascular diseases, compounds which have been proposed to confer protective effects against neurodegenerative diseases by affecting O 2 - release by activated rat microglia. Fourthly, SAR studies which demonstrated that the ionic forms are a contributing factor to the bioactivity of the complex manzamine heterocyclic ring system attached to a β-carboline moiety may explain the potent effect of MZA and D hydrochloride salts on PMA-stimulated O 2 - and TXB 2 . Interestingly, the fused 13-membered macrocyclic amine and octahydroisoquinoline ring system, as well as substitutions in these rings appeared to be a non-factor for the in vitro activity of the manzamines. Finally, the reported pharmacokinetic properties and the lack of significant in vivo toxicity [ 14 ] of MZA, a β-carboline alkaloid whose complete synthesis has been reported [ 11 ], would suggest that MZA is a prime candidate for further investigation of its potential utility as a pharmacophore from which new and novel therapeutic agents for neuroinflammatory diseases might be developed. Methods Reagents LPS B ( Escherichia coli 026:B6) was obtained from Difco Laboratories (Detroit, MI); Wright Giemsa stain (modified), ferricytochrome c type III (from horse heart) (FCC), superoxide dismutase (from bovine liver), phorbol 12-myristate 13-acetate (PMA), zymosan and dimethyl sulphoxide (DMSO) were obtained from Sigma Chemical Co. (St. Louis, MO). PMA was maintained at -80°C as a 10 mM stock solution in DMSO. Opsonized zymosan (OPZ) was maintained at -20°C in a stock solution of 15 mg/ml in PBS and prepared as described [ 65 ]. Dulbecco's modified Eagle medium (DMEM) with high glucose (4,500 mg/l), Hank's balanced salt solution (HBSS), penicillin (P), streptomycin (S), trypsin (0.25%)-EDTA (1 mM) and trypan blue were purchased from GIBCO-BRL (Grand Island, NY); certified heat-inactivated fetal bovine serum (FBS) was obtained from Hyclone (Logan, UT); a LPS stock of 1 mg/ml was prepared in a 0.9% sodium chloride nonpyrogenic solution from Baxter Healthcare Corp. (Toronto, ONT, Canada) and then diluted with DMEM plus 10% FBS plus P and S to the appropriate concentration used in our experiments. Both the LPS stock solution [10 ng/ml] and dilutions were stored at -80°C, thawed prior to each experiment and discarded after use. LPS containment To inactivate extraneous LPS, all glassware and metal spatulas were baked for 4 hours at 180°C. Sterile and LPS-free 75- and 162-cm 2 vented cell culture flasks, 24-well flat-bottom culture clusters, 96-well cell culture clusters and disposable serological pipettes were purchased from Costar Corporation (Cambridge, MA), while polystyrene cell culture dishes (60 × 15 mm) were obtained from Corning Glass Works (Corning, NY). Sterile and LPS-free Eppendorf Biopur pipette tips were purchased from Brinkmann Instruments, Inc. (Westbury, NY). Manzamines A, B, C, D, E and F Manzamine A (MZA) was isolated from a marine sponge species of the genus Haliclona collected off Manzamo, Okinawa in waters at a depth of 30 m in April 1985 [ 10 ]. Manzamine B (MZB), manzamine C (MZC) and manzamine D (MZD), were isolated from a sponge of the genus Amphimedon collected by SCUBA off Manzamo, Okinawa [ 66 ]. Manzamine E (MZE) and manzamine F (MZF) were isolated from a sponge species of the genus Xestospongia collected off the coast of Miyako Island, Okinawa in June 1986 [ 67 ]. All manzamines were dissolved in DMSO to prepare a 10 mM stock and stored at -80°C prior to use in the experiments. Isolation and culture of rat neonatal microglia All experiments were performed with adherence to the National Institutes of Health guidelines on the use of experimental animals and with protocols approved by Midwestern University's Research and Animal Care Committee. To isolate rat neonatal microglia, cerebral cortices of 1–2 day-old Sprague-Dawley rats purchased from Harlan (Indianapolis, IN) were surgically removed and placed in cold DMEM + 10% FBS + 120 U/ml P and 12 μg/ml S, the meninges carefully removed, and brain tissue minced and dissociated with trypsin-EDTA at 36°C for 3–5 min. The mixed astroglial cell suspension was plated in either 75- or 162-cm 2 vented cell culture flasks with DMEM medium supplemented with 10% FBS + 120 U/ml P + 12 μg/ml S and grown in a humidified 5% CO 2 incubator at 36°C for 12–14 days. On day 14 and every 3–4 days thereafter, microglia were detached using an orbital shaker (150 rpm, 0.5 hours, 36°C, 5% CO 2 ), centrifuged (400 × g, 25 min, 4°C), and microglia number and viability assessed by trypan blue exclusion. Microglia were characterized as described earlier [ 23 ]. Depending on the particular experimental protocol (see below), microglia averaging greater than 95% viability were plated in 24-well cell culture clusters, with DMEM supplemented with 10% FBS + 120 U/ml P + 12 μg/ml S, and placed in a humidified 5% CO 2 incubator at 36°C 18–24 hours prior to the experiments. Experimental protocol to study the effect of manzamines A – F on microglia release of TXB 2 and O 2 - To study the effects of manzamines A, B, C, D, E and F on the generation of TXB 2 and O 2 - , rat neonatal microglia (2 × 10 5 cells/24-well cell culture clusters) were treated to the following protocol. Seventeen hours prior to the experiments, microglia cells were treated with LPS (0.3 ng/ml) in a final volume of 1 ml of DMEM supplemented with 10% FBS + 120 U/ml P + 12 μg/ml. Thereafter the media was removed and replaced with 1 ml warm HBSS, one of the manzamines (0.1–10 μM final concentration) or vehicle (DMSO) was added, and the microglia incubated for fifteen minutes in a humidified 5% CO 2 incubator at 35.9°C. After the fifteen minute preincubation period with either manzamines or vehicle, PMA (1 μM) or OPZ (0.5 mg/mL) was added and microglia incubated for 70 minutes in a humidified 5% CO 2 incubator at 35.9°C in the presence of the manzamines or vehicle. The final concentration of DMSO did not affect microglia viability or LDH release. O 2 - , TXB 2 and lactate dehydrogenase (LDH) release were assayed as described below. Assay for TXB 2 Following the incubation of LPS-activated microglia with HBSS, manzamines or vehicle as explained above, PMA- (1 μM) or OPZ- (0.5 mg/mL)triggered TXB 2 generation in the culture supernatants was measured using immunoassays (Cayman Chemical, Ann Arbor, MI) as indicated by the manufacturer's protocol. Results were expressed as pg/mL produced after 70 min of PMA or OPZ stimulation. Assay for O 2 - O 2 - generation was determined by the SOD-inhibitable reduction of FCC [ 23 ]. Briefly, PMA (1 μM) or OPZ (0.5 mg/mL)- triggered O 2 - release from LPS-activated microglia was measured in the presence of FCC (50 μM) and HBSS, with or without SOD (700 Units) which inhibited >95% of FCC reduction, during the 70 min incubation described above. All experimental treatments were run in triplicate and in a final volume of 1 ml. Changes in FCC absorbance were measured at 550 nm using a Beckman DU-650 spectrophotometer. Differences in the amount of reduced FCC in the presence and absence of SOD were used to determine O 2 - generation and expressed in nmol by employing the molecular extinction coefficient of 21.0 × 10 3 M -1 cm -1 . Experimental protocol to study the effect of manzamines on superoxide anion by the hypoxanthine-xanthine oxidase system A standard hypoxanthine-xanthine oxidase system was used as a cell-free source of O 2 - . O 2 - was generated by incubation of purified xanthine oxidase (0.02 Units/ml) with hypoxanthine (1.5 mM) at 37°C [ 21 ]. O 2 - formation was assessed spectrophotometrically as the increase in absorbance at 550 nm associated with the SOD (30 U/mL)-inhibitable reduction FCC (50 μM) as described above for rat microglia O 2 - generation and expressed in nmoles/30 minutes. Lactate dehydrogenase assay Lactate dehydrogenase (LDH) release from microglia was determined spectrophotometrically as described elsewhere [ 9 ]. Microglia LDH release was expressed as a percentage of total LDH. Total LDH resulted from 0.1% Triton X-100-lysed microglia cells (intracellular LDH) plus LDH released to the extracellular medium. Statistical analysis of the data Data were analyzed with the Prism ® software package purchased from GraphPad (San Diego, CA.). One-way analysis of variance followed by Dunnett's test was performed on all sets of data. Manzamine-treated groups were compared with the vehicle-treated group, shown as 0 or control in the corresponding figures. Differences were considered statistically significant at p < 0.05 and reported in each figure legend. Abbreviations CNS, central nervous system; DMEM, Dulbecco's modified Eagle medium; DMSO, dimethyl sulphoxide; FBS, fetal bovine serum certified; FCC, ferricytochrome c type III; HBSS, Hank's balanced salt solution; LPS, lipopolysaccharide; MZA, manzamine A; MZB, manzamine B; MZC, manzamine C; MZD, manzamine D; MZE, manzamine E; MZF, manzamine F; O 2 - , superoxide anion; OPZ, opsonized zymosan; P, penicillin; PBS, phosphate buffered saline; PMA, phorbol-12-myristate-13-acetate ; S, streptomycin; SOD, superoxide dismutase; TXB 2 , thromboxane B 2 . Authors' contributions A.M.S. M. designed and conducted the experiments described and prepared the manuscript draft. M.L.H. , performed the statistical analysis of the data and prepared Fig. 2 , 3 and 4 . S. M. L. helped conduct the xanthine oxidase studies with Manzamines A, B, C, D, E and F. S.P.G. supplied pure Manzamines A, B, C, D, E. and F from the Division of Biomedical Marine Research depository, prepared Fig. 1 , and contributed to various sections of the manuscript. S.A.P. provided financial support and contributed to various sections of the manuscript. S.H.S. contributed to various sections of the manuscript. All authors read and approved the manuscript.

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1079882

Low dose docosahexaenoic acid protects normal colonic epithelial cells from araC toxicity

Background The nucleoside analogue arabinosylcytosine (araC) has been used for many years in the treatment of acute leukemia. Evidence in the literature suggests that araC may inhibit the growth of human colon carcinoma cell lines as well. Because araC action interferes with normal nucleoside metabolism, it is highly toxic to a number of normal cell types including bone marrow and intestinal mucosa cells. Here we investigate whether the omega-3 fatty acid docosahexaenoic acid (DHA) could selectively target araC toxicity toward colonic tumor cells while protecting the normal cells in vitro . Results Cultures of normal rat colonic epithelial cells (4D/WT) and those transformed by v-src (D/v-src) were supplemented with graded concentrations of DHA or arachidonic acid (AA) alone or in combination with araC. AraC was only 1.6 fold more toxic to D/v-src than 4D/WT in cultures without added fatty acids. Supplementing with as little as 3 μM of either AA or DHA increased araC toxicity by more than 30-fold in the tumorigenic cells. The toxic effect of araC on the normal cells was also increased by the fatty acid supplementation. IC 50 values were decreased 1.7 fold by DHA in the 4D/WT cells but a more than 7-fold decrease was observed during AA supplementation. As a result, the therapeutic index of araC (IC 50 normal/IC 50 tumor) was more than 3-fold higher in the DHA than the AA supplemented cells. The expression of protein kinase C isoform epsilon was decreased in AA alone supplemented D/v-src cultures but in combination with araC decreased only in DHA supplemented 4D/WT cells. Conclusion Low dose DHA supplementation may enhance araC chemotherapy in colon cancer while protecting normal tissues, possibly through control of PKC signalling pathways.

Background Colorectal cancer is the second most common cause of death among men and women in North America [ 1 ]. This disease may evolve from genetic alterations in protooncogenes or tumor suppressor genes. There has also been evidence indicating dietary factors, such as fat and fiber, affect several stages of colon carcinogenesis. Japanese have a lower incidence of colon cancer as compared to North Americans, which could be partially attributed to higher amounts of n-3 fatty acids and low amounts of saturated, monounsaturated and n-6 fatty acids in their diets [ 2 ]. Arabinosylcytosine (araC) has been used for many years in the treatment of acute leukemia. Some later studies have suggested that araC and the related drug gemcitabine may be useful in inhibiting the growth of human colon carcinoma cell lines [ 3 ]. Because araC action interferes with normal nucleoside metabolism, a number of normal cell types particularly dependent on salvage of nucleosides, are extremely sensitive to araC; these include the bone marrow and intestinal mucosa. In patients with leukemias and non-Hodgkins lymphoma treated with araC, gastrointestinal and hematopoietic toxicities predominate and frequently limit dose escalation [ 4 ]. We have previously demonstrated that docosahexaenoic acid (DHA) can enhance araC and doxorubicin toxicity in leukemia cells and araC toxicity in transformed rat fibroblasts in vitro [ 5 , 6 ]. More recently, we showed that at least part of this differentially selectivity was due to effects of DHA on two metabolic enzymes cytidine kinase and cytidine deaminase [ 7 ]. In vivo we showed that DHA had beneficial effects on both the bone marrow compartment and gastrointestinal tract of tumor-bearing, araC treated rats [ 8 ]. We also demonstrated that a low dose of dietary DHA supplementation could prolong the life span and limit the occurrence of toxicity in L1210 leukemic mice [ 9 ]. The current study was designed to investigate whether low dose DHA could selectively target araC toxicity toward rat colon tumor cells in vitro . Studies show that araC engages in an array of signaling events including activation of protein kinase C (PKC) [ 10 ]. Essential fatty acids of the omega-3 and omega-6 series are also known to have effects on cell membrane signaling which includes PKC [ 11 , 12 ]. Because both araC and DHA could potentially modulate cell death and drug toxicity in overlapping pathways, we also examined the expression of various PKC isoforms during fatty acid supplementation and drug treatment. Results Using the sulforhodamine dye binding assay the IC 50 values for arachidonic acid (AA), DHA, and araC were examined in the normal colonic epithelial cell line 4D/WT and in the v-src transformed variant, D/v-src (Table 1 .). AA and DHA had approximately the same toxicity profile in both cell types and showed 5-7-fold higher toxicity in the tumorigenic versus the normal epithelial cells. The IC 50 value for araC in 4D/WT was 67 μM and only slightly lower in the D/v-src cells at 42 μM. Pre-incubating 4D/WT cells with 3 μM DHA (well below the IC 50 values for either cell type) only slightly increased araC toxicity (1.7-fold) while pre-incubation with 3 μM AA increased araC toxicity 7-fold in the normal 4D/WT cells. Pre-incubation of the tumorigenic cell line, D/v-src with either 3 μM DHA or AA resulted in a dramatic 40-fold increase in araC toxicity. Therefore the improvement in therapeutic index is 30-fold in the presence of 3 μM DHA (p < 0.03). Toxicity of other fatty acids (saturated, unsaturated, series n-9, n-6 and n-3) either alone or in combination with araC were not different between normal and tumor cells in culture (data not shown). Table 1 Toxicity of fatty acids or araC alone and in combination on normal (4D/WT) and transformed (D/v-src) rat colonic epithelial cells 1 DHA (n = 3) AA (n = 3) AraC (n = 3) AA-araC (n = 3) DHA-araC (n = 4) IC 50 μM 4D/WT cells 87.8 ± 10.2 91.1 ± 18.4 67.4 ± 11.1 9.3 ± 1.4 39.1 ± 10.4 D/v-src cells 14.9 ± 5.9* 12.2 ± 5.0* 41.8 ± 2.8 1.0 ± 0.6 1.3 ± 0.5* 1 For the drug and fatty acid toxicity assays, cells were cultured in media supplemented with graded concentrations of araC or the fatty acids for 24 hrs. For the fatty acid supplemented drug toxicity assays, cells were treated with 3 μM DHA or AA for 24 hrs. Cells were then cultured in media supplemented with DHA or AA and graded concentrations of araC for an additional 24 hrs. Values are means ± SEM with sample sizes indicated in parenthesis. Values followed by * are significantly different from the values within the same column (p < 0.05). The survival curves in araC treated cultures with or without fatty acid supplementation are shown in Figure 1 . The survival rates were not different between the normal and the transformed cells at very low araC concentrations (0.1 and 0.2 μM). At 0.4 μM araC the surviving fraction of normal cells is statistically higher for 4D/WT cells in either DHA or AA supplemented media (> 97%) compared to D/v-src cells (<60%). At about 6 μM araC there is no therapeutic benefit of AA treatment given that the level of kill is equivalent between 4D/WT and D/v-src cells at this point in the dose response curve. However, in DHA-treated 4D/WT cells, the survival curves were statistically higher than D/v-src cells at all araC concentrations and never dropped below 50% over the range of concentrations used in this experiment. Figure 1 Survival curves for cells cultured in media supplemented with DHA or AA and treated with graded concentrations of araC. 4D/WT and D/v-src cells were cultured in media supplemented with 3 μM DHA or AA for 24 h. Fresh media was added to include both fatty acids and graded levels of araC for an additional 24 h. Cell viability was determined by the SRB dye binding assay by comparing to control cells not treated with fatty acid or araC. Values are means ± SEM of 3 – 4 separate experiments. Significant differences were detected between tumor and normal cells at all concentrations of drug tested up to 10 μM araC, beyond which only normal cells co-supplemented with DHA grew significantly better than all other cell cultures. DHA and AA supplemented tumor cell cultures were not statistically different from each other at any araC concentration tested. DHA and AA supplemented normal cells were not significantly different at 0.1, 0.2 and 3 μM araC. AraC was significantly more toxic to the AA supplemented normal cells compared to DHA supplemented normal cells at all other concentrations of drug. The effect of araC on the morphology of 4D/WT and D/v-src cells treated with or without DHA or AA is shown in Figure 2 . AraC at 10 μM, killed only a few 4D/WT cells growing in 10% FBS containing medium (panel A). More 4D/WT cells were killed when cultures were supplemented with DHA (panel C), however less than half of the cells survived when AA replaced DHA in the araC treated cultures (panel B). In contrast, 2 μM araC caused potent killing in D/v-src cultures supplemented with either AA (panel E) or DHA (panel F) compared to araC only treated cultures (panel D). Figure 2 The effect of araC on the morphology of 4D/WT and D/v-src cells treated with or without 3 μM AA or DHA. 4D/WT cells ( A , B , C ) or D/v-src cells ( D , E , F ) were cultured in medium supplemented with 3 μM AA ( B , E ) or DHA ( C , F ) or a medium without the fatty acid supplementation ( A , D ) for 24 h. Cells were then treated with either 10 μM (4D/WT cells, A , B , C ) or 2 μM (D/v-src cells, D , E , F ) araC for an additional 24 h. Dead cells are indicated by the arrow signs. All photographs are at 200× magnification. The expression of the PKC isozymes α, δ, ε and ζ in 4D/Wt and D/v-src cells treated with or without araC or the fatty acids or a combination of the fatty acids and araC are shown in Figure 3 . PKC δ expression was higher in normal colonic epithelial cells than in the transformed variant. Treatment with araC decreased PKC δ expression in the D/v-src cells in both FBS only and DHA/AA supplemented conditions. Less PKC ε was detected in the AA supplemented D/v-src compared to all other treatments. Densitometry and statistical analysis revealed an interaction between araC and fatty acid treatment in both the 4D/WT and D/v-src cells. In the expression patterns of PKC isoforms ε and ζ, the expression of both PKC isoforms was decreased by araC only when DHA was supplemented. There was no statistically significant change in PKC α expression under any treatment conditions. Figure 3 PKC isoform expression as determined by Western blot. 4D/WT and D/v-src cells were cultured in media supplemented with 3 μM DHA or AA for 24 h. Fresh media with/without the fatty acids and 10μM araC was added. Cell lysates containing 30 μg protein/lane were loaded and separated on 10% SDS-PAGE gels. Gels were transferred and probed as described in the Materials and Methods. Densitometric values are means ± SEM of 3 – 4 separated experiments. Bars with different letters within the same PKC isoform and cell type are significantly different (Fisher's protected LSD test). Discussion There is considerable evidence that nutrition and lifestyle factors have major impact on the early stages of carcinogenesis in the colon and other tissues. What is less clear is the role, if any, for nutritional supplementation as part of an adjuvant therapeutic strategy in colon cancer treatment. Given that colon cancer is particularly resistant to existing chemotherapeutic drugs, and at best an increase of 20% in five-year survival rate is achieved with combination 5-fluorouracil (5-FU) and levamisole/leucovorin, improvements in drug toxicity/selectivity would certainly be helpful. Nucleoside chemotherapeutic drugs including araC, gemcitabine and 5-FU all have intracellular targets and interfere with the metabolism of normal nucleosides to disrupt cellular growth. Tissues with high turnover rates such as bone marrow and intestinal mucosa are particular sensitive to those drugs. The present study demonstrated that low dose DHA substantially enhanced araC's ability to kill tumorigenic colonic epithelial cells while only marginally increasing toxicity toward the normal mucosal cell line. While AA also had sensitizing activity, it was much less selective than was DHA. While the concentration of DHA in normal FBS, or in the blood of individuals consuming habitual North American diets is very low, we and others have shown that levels as high as 100 μM can be achieved in blood plasma through dietary supplementation of mouse diets [ 9 ]. In several in vitro and in vivo systems we, and others, have demonstrated that long chain polyunsaturated fatty acids, particularly those found in fish oils, can enhance tumor kill by chemotherapeutic drugs and protect normal tissues [ 5 , 8 , 13 ]. In leukemia treatment, where araC is often used in combination therapy, the plasma levels reach up to 2 μM during high dose or low dose infusions. The observation here, that distinct differences in toxicity were observed between 0.1 and 1 μM, supports the idea that DHA will improve the clinical efficacy of araC at therapeutically relevant doses. Enterocytes and colonocytes express many PKC isoforms including α, β I , β II , δ, ε, η, θ, ζ, and ι. most of which are activated in the post-mitotic compartment. Colon tumors from both humans and rats tend to show decreased abundance of α, β I , δ, ε, ζ and/or η. Perlletti and co-workers have demonstrated a lower PKC δ expression in rat colonic epithelial cells [ 14 ]. Consistent with those previous reports, we showed significantly lower expression of PKC δ in D/v-src cells as compared to 4D/WT cells. Over-expression of δ in CaCo-2 human colon tumor cells has been shown to decreased growth [ 15 ] and at least partially reverses the malignant phenotype of src transformed rat colonic epithelial cells [ 14 ]. PKC is an important modulator of araC toxicity [ 10 , 16 ] and a global reduction in PKC expression correlates with increased araC cytotoxicity [ 16 ]. Our study showed a potent down-regulation of PKC δ with araC treatment in tumor (CLD/src) but not in normal cells (CLD/WT), which may be related to the higher cytotoxity of araC to the transformed cells, in accordance with the previous report. Chapkin's group has shown that dietary fat type can modulate PKC isozyme expression patterns in the colon [ 17 ]. A recent study by Mirnikjoo [ 18 ] showed that EPA and DHA, but not AA, were active inhibitors of the catalytic domains of several kinases including PKC. Nair and coworkers [ 19 ] showed similar selectivity of n-3 fatty acids to modulate PKC activity and subcellular redistribution in cardiac myocytes. In the present study, PKC ζ expression was decreased by the presence of DHA but not by AA in the normal colon cells with or without araC treatment, suggesting DHA can modify the PKC expression in normal cells. However, our results didn't show a significant effect of DHA on the levels of this PKC isozyme expressed in the transformed cells, suggesting an interaction between the cell type and the effect of DHA on PKC expression. PKC ε content was also influenced by the fatty acids and drug in a complicated way. The decreased expression of ε in the D/v-src cells in the presence of AA was not observed when cells were co-treated with araC. In the 4D/WT cells, on the other hand, AA and DHA alone had no effect on PKCε expression but the DHA-araC combination did down regulate this isozyme. If one looks for correlations between the toxicity profiles and PKC expression profiles, an ability to down regulate PKCε expression in 4D/WT is correlated with lower toxicity of araC. An obvious question is how this might be achieved at a molecular level, particularly since individually DHA and araC had no effect on expression, and AA had no combinatorial effect. Though there does not appear to be a single dominant effect of one PKC isoform over another, it could be the combination pattern of expression of the various PKCs that is critical in determining the final response. For example, the high expression of PKC ε and low expression of PKC δ in D/v-src may be a marker of susceptibility. Further experiments are required to determine the molecular nature of these interacting signalling pathways. Conclusion In summary, the present study demonstrated that low dose DHA or AA supplementation synergistically interacts with araC to achieve therapeutic gain against the colon tumor cells. Because DHA, but not AA showed tumor cell selectivity, this suggests that dietary supplementation protocols including DHA with araC, gemcitabine or other drugs should be tested in pre-clinical models of colon cancer. Methods Cell culture The cell lines used in the present research were a generous gift from Dr. Susan E. Pories, New England Deaconess Hospital and Harvard Medical School. These cells developed by Pories et al [ 20 ], consist of two mycoplasma-negative rat colonic epithelial cell lines and the immortalized nontumorigenic cell line referred to as FRC TEX CL 4D (referred throughout the text as 4D/WT) and the derivative transformed cell line FRC TEX CL D/v-src (referred to as D/v-src), established by transfection of a v-src sequence into the 4D/WT parental line. D/v-src cells exhibit the neoplastic phenotype while 4D/WT do not display anchorage independent growth or form tumors in vivo . 4D/WT and D/v-src cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (FBS), hydrocortisone (0.02 ug/mL), insulin (0.25 ug/mL), transferrin (0.12 ug/mL), polybrene (1 ug/mL) and penicillin-streptomycin (50 U/mL) in a humidified environment at 37°C in the presence of 10% CO 2 . Toxicity assays IC 50 values were determined for 4D/WT and D/v-src cells treated with or without DHA or AA or a combination of araC and the fatty acids by protein binding dye sulphorhodamine B (SRB) assay as we have previously described (5,6). DHA or AA as the free fatty acids were first bound to albumin in serum for 1 h at 37°C before diluting into culture media at 0–500 :M. For the drug (0–1 mM) or fatty acid toxicity assays, approximately 1000 cells/well were plated in 96-well plates and cultured for 24–48 h. Cells were allowed to adhere for a minimum of 6 h before the fatty acid or drug was added. PKC protein expression Sub-confluent monolayers of 4D/WT and D/v-src cells were grown in media supplemented with or without 3 μM DHA or AA in 100mm plates for 24 h. Media was then replaced with fresh media ± fatty acid, and/or ± 10 μM araC and cultured for an additional 24 h. Cells were harvested, washed three times in cold phosphate buffer saline (PBS) and resuspended in a small volume of PBS containing protease inhibitor cocktail (Boehringer Mannheim). Samples were sonicated, 10 μL of lysate recovered for protein assay (Bio-Rad Laboratories, Inc., Hercules, CA), and an equal volume of 2× hot Laemmli sample buffer was added. Proteins (30 μg/well) were separated by SDS-PAGE on 10% acrylamide gels and transferred to nitrocellulose membrane on a semidry transfer apparatus. Membranes were blocked in 5% milk powder in Tris-buffered saline (TBS) and probed with the respective primary monoclonal antibody (Transduction Laboratories, Lexington, KY) at concentration range from 1:500 to 1:1500 dilution in 1% BSA. Membranes were washed and then probed with secondary antibodies conjugated to horseradish peroxidase diluted in 5% milk. Blots were developed using the enhanced chemiluminescence method (Amersham) and density of bands quantified by an imaging densitometer using Northern Eclipse software (Empix Imaging Inc, Missisaga, ON). Statistical analysis Analysis was performed using SPSS general linear model program version 7.5 (SPSS Inc. Chicago, IL). The effect of treatment was determined by one-way ANOVA and Fisher's protected LSD post-hoc test with significance determined to be p < 0.05. For the cell viability analysis, data were log transformed to obtain a normal distribution and plotted and IC 50 values determined using a CurveFit computer software. Authors' contributions MC carried out the PKC protein expression assay and drafted the manuscript. AL carried out the toxicity assay. KM conceived of the study and participated in its coordination. All authors read and approved the final manuscript.

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1079883

Effects of first and second generation antihistamines on muscarinic induced mucus gland cell ion transport

Background The first generation antihistamines, such as diphenhydramine, are fairly potent muscarinic antagonists in addition to being H1 selective antihistamines. The antimuscarinic action is often not desirable since it is in part responsible for the drying of secretions in the airways and the sedative effect. We therefore examined a number of antihistamines for antimuscarinic effects on ion transport by mucus gland cells isolated from the airways of swine. Enzymatically isolated airway mucus gland cells were purified utilizing density gradients and grown in culture on porous inserts (Millicell HA™) at an air interface. Cells grown in this manner maintain phenotype and polarity. Transport of ions, as short-circuit current measured under voltage-clamp, was measured in response to acetylcholine (ACh) or histamine applied to the serosal side of the gland cell layers. Concentration-response relationships for ACh or histamine were generated in the presence and absence of various drugs. The potencies against muscarinic receptor activation were estimated using the dose-ratio method of Schild. Results Three known muscarinic antagonists were used to validate the system. Atropine had a pA 2 of 9.4 ± 0.1 (n = 9). 4-DAMP and methoctramine had pA 2 values of 8.6 ± 0.1 and 5.6 ± 0.1, respectively (n = 12, 11) all consistent with inhibition of an M3 subtype muscarinic receptor. The rank order of potency of the antihistamines against the inhibition of M3 receptors was desloratadine = diphenhydramine > hydroxyzine (pA 2 ; 6.4, 6.2, 4.8, respectively). pA 2 values for fexofenadine, loratadine and cetirizine were not determined since they had no effect on the cholinergic response at the highest drug concentrations tested (10, 10 and 100 μM, respectively). The pA 2 values for the antihistamines against the histamine response could not be calculated, but the estimates of the rank order of potency were estimated to be desloratadine> cetirizine ≈ hydroxyzine > fexofenadine > loratadine > diphenhydramine. Conclusion The rank order of selectivity for histamine receptors over muscarinic receptors was estimated to be cetirizine ≈ fexofenadine > loratadine > desloratadine ≥ hydroxyzine ≥ diphenhydramine.

Background The airways are lined by epithelium and the upper airways have mucus gland acini, all of which contribute to secretion of both water and mucus coating the surface. The epithelium forms a physical barrier to inhaled substances and, actively secretes and absorbs fluid to provide an appropriate thickness hydrated layer on the surface of the airways. The epithelium clears particulates from the airways by ciliary action and ingestion by macrophages. There is a local immune response to inhaled antigens in part through resident macrophages and dendritic cells. Epithelial function is controlled by neurotransmitters (ACh for example) and blood born substances (epinephrine, norepinephrine, hormones) and substances released from inflammatory cells (histamine and other substances from mast cells). Several serious diseases are linked to disfunction of the epithelium such as cystic fibrosis and asthma. Therefore proper functioning of the epithelium is critical for normal lung function. Cholinergic stimulation of muscarinic receptors is known to increase mucus secretion from submucosal gland cells [ 1 , 2 ], fluid transport by submucosal gland cells [ 3 ], and ciliary beat frequency of ciliated epithelium [ 4 - 6 ]. The secretory functions are transient (ion, water and mucus), occurring for several minutes during continuous stimulation of the cells by ACh [ 1 , 7 ]. This synchrony makes sense from a functional standpoint since mucus that is secreted must be hydrated by secretion of fluid. The increase in cilia beat frequency caused by muscarinic receptor activation can then clear the ejected and secreted mucus. Histamine can also stimulate the release of mucus and fluid by submucosal gland cells. The effects of ACh and histamine on short circuit current (a measurement of ion transport and therefore fluid movement) are transient, reflecting the transient nature of the increase in secretion of fluid. Stimulation of these cells by histamine probably does not support normal secretions, but represents mast cell degranulation, typically associated with the symptomatology of a pathological state such as asthma or allergies. The symptoms of airway irritation and hypersecretion are commonly treated with antihistamines except for the case of asthma, where excessive drying of the mucus membranes by first-generation antihistamines is considered a contraindication. The antimuscarinic actions of first generation H1 selective antihistamines are well known [ 8 ]. In fact, some of the therapeutic efficacy of these drugs (e.g., drying of mucus membranes) and side effects (e.g., drowsiness, thickening of mucus, accelerated heart rate) may also be attributable to these actions. Generally, the antimuscarinic actions of the H1 selective antihistamines are undesirable, in particular in people with high blood pressure, arrhythmias or asthma. H1 selective antihistamines devoid of antimuscarinic properties should be useful in the treatment of asthma since mast cell degranulation occurs during the early phase of an asthma attack releasing histamine causing mucus secretion, inflammatory reactions in the airway epithelium, vasodilation in the mucosa and contraction of airway smooth muscle [ 9 ]. Each of these events leads to a narrowing or stiffening of the airways and increased resistance to air flow. Theoretically, H1 selective antihistamines devoid of antimuscarinic properties would decrease the pathological effects of histamine without altering the normal control of the cells in the airway by ACh at muscarinic, M3, receptors. Yanni et al. [ 10 ] suggested that an antihistamine, effective at both H1 and H2 receptors, lacking antimuscarinic actions, would be useful in the treatment of asthma. Lee et al. [ 11 ] proposed that selective H1 antihistamines could be useful in the treatment of mild to moderate asthma and have additive effects with leukotriene antagonists. Therefore, the following experiments were performed to determine the antimuscarinic actions of several antihistamines on muscarinic receptor induced increases in ion transport by mucus gland cell epithelium grown in culture or porous bottom inserts as measured using Ussing chamber methodology. Results Antimuscarinic effects After two days in culture the mucus gland cells have formed confluent cell layers on the Millicell inserts and current flow can be measured across the inserts. In Figure 1A the short circuit currents (ΔI sc ) evoked by cumulative addition of ACh are shown. The currents were used to generate concentration-response relationships for agonists on changes in ΔI sc (Fig 1B ). The responses to both ACh and histamine are phasic, particularly obvious at higher concentrations of agonist (>10 -5 M). The maximum increase in current was 19 ± 1 μA/cm 2 (n = 21) for histamine and 15.5 ± 1 μA/cm 2 (n = 32) for ACh. Also, illustrated in this figure is the basic protocol used for all experiments. 10 μM amiloride is added to the mucosal side of the mucus gland cells to block fluid reabsorption. A small decrease in basal current occurs indicative of the reabsorption of sodium and fluid by the monolayer. The addition of the test drug to the serosal chamber, in this case the prototypical muscarinic antagonist atropine, causes little change in baseline current. After five minutes ACh is added cumulatively to the serosal side of the cells. The concentrations are indicated at the arrows. ACh was added at 5 to 6 second intervals into each of the 4–6 Ussing chambers being used during an experiment. Thus, the apparent "shift to the right" that occurs in the response in the presence of atropine in this figure is indicative of inhibition of the muscarinic receptors. The parallel shifts in the concentration-response relationships to the right for ACh caused by atropine are illustrated in Fig 1B for averaged peak data at each agonist concentration normalized to the maximal response for each insert. The method of Schild was used to determine the apparent K i for atropine as shown in Fig 2 . The slope of the line through the data points is set to 1 (and not significantly different from 1) with the X intercept giving an estimate of the pA 2 (-log K i ). The pA 2 for atropine (determined by this method) was 9.4 ± 0.1(n = 9). Subtype selective inhibitors of muscarinic receptors were used to suggest which subtype of muscarinic receptor was being activated by ACh to induce the increases in ΔI sc . 4-Diphenylacetoxy-N-methylpiperidine (4-DAMP) is a relatively selective antagonist for M3 muscarinic receptors and methoctramine is a relatively selective antagonist for m2 muscarinic receptors. The pA 2 for these drugs was: 4-DAMP 8.6 ± 0.1(n = 12); Methoctramine 5.6 ± 0.1(n = 11). These values are consistent with M3 receptor inhibition [ 12 ]. Therefore, the inhibitory constants given for antihistamine block of muscarinic receptors are considered to be against the M3 subtype in the following experiments. Figure 1 A . This is a recording from a typical experiment to measure short-circuit current (ΔI sc ) across the confluent epithelial monolayers grown in Millicell inserts. Short circuit current (ΔI sc ) is measured under voltage-clamp that maintains the potential difference across the epithelium at 0 mV. 10 μM amiloride was added in all experiments to the lumen side of the inserts to block absorption, which caused a decrease in the basal current as shown in Fig. 1A. In this recording, five minutes prior to cumulative addition of ACh, 3 inserts were exposed serosally to 10 -9 , 3 × 10 -9 , or 10 -8 M atropine (red, green, and blue traces respectively). The response to ACh reaches a peak and then declines. The difference of peak response to baseline line at each concentration of agonist was measured as the response to each concentration of ACh (n = 9) B . Concentration-response relationships were generated as the peak response to each concentration of ACh (normalized to the maximal response of the epithelium to ACh) plotted versus the ACh concentration. Atropine causes parallel shifts to the right in the concentration-response relationships with increases in atropine concentration. Average data from 9 experiments. Data were then used to generate Schild plots shown in the next figure. Figure 2 Schild analysis of the concentration response data for the muscarinic antagonists atropine, 4-DAMP, and methoctramine. The Log (DR-1) versus log inhibitor concentration is plotted in this figure. DR is the dose ratio, the ratio of concentrations of ACh giving equal responses (EC50 for this plot) in the absence and presence of test compound. The slope of the lines drawn through the data shown is 1. The X-intercepts of the lines equal pA 2 values, which are equal to the -log K i values, where K i is the affinity of the antagonist. Schild plots are shown for atropine (red) a nonselective muscarinic antagonist (n = 9), 4-DAMP (green, n = 12), M3 selective, and methoctramine (black), m2 selective (n = 11). Six antihistamines were tested for inhibition of the muscarinic receptor response in gland cells. The antihistamines are: desloratadine, diphenhydramine, hydroxyzine, loratadine, cetirizine, and fexofenadine. Each was examined using the protocol described for atropine. In addition, the antihistaminic activity of each compound was confirmed by the ability of these drugs to block histamine-induced increases in ΔI sc . In figure 3A the effects of desloratadine on the ΔI sc induced by ACh are shown. As with atropine there is a shift to the right in the concentration-response relationships (Fig 3B ) that, when plotted using the dose-ratio method of Schild, yields a linear plot with slope of one (Fig 3C ) and an estimated pA 2 of 6.4 ± 0.1(n = 13). Figure 3 A . The effects of desloratadine on gland cell responses to ACh are shown here. ΔI sc in response to ACh are inhibited by desloratadine as shown by a shift to higher concentrations of ACh in the presence of increasing concentrations of desloratadine. B . The concentration response relationships derived from data in A. desloratadine causes a parallel shift to the right in the concentration response relationships for ACh. C . Schild analysis of these data and those for diphenhydramine and hydroxyzine are presented. The Schild plot for diphenhydramine and hydroxyzine is also shown in Fig 3C . Their pA 2 values are 6.2 ± 0.1(15) and 4.8 ± 0.1(11), respectively. Hydroxyzine is much less potent antagonist at muscarinic receptors than desloratadine and diphenhydramine (p < 0.01). The active metabolite of hydroxyzine, cetirizine, has no effects on muscarinic receptor activation (Fig 4A, 4B ) even at very high concentrations (10 -4 M). The precursor of desloratadine, loratadine, had no antimuscarinic action (data not shown). Fexofenadine had no antimuscarinic action at concentrations up to 10 μM (Fig 4C, 4D ). A compilation of the pA 2 values is presented in Table 1 . Figure 4 A and C show that both cetirizine and fexofenadine had no effect on the response of gland cells to ACh. B and D : Concentration-response data generated from the peak increase in ΔI sc induced by ACh. Note the lack of effect of cetirizine and fexofenadine. Table 1 Drug a Muscarinic inhibition (pA 2 ) N H1 receptor inhibition (~1/2 max-μM) N Atropine 9.4 ± 0.1 9 ND Methoctramine 5.6 ± 0.1 11 ND 4-DAMP 8.6 ± 0.1 12 ND Diphenhydramine 6.2 ± 0.1 15 0.3 12 Desloratadine 6.4 ± 0.1 13 0.02 15 Hydroxyzine 4.8 ± 0.1 11 0.14 12 Cetirizine No effect (at 100 μM) 13 0.12 14 Loratadine No effect (at 30 μM) 3 12 8 Fexofenadine No effect (at 10 μM) 3 0.25 11 a- all estimates for K i are statistically different (p < 0.001) except diphenhydramine and desloratadine (p = 0.135) ND: Not done Antihistaminic effects We did not perform Schild analysis on the antihistaminic effects of the antihistamines because the underlying assumptions were not met. The basic underlying assumptions of Schild analysis are: 1) the inhibition of a response by the antagonist is competitive and 2) that during the response, equilibrium of the antagonist and agonist for the receptor is reached. While these requirements seem to be met at least tacitly for muscarinic receptors, they do not appear to be met for antagonism of the effects of histamine. Fig 5A shows the ΔI sc response to histamine. The ΔI sc response declines rapidly after reaching a peak, even at low concentrations of histamine. This provides little time for equilibrium conditions to be met during the response. In the case of diphenhydramine there is a shift to the right in the concentration-response relationships for histamine with little decrease in maximum response, as shown in Fig 5A and inset. The inset shows the resulting Schild plot with an estimated pA 2 of 6.5 ± 0.2 (n = 12) when the slope the line shown in the inset was set to1. However, the least squares fit to these data had a slope of 1.6 (statistically different from 1 (p < 0.05) and was curvilinear with estimated pA2 of 10.8 ± 2.4. This finding suggested that the underlying assumptions of the Schild analysis were not met. This is more clearly illustrated for the case of fexofenadine as shown in Fig 5B . Fexofenadine causes a decrease in the maximum response to histamine and a shift to the right in the concentration response relationship. Using data such as shown in Fig 5B inset for inhibition of peak response, we computed the "% of maximal control ΔI sc " response (control inserts were run simultaneously with each drug treated insert) for each of the antihistamines; hydroxyzine, cetirizine, fexofenadine, loratadine and desloratadine. A graph of these data is shown in Fig 6 . Desloratadine is the most potent antihistamine in this group (50% reduction in peak ΔI sc at approximately 2 × 10 -8 M) and loratadine the least potent (50% reduction in peak ΔI sc at 10 -5 M). Table 1 gives a compilation of the estimates of potency for all compounds tested. Figure 5 A . The effects of diphenhydramine on the response of gland cell epithelium to histamine. The responses to histamine, like those to ACh, are transient. However, the response to histamine decays more rapidly than that for ACh. Note that diphenhydramine causes increasing inhibition with increasing concentration of inhibitor ( A and inset A.1 ) without a decrease in maximum response of the histamine action. The range of concentrations at which inhibition occurs is similar to those that inhibited the ACh response (n = 9). Inset A.2 shows the Schild plot generated from diphenhydramine data. Note that when the slope of the line (red) shown in the inset was set to1, the calculated pA 2 is 6.5 ± 0.2. However the least squares fit to these data had a slope of 1.6 (blue line) with estimated X intercept of -10.8 ± 2.4, suggesting that the underlying assumptions of the Schild analysis were not met. B . The effects of fexofenadine on gland cell responses to histamine. ΔI sc in response to histamine are blocked by fexofenadine. Fexofenadine decreased the maximum ΔI sc and caused a small shift to the right. Inset B shows the concentration response relationships derived from these data (n = 8). Figure 6 This figure shows the inhibition of histamine induced short circuit current (ΔI sc ) by different antihistamines at various concentrations. These antihistamines concentration- dependently inhibited maximum responses induced by histamine, the order of apparent efficiencies of histamine blockade were desloratadine > cetirizine ≈ hydroxyzine > fexofenadine > loratadine. Discussion In order to test the validity of using short-circuit current responses for the measurement of pA2 values for inhibition of muscarinic receptors, we first examined the effect of classical muscarinic receptor inhibitors on the ACh-induced currents. As shown in Table 1 the pA 2 estimated for atropine was 9.4. This value is in good agreement with estimates from many other tissues (range 8.9–9.8, [ 12 ]. The pA 2 values suggest that the muscarinic receptor primarily responsible for the increase in short-circuit current is the M3 subtype since the values estimated for methoctramine and 4-DAMP were 5.6 and 8.6, respectively. The rank order of these values is similar to those given by Caufield and Birdsall [ 12 ] for inhibition of M1 and M3 receptors, although closer to the ranges given for M3 than M1. We have previously demonstrated two receptor subtypes in submucosal gland cells, which at the time were designated M1 and M2 G [ 13 ], corresponding to the current designations of M1 and M3. Culp et al. [ 14 ] concluded that both M1 and M3 receptors were capable of inducing secretory responses in mucus glands cells from sublingual glands and that M3 receptors were sufficient to give a maximal response. We suggest that the increase in short-circuit current is predominantly driven by M3 receptor activation, however these data do not rule out a role for the M1 subtype. These data do demonstrate the validity of this preparation as a model system for determination of the inhibition of muscarinic activity by drugs. Fexofenadine, loratadine and cetirizine had no effect on the concentration response relationships for ACh demonstrating a lack of interaction with M3 receptors. Handley et al., [ 15 ] also found that loratadine did not bind to muscarinic receptors. All other compounds tested in our experiments competitively inhibited the increase in short-circuit current caused by ACh. Our estimate of the K i for hydroxyzine is 15 μM. Kubo et al. [ 16 ] found that hydroxyzine had a K i of 3.8 μM against muscarinic receptors in the cerebral cortex using radioligand binding assays. The estimated K i for desloratadine and diphenhydramine inhibition of muscarinic receptors were not statistically different (p=.135) with estimated K i of in the range of 0.3 to 0.6 μM. Kubo et al. [ 16 ] measured a K i for diphenhydramine against QNB binding in the cerebral cortex, indicative of all muscarinic receptor subtype binding, of 0.28 μM similar to our value of 0.6 μM for M3 muscarinic receptor induced secretion from mucus gland cells. Cardelus et al. [ 17 ] estimated a similar potency for desloratadine of approximately 0.2 μM (pA 2 = 6.7 ± 0.1) against muscarinic-induced contraction of rabbit iris. By contrast, estimates of affinity using the dose-ratio method for the inhibition of the histamine receptor were not done except for diphenhydramine. The underlying assumptions of Schild analysis were clearly not met. Two of these assumptions are: 1) the inhibition of a response by the antagonist is competitive and 2) that during the response, equilibrium of the antagonist and agonist with the receptor is reached. These requirements are at least tacitly met for antihistamine binding to muscarinic receptors and one or both are clearly not met in the case of histamine receptors. The antagonists are known to bind competitively with histamine receptors as shown in receptor binding assays [ 16 , 18 ]. Therefore, it seems reasonable that the second condition is not met. As shown for the ΔI sc response to histamine in Fig 5B , the responses decline rapidly after reaching a peak (within ~15 minutes at the highest concentration the response is near baseline). The decline occurs even at low concentrations of histamine. At least quasi-equilibrium conditions need to be established within a very few minutes. Anthes et al. [ 18 ] demonstrated that for desloratadine the off-rate for binding was quite slow with only 37% of the desloratadine released from the receptor in 6 hours. Christophe et al. [ 19 ] determined the t1/2 for dissociation of cetirizine from the H1 receptor to be 142 min. The slow rate of dissociation of the antihistamine from the receptor resulted in a decreased maximum response, with little apparent shift in the concentration response relationship. This is reminiscent of non-competitive inhibition, since equilibrium cannot be reached during the transient response. Thus, the calculation of pA 2 , using the method of Schild, is not valid for antihistamine inhibition of histamine-induced increases in short-circuit current. This conclusion was also drawn by Miller et al. [ 20 ] for inhibition of the initiation of calcium transients. However, qualitatively the potencies of the antagonists can be estimated from the concentration required to reduce the peak response by 50%, as shown in Fig 5B . The rank order of potency using this method is: desloratadine > cetirizine ≈ hydroxyzine > fexofenadine > diphenhydramine > loratadine. This differs from the rank order of affinities determined by Anthes et al. [ 18 ] using radioligand binding assays: desloratadine > diphenhydramine > hydroxyzine > cetirizine > loratadine (K b (nM):0.9, 2.5, 10, 47, 138, respectively). Our estimates of potency are 10 to 100 times higher than the binding constants reflecting the differences in methodology. The difference in the rank order of potency primarily is due to our estimate of the potency of diphenhydramine using the dose-ratio method. We estimated a pA 2 for diphenhydramine of 6.5 (300 nM) much higher than the K i estimates of 2.5–14 nM by others [ 16 , 18 ]. This suggests that even in the case of diphenhydramine equilibrium conditions are not met. It should be noted that the estimate of pA 2 derived in this report assumed a slope of 1 for the Schild plot. The slope by least square fit to the data was 1.6 (p > 0.05 compared to unity) and curvilinear. Therefore, diphenhydramine inhibition is also non-equilibrium, and we expect that the actual pA 2 for diphenhydramine is in the range of 10.8 ± 2.4 (35 pM ~3.9 nM). This conclusion was also drawn by Miller et al. [ 20 ]. They found that diphenhydramine inhibited the histamine-induced transient calcium response in an apparent non-competitive manner. Our findings suggest that of the antihistamines tested, the rank order of selectivity for histamine over muscarinic receptors is: cetirizine ≈ fexofenadine > loratadine > desloratadine > hydroxyzine ≥ diphenhydramine. This was derived from the ratio of the estimated potencies of muscarinic inhibition and histaminergic inhibition. Since fexofenadine, cetirizine and loratadine did not affect the muscarinic response they were assumed to be the most selective with cetirizine having the higher potency toward histamine receptors. Conclusion It is likely that antihistamines with significant antimuscarinic effects in this assay might show some antimuscarinic actions in vivo. However, the antimuscarinic actions probably will occur early after dosing and be short lived, since as the plasma levels of the drugs decrease muscarinic inhibition would readily reverse, compared with the comparatively slow release of antihistamines from the histamine receptors. This would be true for most second generation antihistamines and hydroxyzine, but would not be true for diphenhydramine. This is born out by the well known antimuscarinic action of diphenhydramine. Thus, of the agents tested, those with the least antimuscarinic action such as fexofenadine and cetirizine, may be the most useful for treatment of allergic rhinitis and possibly as an adjunct drug in the treatment of asthma. Methods Isolation and culture of gland cells Tracheal submucosal gland cells were isolated from the tracheal epithelium of Yorkshire white male swine (30–40 kg) were. Animals were euthanized by exsanguination after anesthesia with 5% isoflurane. The epithelium was removed from the airway and gland cells were enzymatically dissociated and isolated on discontinuous Percoll ® gradients by the methods of Yang et al. [ 13 ] and Chan and Farley [ 21 ] with little modification. The cells were plated on Millicell ® inserts (0.04 μm pore size, 0.6 cm 2 area) coated with human placental collagen (20 μg/cm 2 ) at a density of ~10 6 cells per insert in PC-1 medium. After one day in culture the medium from within the insert was removed and the cells were grown at an air interface [ 22 , 23 ]. As we have shown before after two days in culture the epithelium had become confluent. Experiments were performed after 3–5 days in culture. Measurement of short-circuit current Inserts were mounted in Ussing chambers (Costar) modified to accept the Millicell inserts. The chambers were maintained at 37°C and continuously bubbled with 95% O 2 /5% CO 2 . The bubbling also served to drive bubble-lift circulation that quickly mixed drugs after addition to the serosal or mucosal chambers, each having a volume of 8 ml. The transepithelial short circuit current was measured using either VCC600 or VCC MC2 voltage clamp amplifiers (Physiologic Instruments) connected to the chambers via salt bridges and silver/silver chloride pellet electrodes. 10 μM amiloride was added to the mucosal solution in all experiments to inhibit sodium absorption. All other compounds (ACh, histamine, antagonists) were added to the serosal solution cumulatively from concentrated stock solutions (at least 1000× concentrated). The increases in short circuit current in response to ACh or histamine were measured as the peak currents obtained after addition of agonist at each concentration, subtracted from the baseline current measured prior to the addition of the lowest concentration of agonist. The currents were normalized to the area of the insert. Concentration response curves were generated for each insert and an EC50 determined for each insert by fitting the data with a logistic equation using Origin (Originlab). These data were then used to calculate "dose ratio – 1" values for use in a Schild plot [ 24 ] as discussed below. Solutions and drugs Hepes-buffered physiological saline was used for transport, during dissection of trachea, and dissociation of cells. It contained (in mM): 140 NaCl, 5 KCl, 2 CaCl 2 , 10 Hepes; pH 7.4. A Krebs-Henseleit solution was used in all experiments in the Ussing chamber having the following composition (in mM): NaCl, 113; KCl, 4.8; CaCl 2 , 2.5; NaHCO 3 , 18; KH 2 PO 4 , 1.2; MgSO 4 , 1.2; glucose, 5.5; and mannitol, 30, pH adjusted to 7.4. This solution was bubbled with 95/5% O 2 /CO 2 to give a pH of 7.4. Test compounds and agonists were dissolved in Krebs-Henseleit solution or DMSO. Equivalent volumes (never greater than 0.1%) of DMSO added in control experiments were without effect. All chemicals and drugs were obtained from Sigma Chemical Corporation (St. Louis, MO) except fexofenadine, desloratadine, loratadine and cetirizine, the kind gift of Aventis Pharmaceutical Corporation. Data analysis Cumulative concentration-response relationships were generated by measuring the peak to baseline increases in short-circuit current induced after each concentration of ACh or histamine was added cumulatively to the serosal bath. All data are expressed as mean ± SEM. Data were normalized to the maximum peak current obtained for each insert with the particular agonist being used. Only one concentration-response relationship was generated for each insert. EC 50 values for each agonist were determined by fitting each individual data set with a logistic function using the fitting functions in Origin™ (OriginLab). These values were then used to estimate the shift of the concentration-response relationships for ACh, by the various antagonists used. The shifts in these relationships were plotted as log (dose ratio-1) versus -log (inhibitor concentration) according to the method of Arunlakshana and Schild [ 24 ]. This should yield a plot with a slope of 1 and an X-intercept equal to pA 2 , an estimate of the antagonist affinity for the particular receptor being activated. Slopes not significantly different from unity were found for the inhibition of muscarinic receptors by all compounds. This was not true for the inhibition of histamine receptors. Quasi-equilibrium conditions must be met during the response if the dose-ratio method is to be used. These conditions are most likely never met for binding of antihistamines with the histamine receptor due to the transient nature of the response in comparison to the slow off rate for unbinding of antihistamines such as desloratadine [ 18 ]. An estimate of relative potency of the antihistamines for the histamine receptor was determined from the concentration of drug causing 50% reduction in the maximum current compared with control inserts. Authors' contributions HL carried out the Ussing chamber studies and data analysis in partial fulfillment of requirements for a doctoral degree. JMF was involved in project conception, experimental design, data analysis and manuscript preparation.

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1079884

Effect of testosterone replacement or duration of castration on baroreflex bradycardia in conscious rats

Background In this study, we tested the hypothesis that 17β-estradiol contributes to testosterone-mediated restoration of baroreflex-mediated bradycardia in short-term (3 weeks) castrated rats. Further, a reported increase in serum testosterone after long-term (6 weeks) castration constituted a basis for testing the hypothesis that a spontaneous increase in serum testosterone or androstenedione in this model causes a commensurate increase in baroreflex-mediated bradycardia. Results Testosterone (1 week) replacement enhanced baroreflex-mediated bradycardia in short-term castrated rats without changing 17β-estradiol level. A spontaneous recovery of baroreflex-mediated bradycardia occurred following long-term castration, although circulating testosterone and androstenedione remained suppressed. Conclusion The data suggest: 1) 17β-Estradiol does not contribute to testosterone restoration of the baroreflex-mediated bradycardia in short-term castrated rats. 2) The long-term modulation of baroreflex-mediated bradycardia occurs independent of androgens, or the baroreflex mechanism may become adapted to low levels of circulating androgens.

Background Short-term castration attenuates the vagal component of baroreflex sensitivity in sexually mature male rats [ 1 , 2 ]. Factors influencing baroreflex sensitivity include gender, age, baseline mean arterial pressure and heart rate, anesthesia, and the method of drug administration [ 3 - 9 ]. However, these factors cannot account for the difference in baroreflex sensitivity between intact and castrated rats in reported studies [ 1 , 2 ]. El-Mas et al. [ 1 , 2 ] have shown that testosterone replacement restores baroreflex-mediated bradycardia in short-term castrated rats. Further, we and others [ 4 , 10 ] have demonstrated that 17β-estradiol enhances baroreflex sensitivity. Notably, 17β-estradiol is a product of testosterone aromatization by the aromatase enzyme [ 11 ], which raises its possible contribution to testosterone effects on baroreflex sensitivity. This latter possibility has not been investigated. Further, Ando et al. [ 12 ] demonstrated a reflex increase in circulating testosterone of adrenocortical origin subsequent to long-term (6 weeks) castration. The impact of such an increase in serum testosterone on baroreflex sensitivity is not known. Hence, the main objective of this study was to test the hypotheses that (i) 17β-estradiol, contributes to testosterone restoration of the vagal component of baroreflex sensitivity in short-term (3 weeks) castrated rats, and (ii) the spontaneous increase in serum testosterone, or androstenedione, reported by others [ 12 ] in long-term (6 weeks) castrated rats results in proportionate increase in baroreflex sensitivity. This goal was achieved by determining whether a) Testosterone replacement (1 week) restores the attenuated baroreflex sensitivity in short-term castrated rats, b) Serum 17β-estradiol increases in testosterone-treated rats, and c) An increase in baroreflex sensitivity occurs in long-term castrated rats along with an increase in serum testosterone or androstenedione. These studies were undertaken in conscious unrestrained rats to avoid the confounding effects of anesthetics on the measured variables [ 3 ]. Results Effect of testosterone replacement on baroreceptor reflex control of heart rate Baseline mean arterial pressure values, measured on the day of the experiment, were similar between the orchiectomized/testosterone and orchiectomized/vehicle rats (Table 1 ). However, the orchiectomized/testosterone rats exhibited a significantly (P < 0.05) lower baseline heart rate compared with the orchiectomized/vehicle rats (Table 1 ). Phenylephrine elicited similar rises in mean arterial pressure in all groups (data not shown). However, the similar increments in mean arterial pressure were accompanied by greater reflex bradycardic responses in the orchiectomized/testosterone compared with the orchiectomized/vehicle rats (data not shown). Therefore, baroreflex-mediated bradycardia was significantly enhanced in orchiectomized/testosterone compared with the orchiectomized/vehicle rats (-1.82 ± 0.15 vs. -1.43 ± 0.1 beats min -1 mmHg -1 ; P < 0.05, Figure 1C ). In addition, serum testosterone increased to within physiological levels in orchiectomized/testosterone rats (P < 0.001, Figure 1A ). There was no change in serum 17β-estradiol in orchiectomized/testosterone compared with orchiectomized/vehicle rats (Figure 1B ). Table 1 Baseline mean arterial pressure (MAP) and heart rate (HR) of orchiectomized/testosterone (O/T), orchiectomized/vehicle (O/V), long-term castrated (L.T.C.), and sham-operated (SO) rats. The rats were allowed to acclimatize to laboratory conditions for at least 2 h prior to experimentation and a period of at least 30 min was allowed after connecting the rat to the pressure transducer for stabilization of blood pressure and heart rate. a P < 0.05 vs orchiectomized/vehicle. Group n MAP (mmHg) HR (beats/min) O/T 21 108 ± 2.6 392 ± 6 a O/V 14 105 ± 1.7 427 ± 10 L.T.C. 12 109 ± 4.1 414 ± 9 S.O. 5 102 ± 1.8 421 ± 14 Figure 1 Effect of testosterone depletion and replacement on baroreflex sensitivity and serum hormone levels . Serum testosterone (A) and 17β-estradiol (B) level and baroreflex sensitivity (C) in conscious unrestrained male rats following orchiectomy (3 weeks) and 1-week treatment with testosterone or vehicle. Data are means ± SEM. Numbers in parentheses are number of observations. Orchiectomized/testosterone (O/T), orchiectomized/vehicle (O/V). * P < 0.05, *** P < 0.001 vs. orchiectomized/vehicle. Effect of long-term castration on baroreceptor reflex control of heart rate Baseline mean arterial pressure and heart rate values, measured on the day of the experiment, were similar between the sham-operated and long-term castrated groups of rats (Table 1 ). Phenylephrine elicited similar rises in mean arterial pressure in all groups (data not shown). In addition, at any given rise in blood pressure, the reflex bradycardic response was similar in long-term castrated rats to that of the sham-operated rats (data not shown). Therefore, baroreflex-mediated bradycardia was restored compared with the sham-operated rats (-2.20 ± 0.35 vs. -2.29 ± 0.24 beats min -1 mmHg -1 ; P > 0.05, Figure 2C ). Finally, the restoration of baroreflex sensitivity subsequent to long-term castration occurred in the presence of significantly (P < 0.001) lower circulating levels of testosterone and androstenedione compared with the sham-operated rats (Figures 2A and 2B ). Comparison of the data obtained from short-term vs. long-term castrated rats revealed that the baroreflex-mediated bradycardia underwent a phase of inhibition at the 3 rd week, but such inhibition was no longer evident by the 6 th week after castration (Figure 2C ). Notably, serum testosterone depletion, observed 3 weeks after castration, continued to be evident 6 weeks after castration (Figure 2A ). Therefore, no correlation was observed between baroreflex sensitivity and serum testosterone (r = 0.2, P > 0.05). Figure 2 Effect of long-term castration on baroreflex sensitivity and serum hormone levels. Serum testosterone (A) and androstenedione (B) level and baroreflex sensitivity (C) in conscious unrestrained rats 6 weeks after castration (long-term castration or L.T.C.) or sham-operation (SO). Data are means ± SEM. Numbers in parentheses are number of observations. *** P < 0.001 vs. sham-operated. Discussion The current study presents 3 new findings. First, testosterone replacement (1 week) restores baroreflex sensitivity (baroreflex-mediated bradycardia) to control level, but 17β-estradiol does not contribute to this action in short-term (3 weeks) castrated rats. Second, baroreflex-mediated bradycardia spontaneously recovers to sham-operation level subsequent to long-term (6 weeks) castration, which suggests that the restoration of baroreflex-mediated bradycardia is time-dependent. Third, the spontaneous recovery of baroreflex sensitivity occurs in spite of significantly suppressed circulating levels of testosterone and androstenedione. This suggests that the long-term modulation of baroreflex sensitivity occurs independent of androgens, or the baroreflex mechanism may become adapted to low levels of circulating androgen. In the present study we investigated the possibility that 17β-estradiol derived by the aromatization of testosterone [ 11 ] contributed to the testosterone-mediated increase in baroreflex-mediated bradycardia recently reported by El-Mas et al. [ 1 , 2 ]. Because Saleh and Connell [ 10 ] have shown that 17β-estradiol enhances the baroreflex-mediated bradycardia of male rats, we reasoned that testosterone enhancement of baroreflex sensitivity in castrated rats, might be secondary to the conversion of testosterone to 17β-estradiol. As previously shown [ 1 , 2 ] we demonstrated that testosterone replacement, which resulted in physiological levels of plasma testosterone, increased baroreflex-mediated bradycardia. Results of the present study showed that serum 17β-estradiol level did not change in testosterone-treated rats, which suggests that 17β-estradiol does not contribute to the action of testosterone on baroreflex control of heart rate following testosterone replacement. Hence, testosterone or another androgenic metabolite(s) seems to mediate the enhanced baroreflex response in short-term castrated rats. This notion is consistent with our preliminary finding that demonstrates the importance of the androgen receptor in the enhancement of baroreflex sensitivity in male rats [ 16 ]. The enhanced baroreflex-mediated bradycardia by testosterone replacement observed in this study and by others [ 1 , 2 ] together with the compensatory reflex increase in serum testosterone by the adrenal cortex, subsequent to long-term (6 weeks) castration [ 12 ], raised the interesting possibility that such an increase in endogenous testosterone may result in a proportionate increase in baroreflex-mediated bradycardia. Indeed, baroreflex-mediated bradycardia spontaneously recovered after long-term (6 weeks) castration to the sham-operation level. This finding supported our hypothesis and suggests that the restoration of baroreflex sensitivity after castration is time-dependent. Contrary to our expectation and in disagreement with reported findings in a similar animal model [ 12 ], serum testosterone remained significantly suppressed in our animals. The discrepancy between both studies may be attributed to the antibody used by Ando et al. [ 12 ] to measure serum testosterone, which exhibited 40% cross-reactivity with dihydrotestosterone (a major product of testosterone) vs. 4% in our study. Nonetheless, the spontaneous and complete recovery of baroreflex-mediated bradycardia in the presence of significantly suppressed serum testosterone raised the following possibilities. Androgens other than testosterone (particularly androstenedione) may have enhanced baroreflex-mediated bradycardia subsequent to long-term castration (6 weeks), because (i) serum androstenedione increased to pre-castration levels 6 weeks after castration [ 12 ], and (ii) Labrie et al. [ 16 ] demonstrated that flutamide, a competitive nonsteroidal antiandrogen, which attenuated baroreflex-mediated bradycardia in our preliminary study [ 17 ], antagonized the effect of androstenedione at the androgen receptor. Since we observed no change in serum androstenedione subsequent to long-term castration, which again disagrees with reported findings [ 12 ], the present findings suggest that androstenedione, like testosterone, may not play a role in the spontaneous recovery of baroreflex-mediated bradycardia. The discrepancy between both studies may be attributed to a lower recovery rate (<65%) for androstenedione by Ando et al. [ 12 ] vs. a higher recovery rate (>80%) in this study. An alternate explanation for the spontaneous recovery of baroreflex sensitivity in the presence of suppressed testosterone and androstenedione is that the baroreceptor reflex mechanism may have become adapted to the low level of circulating androgens. This would suggest an association between long-term castration, and the activation of compensatory mechanisms possibly involving an increase in androgen receptor density and/or signaling. Yu and McGinnis [ 18 ] reported a decrease in androgen receptor density in the nucleus ambiguus (one of the nuclei implicated in the baroreceptor heart rate response) [ 19 ] at 2 weeks castration, a period that coincides with a significant attenuation of baroreflex sensitivity observed in the present study and by others [ 1 , 2 ]. Whether an upregulation of androgen receptors in the brainstem occurs 6 weeks after castration remains to be investigated. Conclusion Testosterone contributes to the maintenance of baroreflex-mediated bradycardia in adult rats. Such a physiological role for testosterone seems to manifest only on a short-term basis because: (i) Short-term (3 weeks) castration caused a significant reduction in baroreflex-mediated bradycardia, a deficit corrected by testosterone replacement. (ii) Spontaneous recovery of baroreflex-mediated bradycardia occurred following long-term (6 weeks) castration in spite of the continued suppressed testosterone and androstenedione levels, which may argue against a long-term effect of testosterone or androstenedione on baroreflex-mediated bradycardia. Circulating androgens other than testosterone and androstenedione may contribute to the restoration of baroreflex-mediated bradycardia subsequent to long-term castration. An alternate possibility, that remains to be investigated, is that the baroreceptor reflex mechanism may have become adapted to the low level of circulating androgens. Methods Preparation of the rats Male Sprague-Dawley rats (Harlan Farms, Indianapolis, IN) were used in this study. Arterial blood pressure was measured according to the method used in our previous studies [ 4 ]. Briefly, the rats were anesthetized with methohexital sodium (50 mg kg -1 , i.p.). Catheters (polyethylene 10 connected to polyethylene 50), which were filled with heparinized saline (100 U ml -1 ), were placed in the abdominal aorta and vena cava via the femoral artery and vein for measurement of blood pressure and i.v. administration of drugs, respectively. The catheters were inserted about 5 cm into the femoral vessels and secured in place with sutures. Finally, the catheters were tunnelled s.c., exteriorized at the back of the neck between the scapulae, and plugged by stainless steel pins. Incisions were closed by surgical staples and swabbed with povidone-iodine solution. Each rat received s.c. injection of buprenorphine hydrochloride (Buprenex; 0.3 μg rat -1 ) to control pain and an i.p. injection of 50,000 U kg -1 of penicillin G benzathine and penicillin G procaine in an aqueous suspension (Durapen) and was housed in a separate cage. The experiment was started 48 h later, which involved the connection of the arterial catheter to a Gould-Statham pressure transducer (Oxnard, CA). The blood pressure was displayed on a Grass polygraph (model 7D, Grass Instruments Co., Quincy, MA). Heart rate was computed from blood pressure waveforms by a Grass tachograph and was displayed on another channel of the polygraph. Experiments were performed in strict accordance with institutional animal care and use guidelines, and in accordance with the principles and guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Orchiectomy Bilateral orchiectomy was performed as described [ 13 ] and according to an approved protocol by the institutional animal care and use committee. Under methohexital sodium (50 mg kg -1 , i.p.) anesthesia, a small surgical incision was made in the center of the scrotum. Each testicle was exposed through the surgical orifice. The ductus deferens and main arteries and veins were isolated and ligated. Subsequently, the duct and blood vessels were severed allowing the testicle and epididymis to be removed. The incision was then closed, sutured and swabbed with povidone-iodine solution. The sham operation involved the exposure of the testes without isolation. The post-operative procedure was implemented as previously described. Finally, the rats were housed in separate cages and allowed free access to food and water. Radioimmunoassays The commercially available radioimmunoassay "Coat-A-Count Total Testosterone", "Coat-A-Count Direct Androstenedione" and "Double Antibody Estradiol" kits were used for the analysis of serum testosterone, androstenedione, and 17β-estradiol, respectively, and were purchased from Diagnostic Products Corporation (Los Angeles, CA). Protocols and experimental groups Effect of testosterone replacement on baroreceptor reflex control of heart rate Two groups of conscious unrestrained rats (orchiectomized 3 weeks earlier at 250–275 g; orchiectomized/testosterone: n = 21 and orchiectomized/vehicle: n = 14), weighing 325–350 g, were used in this experiment to investigate whether (i) testosterone replacement restores the baroreflex heart rate response, and (ii) 17β-estradiol contributes to testosterone restoration of baroreflex sensitivity. Silastic implants (inner diameter, 2.6 mm; length, 25 mm; outer diameter, 3.1 mm; Konigsberg Instruments, Inc, Pasadena, CA) containing crystallized testosterone (or empty implants for controls) were subcutaneously implanted (for 1 week) within the orchiectomized rats according to the method used in previous studies [ 14 ]. The post-operative procedure was implemented as previously described. On the day of the experiment, the rats were allowed to acclimatize to laboratory conditions for at least 2 h prior to experimentation. Subsequently, the arterial catheter was connected to a pressure transducer for measurement of blood pressure and heart rate. Blood samples (0.6 ml) were collected, from the femoral artery, for measurements of serum testosterone and 17β-estradiol levels. A period of 30 min was then allowed for further stabilization of blood pressure and heart rate. Baroreflex curves were constructed in all rats by the i.v. bolus injection of randomized doses of phenylephrine (1–16 μg kg -1 ) at 5-min intervals. Phenylephrine was dissolved in saline and administered in varying volumes of a stock concentration (36 μg ml -1 ) of phenylephrine to achieve the desired doses. Each experiment lasted approximately 1 h. The peak changes in mean arterial pressure and heart rate, obtained following phenylephrine injections, were used for the construction of the baroreflex curves. Effect of long-term castration on baroreceptor reflex control of heart rate Two groups of rats (orchiectomized n = 12, or sham-operated n = 5, 6 weeks earlier at 175–200 g), weighing 325–350 g on the day of the experiment, were used to investigate whether a reflex adrenocortical increase in testosterone or androstenedione, subsequent to long-term castration, results in a proportionate increase in baroreflex sensitivity. Blood samples (0.6 ml) were collected, from the femoral artery, prior to each experiment for the analysis of serum testosterone and androstenedione levels. Baroreflex-mediated bradycardia was assessed as previously described. Drugs Phenylephrine hydrochloride (Sigma Chemical Co., St. Louis, MO), crystalline testosterone (Sigma Chemical Co., St. Louis, MO), methohexital sodium (Eli Lilly and Company, Indianapolis, IN), Buprenex (buprenorphine hydrochloride; Rickitt & Colman, Richmond, VA), povidone-iodine solution (Norton Co., Rockford, IL), and Durapen (penicillin G benzathine and penicillin G procaine; Vedco, Overland Park, KS) were purchased from commercial vendors. Statistical analysis Values are expressed as means ± SEM. The relationship between increases in mean arterial pressure (Mean Arterial Pressure = diastolic pressure + one-third {systolic - diastolic pressures}) and associated decreases in heart rate was assessed by regression analysis for individual animals as described in our previous studies [ 4 ]. The regression coefficient (slope of the regression line) expressed as beats min -1 mmHg -1 was taken as an index of baroreflex-mediated bradycardia [ 4 ]. Analysis of variance (ANOVA) followed by Fisher's Least Significant Difference post hoc analysis was used for multiple comparisons. The Student's t-test was used in the analysis of unpaired data, with the level of significance set at P < 0.05. In accordance with reported criteria, control rats possessing (i) baroreflex sensitivity values with significant (P < 0.05) correlation coefficients greater than or equal to 0.8 [ 8 ], and (ii) serum testosterone levels greater than or equal to 60 ng/dl [ 15 ] were included in the data analysis. Authors' contributions ARA conceived of the study and, along with GRW, participated in the design and coordination of the study as well as the drafting of the manuscript. GRW carried out all of the experiments and the statistical analyses. Both authors read and approved the final manuscript.

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1079885

Exercise responsive genes measured in peripheral blood of women with Chronic Fatigue Syndrome and matched control subjects

Background Chronic fatigue syndrome (CFS) is defined by debilitating fatigue that is exacerbated by physical or mental exertion. To search for markers of CFS-associated post-exertional fatigue, we measured peripheral blood gene expression profiles of women with CFS and matched controls before and after exercise challenge. Results Women with CFS and healthy, age-matched, sedentary controls were exercised on a stationary bicycle at 70% of their predicted maximum workload. Blood was obtained before and after the challenge, total RNA was extracted from mononuclear cells, and signal intensity of the labeled cDNA hybridized to a 3800-gene oligonucleotide microarray was measured. We identified differences in gene expression among and between subject groups before and after exercise challenge and evaluated differences in terms of Gene Ontology categories. Exercise-responsive genes differed between CFS patients and controls. These were in genes classified in chromatin and nucleosome assembly, cytoplasmic vesicles, membrane transport, and G protein-coupled receptor ontologies. Differences in ion transport and ion channel activity were evident at baseline and were exaggerated after exercise, as evidenced by greater numbers of differentially expressed genes in these molecular functions. Conclusion These results highlight the potential use of an exercise challenge combined with microarray gene expression analysis in identifying gene ontologies associated with CFS.

Background In a state of health, physical exercise has a quantifiable effect on neuroendocrine, autonomic, and immune systems influencing metabolic and immune responses. However, in the initial phase of acute illness, there is an avoidance of physical stressors so energy can be dedicated to healing and a return to homeostasis. While physiologic disturbance in acute illness is transient, chronic illnesses, such as chronic fatigue syndrome (CFS), have prolonged disturbances that have a debilitating effect both physiologically and psychologically. Consequently, activities that are physiologic stressors, such as physical exercise, exacerbate the symptoms that define CFS. CFS is a complex, multifactorial illness whose etiology and pathophysiology remain unclear [ 1 ]. CFS is defined by a characteristic symptom complex in the absence of other medical or psychiatric conditions with similar clinical characteristics [ 2 , 3 ]. Subtle differences in hypothalamic-pituitary-adrenal axis function [ 4 ], immune system function [ 5 ], and psychological profiles [ 6 ] between CFS patients and controls have been reported; however, no consistent distinguishing difference or frank abnormality has been confirmed [ 7 , 8 ], and it remains unclear whether CFS represents a unique disease or a common illness response to a variety of insults. Perhaps the greatest methodological problem with studying CFS is that many individuals identified in population studies have been sick for at least 5 years [ 9 ]. During this time, the illness waxes and wanes, making it difficult to identify biomarkers or define pathogenesis. Physical, mental, and emotional stress exacerbate CFS and result in case-defining post-exertional fatigue [ 2 ] with measurable physiologic differences [ 10 ]. Therefore, exercise challenge of people with CFS is an effective method for calibrating CFS subjects and thus increasing the likelihood of uniformly identifying biomarkers and/or physiologic abnormalities. We used gene expression profiling of peripheral blood to evaluate differences between CFS subjects and sedentary healthy controls both before and following an exercise challenge. Overall, we found the gene expression profiles to be quite similar, and of importance, most differences were present prior to exercise challenge. These differences were in G protein-coupled receptor and ion transport and ion channel activity ontologies. The latter was exaggerated after exercise as evidenced by differential expression of a greater number of genes involved in these molecular functions. Differences were also evident in exercise response, including chromatin and nucleosome assembly, cytoplasmic vesicles, membrane transport and G-protein coupled receptor ontologies. These differences may help explain the symptoms of CFS. Results Exercise response genes were evaluated using a random variance t test in a paired, class comparison analysis of control subjects before and after exercise, and 21 genes were identified as being differentially expressed (Table 2 ). The probability of identifying these 21 genes by chance if there are no real differences between the classes was 0.056 as determined by the multivariate permutation test. Among the 21 genes, 16 could be categorized in the Gene Ontology (GO) of biological process and 15 in molecular function (results not shown). The most significant categories or "themes" of these exercise-responsive genes as assessed by an EASE score of <0.10, pertained to the biological process of transport (both vesicle-mediated and protein transport). 5 of the 21 genes were involved in this process. Since these 21 genes reflect a healthy subject's peripheral blood gene expression response to exercise challenge, we reasoned that the expression of these would be altered in CFS subjects. To have a visual representation of these differences, the gene list from Table 2 (differentially expressed genes in control subjects, compared before and after exercise challenge) was used in a two-way hierarchical cluster analysis (Figure 1 ). The response of 10 of the 21 genes was quite similar in terms of magnitude and direction for both CFS and control subjects (Figure 1 , marked in blue). For the other 11 genes, the magnitude of the exercise change was considerably smaller in CFS subjects (Figure 1 , subject clusters 2 and 4) than in control subjects (clusters 1 and 3). With regard to the GO categories of these 21 genes, 10 genes were associated with binding and 8 with metabolism, all of which were equally distributed between the two response types (denoted by # and in Figure 1 ). However, 5 genes classified in vesicle-mediated and protein-transport ontologies differed between CFS and control subjects (Figure 1 , highlighted in yellow). No differentially expressed genes were identified by class comparison analysis (at a significance level of p > 0.005) for CFS subjects before and after exercise, for CFS subjects before exercise compared with controls before exercise, or for CFS subjects after exercise compared with controls after exercise. Because differentially expressed genes were identified by class comparison prior to the exercise challenge, we reasoned that a comparison of gene expression in CFS and control subjects, using genes categorized by ontology, would more efficiently reveal perturbed physiological pathways. Figures 2 and 3 show the results of these analyses. GO terms with defined parent-child relationships are grouped together in the figures and are color-matched. Only two exercise-responsive GO categories [phospholipid binding (orange) and chromatin architecture (pale green)] were common to controls and CFS subjects (Figure 2a and 2b ). However, for the chromatin architecture category, the CFS comparison highlighted 7 overlapping ontologies (containing 59 unique genes), compared with 1 ontology of 33 genes in the control comparison. The 33 genes overlap with the 59 identified in the CFS comparison. The phospholipids-binding ontologies were identical in gene composition. Exercise-related changes that were identified as significant only in control subjects were associated with genes involved with vesicle (yellow, Figure 2a ), dehydrogenase (grey, Figure 2a ), ATPase (pink, Figure 2a ), and transporter (blue, Figure 2a ), activities. Exercise-related changes that were seen only in CFS subjects were related to G-protein-coupled receptor signaling (purple, Figure 2b ). Gene ontology comparison was also used to evaluate differences between control and CFS subjects before (i.e. baseline, Figure 3a ) and after (Figure 3b ) exercise. Baseline differences between CFS subjects and controls that continued after exercise involved GO terms relating to ion transport (blue, Figure 3a ). After exercise, these differences appear to be amplified, as evidenced by increased numbers of genes present in these GO categories and also by inclusion of more GO terms pertaining to ATPase transmembrane movement of ions (pink, Figure 3b ). G-protein-coupled receptor binding (purple, Figure 3a ), part of the broad functional category of signal transduction, differed between CFS subjects and controls prior to exercise. This baseline difference between controls and CFS subjects was not significant after exercise. Interestingly, complement activation (dark green, Figure 3b ) was one of the exercise-induced differences between subjects and controls that was present only after challenge. Genes in most of the ontologies identified as different between CFS and control subjects had lower expression levels in CFS subjects. Discussion Gene expression profiling affords a unique opportunity to characterize CFS at a systems biology level. Changes in gene expression underlie many biologic processes and may provide insight into disease-specific gene expression and the response of genes to environmental stimuli. In a proof-of-concept study, we found that CFS patients had different blood mononuclear cell gene expression patterns than non-fatigued controls [ 11 ] and that CFS is a heterogeneous illness as evidenced by different gene expression profiles for patients reporting gradual onset of their illness compared with those reporting sudden onset of illness [ 12 ]. In addition, differential display polymerase chain reaction on a small number of CFS and control subjects identified candidate biomarkers in the peripheral blood [ 13 , 14 ]. CFS is defined by a post-exertional fatigue that does not subside 24 hours following physical stress. In contrast, exercise in healthy, untrained people induces changes in cellular homeostasis in 1 to 4 hours and a return to basal levels within 24 hours, as measured in muscle [ 15 ]. Analysis of peripheral blood gene expression in the healthy control subjects confirmed this observation since the majority of gene expression levels were the same before and 24 hours following exercise challenge. This implied that expression either returned to basal levels or was unchanged as a result of the exercise challenge. And indeed, many of the 21 exercise-induced, differentially expressed genes in control subjects were characterized by GOs that reflect a diverse set of molecular functions necessary for cell function and viability. (These ontologies overlapped with those identified in the GO comparison analysis given in Figure 2a ). Figure 1 clearly illustrates the reciprocal pattern of gene expression in the 21 genes for most of the control subjects. In contrast, 11 of the genes were unchanged in CFS subjects before and after exercise; with 5 being classified in a transport-related ontology. Because this difference in gene expression is so dramatic, it implicates a fundamental perturbation in the biochemical activity of lymphocyte and monocyte peripheral blood fractions from CFS subjects compared with control subjects that does not affect classical immunologic markers (i.e, CD45) that have been shown to be unaffected in CFS patients [ 16 , 17 ]. Rather, low expression of these genes may have subtle effects on immune function. Immune dysfunction has been inconsistently implicated in CFS pathogenesis [ 18 ]. Class comparison was used to identify these 21 differentially expressed genes, which indicated the possible disturbance of biologic pathways (Figure 1 ). To explore this possibility, we used the GO comparison that is based on the knowledge that gene expression levels are dependent variables in biological processes, cellular components, and molecular functions. In this way, multiple genes in the same category reinforce each other and enhance the power for identifying the significance of the category. The GO categories considered significantly different (p < 0.005) when comparing CFS subjects with controls after exercise challenge were those pertaining to ion transporter activity (a total of 87 genes applied to this category in the comparison of CFS and controls after exercise) and ATPase activity coupled to transmembrane movement (42 genes). When the CFS and control classes are compared prior to exercise, ion transport activity and voltage-gated, ion channel activity are identified (38 and 44 genes within the GO categories, respectively). It is evident that ion transport and ion channel activity segregate cases from controls and that exercise seems to intensify these differences. Several other conditions have been reported in which fluctuating fatigue occurs that are known to be caused by abnormal ion channels. These conditions include genetically determined channelopathies and acquired conditions such as neuromyotonia, myasthenic syndromes, multiple sclerosis, and polyneuropathies [ 19 , 20 ]. There are other transmembrane functions associated with differences between controls and CFS patients, including signal transducer activity through receptor binding/activity (Figure 3a ). Signal transduction of transmembrane receptors occurs by a number of mechanisms, including structural changes, ion channels, and changes of transmembrane potentials. The G-protein-coupled receptors play an important role in the membrane trafficking machinery [ 21 ]. The most obvious exercise-induced changes in CFS cases pertain to gene regulation at the point of chromatin structure; whether these changes reflect the differences seen in the mRNA transcripts relating to membrane trafficking differences between cases and controls has not yet been determined. One interesting correlate of this study was the finding that the complement pathway showed significant differences between CFS and control subjects after exercise. This has been reported previously in the analysis of these same exercise challenge-derived specimens. Sorensen et al. [ 22 ] measured levels of complement split products in the sera of these subjects and found differences between CFS and control subjects in C4a after exercise challenge. Complement activation was identified as an ontology that was significantly different between CFS and control subjects after exercise. The correlates on the data are interesting as their study measured protein levels (i.e., gene product levels) and this study measured the transcript levels. The class comparison analysis performed in this study accounted for multiple testing and the over fitting problems of microarray data analysis. The lack of statistical significance in the 3 other class comparison analyses performed (CFS cases compared before and after exercise, comparison of cases to controls at baseline, and the comparison of cases to controls 24 hours after exercise) reflects low experimental sensitivity, most likely due to a small number of subjects, rather than an absence of biological effect. This is accounted for in the gene ontology comparison tool where classes are compared by GO category rather than with regard to individual genes. The next line of research will detail larger numbers of subjects in the expression arrays. The emphasis in such studies will be on developing a gene expression-based multivariate function, or predictor, that accurately predicts the class membership of a new sample on the basis of the expression levels of key genes. Class discovery tools will also be applied to CFS subjects' expression profiles in an attempt to further describe discrete subsets of this disease on the basis of gene expression as we have done for gradual and sudden onset of illness [ 12 ]. However, the methods used in this study will be applied to these data sets too, as these analytical tools will prove to be very helpful in defining the pathophysiology of CFS. It is hoped that this broader, more fully encompassing approach to CFS research will open many doors to the understanding of this syndrome and perhaps of fatigue and un-wellness in general. Methods Study subjects This study adhered to human experimentation guidelines of the U.S. Department of Health and Human Services and the Helsinki Declaration. All participants were volunteers who gave informed consent. Study parameters have been described [ 22 ]. In brief, women who attended a CFS outpatient fatigue clinic volunteered for the study. All of these patients met the current working definition for CFS [ 2 ] that includes the following criteria: fatigue lasting longer than 6 months or more, no other illness that could explain the fatigue, 4–8 concurrent symptoms, fatigue that is not relieved by rest, and fatigue that interferes with occupational, educational and social or personal activities. Healthy controls were recruited through advertisements. They were similar in age, sex, and activity level (sedentary to moderately active) to the CFS patients. All women, were scheduled for exercise challenge 5 to 10 days after the first day of their menstrual cycle. All subjects were asked not to use inhaled or systemic corticosteroids, anti-histamines, or anti-inflammatory medication for 7 days prior to the exercise challenge. Subjects performed a submaximal (70% predicted maximum work load), steady-state exercise for 20 minutes on a stationary bicycle ergometer. All subjects met these challenge criteria. Blood samples were obtained immediately before and 24 hours following exercise. The selection of subjects for inclusion into the gene expression pilot study, focused on those without allergies, to whom an exercise challenge was given: 5 women with CFS and 5 female controls. RNA isolation Immediately following blood collection, peripheral blood mononuclear cells were isolated, using Ficoll gradients and stored in RNAqueous™ lysis buffer (Ambion Inc., Austin, TX) at -70°C. Total RNA was extracted using the RNAqueous™ kit (Ambion), with quality and quantity assessed by agarose gel electrophoresis as described previously [ 23 ]. Preparation and hybridization of labeled cDNA Biotinylated cDNA synthesis from 1 μg of total RNA, microarray hybridization, and detection were performed as previously described [ 23 ]. Atlas™ Human 3.8I oligonucleotide glass microarrays (Clontech Laboratories, Palo Alto, CA) were used. The slides were archived and images captured using the GSD-501™ scanner (Invitrogen Life Technologies, Carlsbad, CA). Data analysis Preprocessing data The scanned TIFF images were processed using ArrayVision™ (Imaging Research Inc., Ontario, Canada) as previously described [ 23 ]. A median background value was calculated around each of the 3757 features and subtracted from the mean feature signal to give the net signal for the respective gene. Values were log 2 -transformed, and a global normalization was used to median center the log intensity values. Genes whose expression differed by at least 1.5 fold from the median in at least 20% of arrays were retained (3699 genes), thus excluding genes showing minimal variation across the set of arrays. If an expression value was missing or filtered out in more than 50% of the arrays, that feature was not included, leaving a total of 3682 genes for analysis. Analysis We used several analytical approaches to look for differential expression among four predefined classes: control subjects before exercise, control subjects after exercise, CFS subjects before exercise, and CFS subjects after exercise. Comparisons included paired analyses of controls before versus after exercise and CFS cases before versus after exercise. In addition, we examined pre-exercise (baseline) controls versus pre-exercise CFS subjects and post-exercise controls versus post-exercise CFS subjects. Analytical approach 1 The more commonly used approach to microarray gene expression analysis is to establish if gene expression profiles differ between subjects in predetermined classes and to identify the genes responsible for the differences. The Class Comparison analysis method in BRB ArrayTools [ 24 ] uses this approach, applying a random variance t-test to the data. This is an improvement over the standard t-test as it permits sharing information among genes about within-class variation without assuming that all genes have the same variance [ 25 ]. It is an advisable modification when the numbers of samples per class are small. Genes were considered statistically significant if the parametric p-value was less than 0.005. Next a global, multivariate permutation test was performed to determine whether the expression profiles differed between the classes by permuting the array classification. We used this test to provide a median false discovery rate of 10%. The EASE software package [ 26 ] was then used to evaluate the biologic significance of the ontology of genes identified as differentially expressed by class comparison. EASE performs a statistical analysis of gene categories in a gene list relative to all genes on the array and calculates a conservative variant of the standard Fisher exact probability called the EASE score. The most significant categories as assessed by EASE score are deemed "themes" of the gene list. These themes correspond to the systematic and standardized nomenclature developed by the GO (Gene Ontology) Consortium [ 27 ]. The three organizing principles of GO are molecular function, biological process and cellular component, and presently 22,665 human gene products have been annotated. Associating genes with related GO terms assists in the interpretation of expression patterns. A GO category includes genes described by that term and those included in any subset (or children) of that GO term. A gene may be categorized in more than one ontology category. To demonstrate the distinct gene clusters, we performed a two-way hierarchical cluster analysis on the genes identified by this analytical approach, using the algorithm described by Eisen et al. [ 28 ]. Analytical approach 2 We used the Gene Ontology comparison module of BRB Array tools [ 24 ] as a second analytical approach. In this method, the genes are assigned GO terms prior to analysis. For each GO term the total number of genes on the array belonging to that category was determined. A random variance t-test or F-test [ 25 ] was used to determine the p-value for differences between the predefined classes for each gene in the GO term. Two statistics are computed to summarize the p-values for all genes in the GO term: the Fisher statistic and the Kolmogorov-Smirnov statistic [ 24 ]. These p-values provide a list of GO categories that have more genes differentially expressed among the classes than expected by chance. We considered a GO category differentially regulated if either significance level was less than 0.005. All GO categories with between 5 and 100 genes represented on the array were considered. Competing interests The author(s) declare that they have no competing interests. Authors' contributions TW contributed to the design of the experimental approach, the analysis of the data and drafted the manuscript. JFJ contributed to the conception and design of this study, conducted the exercise challenges and assisted in drafting the manuscript. ERU contributed to the design of the experimental approach, and to the manuscript preparation. SDV was instrumental in the design of the experimental approach, analysis, presentation, discussion of these data and manuscript preparation. All authors read and approved the final manuscript.

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1079886

The asthma epidemic and our artificial habitats

Background The recent increase in childhood asthma has been a puzzling one. Recent views focus on the role of infection in the education of the immune system of young children. However, this so called hygiene hypothesis fails to answer some important questions about the current trends in asthma or to account for environmental influences that bear little relation to infection. Discussion The multi-factorial nature of asthma, reflecting the different ways we tend to interact with our environment, mandates that we look at the asthma epidemic from a broader perspective. Seemingly modern affluent lifestyles are placing us increasingly in static, artificial, microenvironments very different from the conditions prevailed for most part of our evolution and shaped our organisms. Changes that occurred during the second half of the 20th century in industrialized nations with the spread of central heating/conditioning, building insulation, hygiene, TV/PC/games, manufactured food, indoor entertainment, cars, medical care, and sedentary lifestyles all seem to be depriving our children from the essential inputs needed to develop normal airway function (resistance). Asthma according to this view is a manifestation of our respiratory maladaptation to modern lifestyles, or in other words to our increasingly artificial habitats. The basis of the artificial habitat notion may lie in reduced exposure of innate immunity to a variety of environmental stimuli, infectious and non-infectious, leading to reduced formulation of regulatory cells/cytokines as well as inscribed regulatory pathways. This could contribute to a faulty checking mechanism of non-functional Th2 (and likely Th1) responses, resulting in asthma and other immuno-dysregulation disorders. Summary In this piece I discuss the artificial habitat concept, its correspondence with epidemiological data of asthma and allergy, and provide possible immunological underpinning for it from an evolutionary perspective of health and disease.

Background Asthma is a major health problem that has reached alarming proportions in the past two decades in western societies [ 1 ]. What lies behind the recent increase in asthma in affluent societies is still an area of lively debate, but its rapid changing patterns and huge variation across populations favor environmental explanations [ 1 - 3 ]. While assessment of different old and new exposures is continuing, a unifying paradigm remains elusive, so as a guiding principle for prevention. The simultaneous increase of all forms of allergic disease on the other hand, argues for a change in host susceptibility/resistance [ 4 ]. The apparent association between asthma and western lifestyle has lead to numerous studies trying to link novel or increased exposures associated with westernization-modernization, especially those occurring during childhood, to asthma. For example, exposure to gas cooking, tobacco smoke, trans fatty acids, domestic animals, and allergens were found to influence respiratory health, but did not provide a conclusive answer to the current trends in asthma [ 5 - 10 ]. While it is early to discard the role of these and other factors, a major contribution of asthma research during the past two decades lies in the elucidation of its heterogeneous and multi-factorial nature, where different exposures have different roles and relevance depending on the target population, setting, and disease course. Discussion 1- The hygiene hypothesis and asthma Since its introduction in the nineties, the hygiene hypothesis (HH) continues to generate enthusiasm among asthma researchers as the most comprehensive theoretical framework, by which the relation between suspected environmental factors and allergy can be tested [ 11 - 13 ]. This hypothesis originated from the coupling of observations on the allergy-protective effect of sibship size/birth-order with the emerging concept of helper T cell polarization into two counter-regulatory subsets; pro-infection Th1, and pro-allergy Th2 [ 14 , 15 ]. Backed by some experimental and clinical evidence [ 16 - 18 ], the hygiene hypothesis suggests that the recent rise in allergic disease among children in affluent societies is due the preferential programming of the T cell repertoire towards pro-allergy Th2 responses, brought by the decline in infections (increased hygiene, immunization, decreased sibship size, antibiotic use) [ 13 - 15 ]. With the increasing recognition of the role of T regulatory cells (Tregs) and cytokines in the pathogenesis of allergic inflammation, the hygiene paradigm has been extended recently integrating infection's role in generating such cells and mediators [ 19 - 21 ]. Asthma trends however, did not fit well with the hygiene model, which failed to explain the urban predominance of asthma, the increase in non-allergic asthma, the disparity between atopy and allergy in some populations, and the asthma inducing properties of some infections [ 22 - 27 ]. Specifically, studies looking at the infection-asthma relationship failed to yield a consistent pattern so far [ 28 - 34 ], prompting David Strachan, the father of the hygiene hypothesis, to conclude that "the totality of current evidence from cross sectional and longitudinal studies of common specific and non-specific infectious illnesses in infancy and childhood offers no support for the hygiene hypothesis" [ 11 ]. Other studies doubted even the fundamentals of the HH showing that the effect of siblings is not universal, or could have been programmed in utero rather than being a marker of childhood infection [ 35 , 36 ]. Perhaps the hygiene model's major shortcoming lies in its concentration on only one aspect (infection) of the drastic change that touched upon every detail of life in western societies in the past few decades. It also adopted a mechanistic approach for the study of adaptive immune responses and the relation between exposure-outcome, where an array of potential interactions are reduced to a single level; Th1-Th2 counter-regulation, siblings-infection, daycare attendance-infection, dog ownership-endotoxin, farming-endotoxin, etc. In brief, the search for a holy grail in the asthma epidemic may need to be replaced by the conceptualization of a more generalizing notion that allows for the consideration of multitude of factors within an ever changing environment. 2- Asthma and our artificial habitats The hygiene model on the other hand, involved an evolutionary logic alerting us to the negative potential of sudden elimination of exposures that have shaped throughout the ages our organs and systems [ 37 ]. Health from such perspective is not about abstract assessment of the relation between exposure and outcome, or eliminating harmful exposures, but about seeing the whole dynamics of our interaction with our novel habitats [ 38 ]. Because of the slowness of adaptive evolutionary machinery, of particular interest according to this perspective are lifestyle factors that either witnessed a rapid change in recent times or represent an obvious departure from the conditions prevailed for most part of our evolution, i.e. factors likely to exemplify the discordance between our "Stone Age" genes and "Space Age" living conditions [ 37 , 38 ]. General trends of asthma show that populations who conserved elements of ancient lifestyles have low levels of asthma. The north-south, urban rural, gradients in asthma occurrence have been extensively documented [ 24 , 37 , 39 - 43 ]. In additions, studies in Africa show that some populations seem to be protected from asthma regardless of atopic predisposition or parasitic infection (both are Th2-related) [ 20 , 22 , 44 - 46 ]. This indicates that some environmental influences associated with more traditional lifestyles are conferring respiratory resistance to stimuli that could have lead otherwise to clinically relevant airway inflammation. Within western societies furthermore, lower levels of asthma were found in families with traditional lifestyles [ 47 , 48 ] and higher levels of asthma were found among obese or less physically fit children and adults [ 49 - 51 ]. Because not all these observations can be explained by variability in infection, or any other single factor for that matter, a broader concept seems more plausible. Modern life is increasingly placing us in static, artificial, micro-niches optimized for our convenience, but which bear little resemblance to the dynamic inputs provided by the environments that nurtured our evolution. In other words, we are increasingly living within an array of artificial habitats designed to handle us very well, but we may well not be equipped to handle them. Asthma according to this view, becomes a manifestation of our respiratory maladaptation to modern lifestyles. Changes that occurred during the second half of the 20 th century in industrialized countries with the spread of central heating/conditioning, building insulation, hygiene, TV/PC/games, manufactured food, indoor entertainment, cars, medical care, and sedentary lifestyles all seem to be depriving our children from the essential inputs needed to develop normal airway function (resistance). 3- Epidemiology of asthma from a new perspective The suggested view -called here the artificial habitat (AH)- can provide alternative interpretations to existing data starting with the protective effect of sibship size/birth-order, which is one of the landmark observations of the HH that has been ascribed to increased exposure to infection [ 52 ]. It can be postulated that the mechanism of protection of siblings (especially older males) is related to their importance to the child's level of physical activity as well as ability to spend more time outdoors (i.e. in a more dynamic environment). This is particularly relevant to children living in dangerous neighborhoods, such as inner cities in the US, where the spread of asthma represents one of the main challenges to the hygiene paradigm [ 53 ]. Indeed, Andrew-Aligne and colleagues found that the higher prevalence of asthma among inner city black children is not due to race or low income per se, but to their living in an urban setting [ 54 ]. Additional intriguing support to the AH notion comes from the two largest studies looking at the effect siblings on the occurrence of allergy, whereby a stronger protective effect was observed for brothers than for sisters [ 55 , 56 ]. While exposure to infection cannot be expected to be related to sibling's gender, activity and outdoor time may well be influenced by this factor. By the same token, birth order can determine, among other things (e.g. social development, healthy food availability), the child's level of activity (how many playmates the child have) and ability to spent time outdoors. As new evidence are emerging against the HH's assumption considering the infection-related effect of the sibship size/birth-order, by showing for example that the role of birth order is independent of sibship size [ 57 ], and that in the same population sibship size can protect against asthma while infection predispose to it [ 31 , 58 ], the AH concept seems to offer an alternative explanation. Another important observation of the hygiene paradigm concerns the protective effect of early daycare attendance on later development of asthma, which is ascribed to increased exposure to infection [ 28 ]. Alternatively, it can be argued that the daily routine at a daycare center would be different in many aspects from home, in addition to exposure to infection. Reducing potential differences in activity, exposure, socio-behavioral development, and parental attitudes between those who do and don't attend daycare to mere infection seems over-simplistic. The AH concept looks at this observation from a broader angle involving a mixture of lifestyle and developmental factors. Related to this issue is the argued window of opportunity in early infancy for the protective effect of daycare attendance/infection [ 59 , 60 ], which is connected to a critical period of immune education [ 13 ]. By its own nature in contrast, the AH view is consistent with the notion that environmental signals throughout the lifespan can affect the risk as well as the course of asthma and allergy. On a different juncture of asthma research, a multitude of recently published reports show lower rates of asthma and atopy in children raised on a farm [ 61 - 68 ]. Heavily influenced by the HH, these observations were largely attributed to increased exposure to bacterial components found in barns or farm milk (endotoxin in particular) [ 67 - 69 ], forgoing that children raised on a farm have very different lifestyles from children growing in inner cities in the US for example, where infection is also commonplace [ 70 ]. For example, one of the landmark farming studies has shown that endotoxin levels in children's mattresses were inversely associated with the occurrence of hay fever and atopic asthma [ 67 ]. However, leukocytes of children exposed to high levels of endotoxin produces less Th2 suppressing cytokines (mainly interleukin 10), arguing against the endotoxin-hygiene paradigm [ 67 , 71 , 72 ]. From the AH perspective however, a farm is the closest we can get in today's' western societies to conditions prevailed for most part of our evolution. Such an environment can provide ample opportunities of behaviors and exposures different from those of modern urban life. The AH perspective is consistent with the assumption that time spent outdoors and level of physical activity should be protective against the development of asthma. A recent study by McConnell and colleagues however, has shown just the opposite, where the risk of developing asthma was positively associated with number of sports played and time spent outdoors [ 73 ] However, when participating communities were separated according to their level of atmospheric ozone, this association was only seen in the high ozone levels communities, while number of sports played and time spent outdoors seem to be protective in the low ozone communities [ 73 ]. On the other hand, twines in the Odense study who participated in conditioning exercise had a decreased risk of asthma compared to the more sedentary co-twins [ 51 ]. The physiological underpinning of the effect of activity on asthma can be partly elucidated by the work of Feldberg and colleagues and Togias and colleagues, who showed that disruption of dynamic breathing (static breathing without deep breaths or sighing) can lead to bronchial hyper-responsiveness (BHR), the pathophysiological hallmark of asthma [ 74 , 75 ]. Furthermore, recent evidence shows that obesity and weight gain are associated with increased risk of BHR [ 76 ], providing more insight on possible ways by which sedentary life factors can co-interact to predispose to asthma. Finally, the AH concept can offer an explanation for some puzzling observations, such as the protective effect of dog ownership on asthma [ 77 , 78 ]. While the HH proponents looked for explanation in endotoxin levels associated with dog ownership, but with conflicting results so far [ 79 ], one can argue that the change in lifestyle (of children particularly) associated with dog ownership can be responsible (more playing, more time out, emotional interaction, as well as exposure to dog's constituents). Generally, families who opt to have a dog may be different from those who don't in being more active and outgoing. The focus here on activity and outdoor time is because these factors are clearly envisioned. Other aspects of traditional lifestyles may be just as important, such as household air exchange (e.g. affecting allergens, pollutants, humidity), nutritional habits (e.g. breast feeding), as well behavioral adaptations. 4- An innate control of asthma and allergy Now how can the AH view be related to what we know about the immunopathology of asthma and allergy? Asthma is an immunological disorder with a predominant Th2 inflammatory response in the airways. This Th2 response is thought to be a remnant of our ability to expel parasites abundant in the cradle of human evolution, the tropical savannah [ 80 ]. Indeed, evidence exist showing that Th2 pro-inflammatory genetic alleles are more prevalent in populations with a tropical origin than those with a temperate one [ 81 ]. It is possible that the need to deal with a wide variety of pathogens may have meant that the activation threshold of Th2 responses has to be set low, leading to many false alarms to non-pathogenic particles and giving rise to asthma. Evolutionary logic indicates however, that a trait with a potential to endanger air passage into our vital respiratory organs would not have been selected, had some regulatory mechanisms not been in place. Studies on the initial phase of allergic sensitization show that a transient low-level IgE (the atopic antibody) response to inhalant antigen occurs in normal children, with those who do not develop allergy down-regulate it in the first years of life [ 82 - 84 ]. Asthma in this regard, becomes a manifestation of breakdown of regulatory mechanisms at respiratory mucosal surfaces. But let's take one step back to look at another recent puzzling trend; the increase of Th1 autoimmune disorders such as type-1 diabetes and multiple sclerosis in western societies [ 21 ]. Recent evidence suggests that that the two groups (Th1 and Th2 mediated diseases) can be associated in individuals [ 85 - 88 ], arguing against the Th1-Th2 counter-suppression of the HH, and favoring a common ground of faulty regulation. Such developments were picked up by proponents of the HH to suggest that hygiene can work through depriving the immune system from signals necessary for the development of regulatory pathways/cells capable of dampening both Th1 and Th2 responses [ 89 , 90 ]. While this can be true, the focus on infection yet again is a reductionistic view likely to suffer the same shortcomings of the original Th1-Th2 counter-regulation of the HH. At the same time, advances made in immunology were unraveling the central role of the innate immune system in orchestrating immune responses [ 91 ]. In particular, antigen presenting cells, such as dentritic cells (DCs), can engage infectious components with their Toll-like receptors (TLRs) (a group of ancient immune recognition molecules) leading to activation of adaptive immune responses and induction of regulatory cells and mediators [ 92 , 93 ]. In their turn, T regulatory cells (Tregs), which are induced naturally or by elements of innate immunity are able to regulate all types of adaptive immune responses as well as influence DCs activation and regulation [ 94 , 95 ]. Interestingly, it looks that none of the Th1, Th2, or Treg-inducing functions of DCs is an intrinsic attribute that is not sensitive to instructions from the surrounding environment [ 96 , 97 ]. Without getting into the details of this fascinating and still unfolding field, the move from the see/saw mechanistic counter-regulation of adaptive Th1-Th2 responses to elements of innate immunity offers an evolutionary sound and possibly robust checking mechanism (break) against inappropriate responses (e.g. Th2 responses to non-pathogenic elements) at our vital airways. The ability of DCs to be activated in response to danger signals induced by stress, damage, or necrotic cell death [ 98 ], and the role of DCs at the gastrointestinal tract in the development of mucosal tolerance [ 99 ], broadens their possible range of involvement with different environmental stimuli and thus their contribution to immune homeostasis at the respiratory surface. For example, heat-shock proteins (hsps, which are highly conserved cellular proteins that can be produced by thermal stimuli, physical activity, or other stresses) can activate DCs as well as contribute to T cell regulation of inflammatory responses [ 100 , 101 ]. Taken together, it can be suggested according to the AH concept that dynamic/traditional lifestyles with associated exposures can ensure constant challenge of DCs and other elements of innate immunity giving rise to immune responses, but at the same time maintaining adequate turnover of regulatory cells and cytokines and inscribed regulatory pathways. This ongoing activation of regulatory pathways can help maintain healthy control of non-functional Th2 responses at the respiratory surface. The DC-orchestrated dynamic balance between Th2 responses and regulatory mechanisms is likely to influence all phases (initiation, effector) of inflammation in the airways, and throughout the lifespan of the individual. Summary While it offers no specific explanation to different asthma trends and variations, the suggested AH notion provides a generalizing scheme for the study of asthma, and provides novel insights for existing epidemiological observations. According to this perspective there is no single answer to the asthma epidemic, but different factors have different relevance depending on the population and environment in focus. In addition to being free from the HH one-dimensional approach for the relation between exposure-outcome, this view is evolutionary-driven allowing to place the asthma epidemic within the wider perspective of increasing discordance between us and our dramatically changing environments. Sedentary lifestyles, static indoor microenvironments, and automation of the food chain are apparently not only predisposing us to obesity ad cardiovascular disease but also depriving our respiratory system from many stimuli necessary for the development of normal airway resistance. The immunological basis of the AH notion can lie in the centrality of innate immunity and its ability to respond to different types of environmental stimuli, insuring adequate turnover of regulatory cells and mediators. The evolutionary tenet "the more we change the world the more we stay the same" probably lacks accuracy. Newer environments, constantly confront us with new adaptive challenges that should be looked upon, as in the case of asthma, within the evolutionary context of health and disease. Abbreviations HH- hygiene hypothesis AH- artificial habitat DCs- dentritic cells BHR- bronchial hyper-responsiveness IL10- interleukin 10 hsps- heat shock proteins Th1- T helper cell type 1 Th2- T helper cell type 2 Tregs- regulatory T cells IgE- immunoglobuline E TLRs- Toll-like receptors Competing interests The author(s) declare that they have no competing interests. Authors' contributions Dr. Wasim Maziak is the sole author and contributor to this manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079887

Psychometric properties of the Flemish translation of the NEECHAM Confusion Scale

Background Determination of a patient's cognitive status by use of a valid and reliable screening instrument is of major importance as early recognition and accurate diagnosis of delirium is necessary for effective management. This study determined the reliability, validity and diagnostic value of the Flemish translation of the NEECHAM Confusion Scale. Methods A sample of 54 elderly hip fracture patients with a mean age of 80.9 years (SD = 7.85) were included. To test the psychometric properties of the NEECHAM Confusion Scale, performance on the NEECHAM was compared to the Confusion Assessment Method (CAM) and the Mini-Mental State Examination (MMSE), by using aggregated data based on 5 data collection measurement points (repeated measures). The CAM and MMSE served as gold standards. Results The alpha coefficient for the total NEECHAM score was high (0.88). Principal components analysis yielded a two-component solution accounting for 70.8% of the total variance. High correlations were found between the total NEECHAM scores and total MMSE (0.75) and total CAM severity scores (-0.73), respectively. Diagnostic values using the CAM algorithm as gold standard showed 76.9% sensitivity, 64.6% specificity, 13.5% positive and 97.5% negative predictive values, respectively. Conclusion This validation of the Flemish version of the NEECHAM Confusion Scale adds to previous evidence suggesting that this scale holds promise as a valuable screening instrument for delirium in clinical practice. Further validation studies in diverse clinical populations; however, are needed.

Background Delirium, as defined by the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, text revised (DSM-IV-TR), is a disturbance of consciousness with reduced ability to focus, sustain or shift attention, a change in cognition or the development of a perceptual disturbance that occurs over a short period of time and tends to fluctuate over the course of the day [ 1 ]. This transient and etiologically non-specific, organic cerebral syndrome of acute onset, is a common and serious health problem for elderly hospitalized patients [ 2 - 9 ]. Several studies have shown that patients who develop delirium are at increased risk for morbidity, longer and costlier hospitalizations, nursing home placement and death [ 9 - 11 ]. Because of significant and profound negative outcomes of delirium, and its potential reversibility, prompt and effective treatment is dependent upon the early recognition of delirium [ 12 - 14 ]. However, nurses and physicians have been shown to consistently underdiagnosis delirium [ 12 , 14 - 16 ]. The underrecognition and misdiagnosis of delirium is partly a function of the failure of providers to consistently use standardized methods of detection [ 15 ]. Several standardized instruments for the routine, systematic, and comprehensive assessment of changes in cognitive status or detecting delirium have been described in greater detail elsewhere [ 17 - 20 ]. The most common method for assessing cognition is the mental status questionnaire, with the most frequently used bedside test of cognition being Folstein's Mini-Mental State Examination [ 21 ]. However, the usefulness of mental status questionnaires is equivocal since they pose significant response and administration burdens for patient and nurse [ 22 ], especially when repeated administration is needed for monitoring or following the pattern of a delirium episode, thereby limiting their clinical usefulness. Additionally, the interpretation of the resulting score is not always clear and can be misleading, as the severity of the cognitive impairment, level of formal education, fatigue, and characteristics of the testing environment adversely influence performance on mental status testing [ 23 ]. Moreover, nurses favor instruments that are more heavily based on the observation of behavior than formal testing, those that can be used in the course of usual practice, repetitively and without respondent burden [ 24 ]. In response to this criticism, Neelon and colleagues [ 22 , 25 ] developed the NEECHAM Confusion Scale. The advantages of the NEECHAM over standard cognitive testing are that the NEECHAM can be rapidly completed (during 10 minutes) by the nurse at the bedside using a structured database derived during routine nursing assessments and interactions with patients. Because the NEECHAM places a minimal response burden on the patient, and because it is comprised of items that have no learning effect, testing can be repeated at frequent intervals to monitor changes in the patient's cognitive status. This can be of major importance, since symptoms of a delirium vary in intensity and fluctuate diurnally [ 1 , 13 ]. Additional advantages of the NEECHAM are that it can detect delirium in its early stage, and is sensitive to both the hyperalert-hyperactive and hypoalert-hypoactive variants of delirium [ 22 ]. Several studies have reported acceptable psychometric properties of the original English version [ 22 , 25 - 27 ] and of a Swedish translation [ 28 ] of the NEECHAM Confusion Scale (Table 1 ). The aim of the current study was to determine the reliability, validity and diagnostic value of the Flemish translation of the NEECHAM Confusion Scale. Table 1 Overview of psychometric properties of the NEECHAM Confusion Scale Author Sample Reliability Validity Diagnostic Values Champagne et al. [26] n = 35 (21 hospitalized and 14 nursing home patients) age ≥ 69 years Internal consistency = 0.85 Interrater reliability = 0.96 Test-retest reliability = 0.98 Concurrent: Correlation with MMSE = 0.81 Construct: Factor analysis revealed 2 factors (% of variance explained not given) Neelon et al. [25] n = 158 medical patients age ≥ 65 years At least two of three clinical indicators (MSP 1 , MMSE 2 , DSM-III 3 ) = reference criterion; and NEECHAM cut-off = 24/25: sensitivity: 95% specificity: 78% predictive value: 57% false positive: 17% false negative: 3% Neelon et al. [22] Sample 1: n = 168 medical patients age ≥ 65 years Sample 1: Internal consistency = 0.90 Sample 1: Concurrent: * Correlation with MMSE = 0.87 * Correlation with DSM-III-R total score = -0.91 * Correlation with diagnosis of delirium by DSM-III-R diagnostic criteria = -0.70 Construct: Factor analysis revealed 2 factors (explaining 72% of variance) Sample 2: n = 258 medical patients age ≥ 65 years Sample 2: Internal consistency = 0.90 Sample 2: Concurrent: * Correlation with DSM-III-R total score = -0.86 * Correlation with diagnosis of delirium by DSM-III-R diagnostic criteria = -0.54 Construct: Factor analysis revealed 2 factors (% of variance explained not given) Csokasy [27] n = 19 patients on intensive care unit age ≥ 65 years Internal consistency = 0.81 Concurrent: * correlation with oxygen saturation below 91 = 0.93 * correlation with DSM-III-R total score = 0.68 Johansson et al. [28] n = 73 patients with hip fracture age ≥ 60 years Before surgery: Internal consistency = 0.73 After surgery: Internal consistency = 0.82 Before surgery: Construct: Factor analysis revealed 2 factors (explaining 57.4% of variance) and 3 factors (explaining 69% of variance) After surgery: Construct: Factor analysis revealed 2 factors (explaining 61.5% of variance) and 3 factors (explaining 73.6% of variance) 1 MSP = chart report of mental status problem 2 MMSE = Mini-Mental-State Examination 3 DSM-III-R = Diagnostic and statistical manual of mental disorders, 3 rd ed., revised Methods Design and sample This study is a secondary data analysis of the experimental group of a larger intervention study [ 29 ] that was approved by the local ethics committee of the University Hospitals of Leuven, Belgium. Subjects were recruited at the emergency room of the University Hospitals of Leuven (Belgium). Patients were eligible if they had a traumatic fracture of proximal femur (intra- and extra-capsular) and were hospitalized to one of the two traumatological nursing units within 24 hours after surgery (convenience sampling). Subjects had to be 65 years of age or older, Flemish-speaking, and had to give informed consent. Exclusion criteria were multiple trauma, concussion of the brain, pathological fractures, surgery occurring later than 72 hours after admission, aphasia, blindness, deafness and fewer than 9 years of formal education (the latter because of validity concerns of the MMSE with lesser educated subjects [ 23 ]). A total of 60 older patients with a hip fracture were admitted to the study during a 5-month period. Six patients were excluded from analyses because of missing data for demographic variables and/or co-morbidity. From the remaining 54 patients, the majority was female (81.5%) with a mean age of 80.9 years (SD = 7.8) (Table 2 ). Table 2 Demographic variables and co-morbidity (n = 54) Mean age in years (SD) 80.9 (7.8) Gender, n (%) Male 10 (18.5) Female 44 (81.5) Marital status, n (%) Single 6 (11.1) Married 16 (29.6) Widowed 31 (57.4) Type of hip fracture, n (%) Intra-capsular 7 (13.0) Extra-capsular 47 (87.0) Co-morbidities, n (%) Dementia 7 (12.9) Depression 5 (9.3) Cardiac 8 (14.8) Pulmonary 8 (14.8) Hypertension 11 (20.4) Diabetes 9 (16.7) Other 15 (27.7) Instruments The NEECHAM Confusion Scale consists of nine components organized in three subscales, allowing the detection not only of changes in cognitive status, but also changes and different patterns of physiological and behavioral manifestations over short time frames [ 22 ]. Subscale 1 (« Processing ») (0 to 14 points) focuses on the primary components of cognition: attention/alertness, verbal and motor command of information, and memory and orientation. Subscale 2 (« Behavior ») (0 to 10 points) measures behavioral manifestations associated with more physical performance functions: appearance/posture control, sensorimotor performance and verbal manifestations accompanying or heralding deliriumlike syndromes. Subscale 3 (0 to 6 points) consists of physiological control and stability, including vital functions, oxygenation, and continence. These last 3 components in subscale 3 are an attempt to link the cognitive and behavioral symptoms of delirium with alteration in various physiologic parameters to facilitate the identification of the underlying etiolog(y)(ies) [ 22 , 24 ]. The NEECHAM total score is calculated by adding the scores for the three subscales, and ranges from 0 (minimal responsiveness) to 30 (normal function) points. The following cut-points are suggested for clinical practice: 0 to 19 indicates acute and moderate confusion, to severe confusion and/or delirium, to nonresponsiveness; 20 to 24 indicates mild disturbance in information processing, to mild or early development of confusion and/or delirium; 25 to 26 indicates risk for confusion and/or delirium; and scores equal or greater than 27 indicate normal cognitive functioning or the absence of confusion and/or delirium [ 30 , 31 ]. The NEECHAM instrument was translated into Flemish by the first author (KM) and examined by the fourth co-author (JG). Both authors have good knowledge of English and wide experience in assessing forms for grading cognitive and behavioural state. Delirium was assessed by using the English version of the Confusion Assessment Method (CAM) [ 32 ]. The CAM is an easy to use diagnostic algorithm to efficiently and effectively detect delirium that was developed from the DSM-III-R criteria for delirium [ 33 ]. The CAM consists of 9 diagnostic criteria of which 4 are considered core criteria for delirium: (1) acute onset and fluctuation, (2) inattention, (3) disorganized thinking, (4) altered level of consciousness. The first two and at least one of the other two criteria must be fulfilled to classify a patient as delirious. The diagnostic value of the CAM is excellent when using psychiatric diagnosis as the gold standard. More specifically, the CAM has a 94%–100% sensitivity, 90%–95% specificity, 91%–94% positive predictive value and 90%–100% negative predictive value [ 32 ]. Interobserver reliability (Kappa) ranged between 0.81–1.0. The CAM also was shown to have convergent agreement with other mental tests, including Mini-Mental State Examination (k = 0.64) [ 21 ], Visual Analog Scale for Confusion (k = 0.82) [ 34 ], Digit Span test (k = 0.66) [ 35 , 36 ] and a memory recall test (k = 0.59) [ 37 ]. To measure the severity of delirium, scoring of the CAM was modified in consultation with the originator of the instrument [ 29 ]. Six items of the CAM (inattention, disorganized thinking, disorientation, memory impairment, perceptual disturbances, and psychomotor agitation/retardation) were scored as 0, 1 or 2 points indicating absent, present in mild form or present in severe form, respectively, rather than merely present or absent. A seventh item (altered level of consciousness) was scored as alert (0 points), vigilant or lethargic (1 point), and stupor or coma (2 points). The total score ranges from 0 to 14, in which a higher score indicates greater severity of delirium. Cognitive impairment was assessed using the Mini-Mental State Examination [ 21 ], an 11-item instrument measuring orientation, memory, attention, the ability to name, ability to follow verbal and written commands, ability to write a sentence spontaneously, and ability to copy a geometric figure. The total score is obtained by summing the correct responses, yielding a score between 0 to 30 (total score below 24 indicating cognitive impairment). The MMSE is a reliable and valid measurement instrument for screening and rating severity of cognitive functioning, reported to be psychometrically robust across settings, and despite gender, race, ethnicity, and social class [ 19 , 23 ]. Procedures All nurses of the emergency department and both traumatological units were trained by the first author (KM) in the use of the NEECHAM Confusion Scale. Using this instrument in the standard nursing care plans of older patients with hip fracture was part of a larger intervention program for delirium [ 29 ]. Patients were evaluated by the nurses using the NEECHAM within 12 hours of admission to the emergency department, and during the morning shift on the first, third, fifth and eighth postoperative days at the traumatological units. For measuring oxygen saturation and vital function (Subscale 3 of the NEECHAM), a noninvasive pulse-oximeter (NPB-40, Mallinckrodt Belgium NV) and standard clinical methods were used, respectively. The MMSE and CAM were administered by 3 trained research nurses on the same measurement points of the NEECHAM evaluation. Statistical analyses Fifty-four patients were each assessed at 5 data collection measurement points, generating a total of 270 observations. However, not all NEECHAM evaluations by the registered nurses could be used, because of incomplete data. Therefore, only 194 observations (30, 36, 40, 43 and 45 observations on measurement points 1 till 5, respectively) were eligible for statistical analysis. First, the distribution of the total NEECHAM scores and items were examined. Further, the internal consistency of the scale was tested by calculating Cronbach's alpha, item-total and inter-item correlations. To explore the inter-item correlations, principal components analysis was performed. Paired observations of the nurses and research nurses were used to test concurrent validity, meaning that total NEECHAM scores were correlated with total scores on the same measurement points on the modified version of the CAM and on the MMSE, respectively (Pearson correlation coefficient). To explore the diagnostic value of the NEECHAM scale to identify subjects as being delirious or not, a receiver operating characteristic curve (ROC) was constructed and sensitivity, specificity, positive and negative predictive values and accuracy were examined for all possible total NEECHAM scores (0–30) using the CAM diagnostic algorithm as gold standard. Results NEECHAM Scores and item distribution The total score on the NEECHAM scale ranged from 6 to 30, with a mean of 26.7 (SD = 4.99). According to the NEECHAM classification a total score less than or equal to 19 was found for 17 observations (8.8%). Another 17 observations (8.8%) ranged between 20 and 24 and 25 observations (12.9%) ranged between 25 and 26. For all other observations (n = 135, 69.5%) scores were 27 or greater. Further, a full range of scores was observed for all items (Table 3 ), except for the item "command" processing for which an almost full range was observed (score 0 not observed), indicating good responsiveness for all items. Table 3 Total NEECHAM scores and item distribution (n = 54 patients, 194 test occasions) NEECHAM item Mean SD Minimum score Maximum score Attention processing (score 0 to 4) 3.65 0.74 0 4 Command processing (score 0 to 5) 4.56 0.94 1 5 Orientation processing (score 0 to 5) 4.42 1.08 0 5 Appearance behavior (score 0 to 2) 1.77 0.49 0 2 Motor behavior (score 0 to 4) 3.62 0.90 0 4 Verbal behavior (score 0 to 4) 3.69 0.75 0 4 Vital function stability (score 0 to 2) 1.72 0.53 0 2 Oxygen saturation stability (score 0 to 2) 1.74 0.53 0 2 Urinary continence (score 0 to 2) 1.52 0.76 0 2 Total NEECHAM score (score 0 to 30) 26.69 4.99 6 30 Reliability Internal consistency of the scale was tested by calculating Cronbach's alpha, inter-item and corrected item-total correlations. The alpha coefficient for the total NEECHAM score was high (0.88) and increased by omitting the items 'vital' and 'oxygen' (Table 4 ). Table 4 Pearson Item-total correlation coefficients of NEECHAM Confusion Scale (n= 54 patients, 194 test occasions) NEECHAM item Corrected item-total correlation Total alpha if item is deleted Attention 0.8148 0.8550 Command 0.8827 0.8448 Orientation 0.8579 0.8484 Appearance 0.7113 0.8701 Motor 0.8031 0.8537 Verbal 0.8689 0.8499 Vital -0.0403 0.9091 Oxygen 0.1799 0.8982 Continence 0.5408 0.8777 For item-total correlations (Table 4 ), the processing (attention, command, orientation) and behavior (appearance, motor, verbal) items correlated strongly with the sum of the other items (Pearson r > 0.7113), while urinary continence and both vital function and oxygen saturation correlated modestly (Pearson r = 0.5408) and weakly (Pearson r < 0.1799), respectively. For the item vital function, the correlation was even negative. Pearson inter-item correlation coefficients among the items ranged from 0.0049 to 0.8830 (Table 5 ). For oxygen saturation, low correlations were found between all other items (Pearson r from 0.0766 to 0.1995). Also vital function showed low and negative correlations with most of the other items (Pearson r from -0.0434 to -0.1329) except for the items appearance, oxygen and continence which were still low but positive (Pearson r = 0.0049, 0.1995, 0.0856, respectively). All other items correlated moderately to highly (Pearson r from 0.3791 to 0.8830). Table 5 Pearson Inter-item correlation coefficients of NEECHAM Confusion Scale (n = 54 patients, 194 test occasions) ITEMS Attention Command Orientation Appearance Motor Verbal Vital Oxygen Continence Attention 1.0000 Command 0.8428 1.0000 Orientation 0.7955 0.8421 1.0000 Appearance 0.6190 0.7076 0.6670 1.0000 Motor 0.6998 0.7811 0.7526 0.6631 1.0000 Verbal 0.8155 0.8830 0.8223 0.6831 0.7805 1.0000 Vital -0.1329 -0.0950 -0.0893 0.0049 -0.0434 -0.0691 1.0000 Oxygen 0.1274 0.1346 0.1725 0.0869 0.1815 0.1321 0.1995 1.0000 Continence 0.4756 0.5056 0.5384 0.3791 0.4620 0.4748 0.0856 0.0766 1.0000 Construct validity To further explore the inter-item correlations, principal components analysis was performed. Two eigenvalues greater than 1 were found, suggesting that, at most, only 2 sources contributed substantially to the shared variance among the items. A two-component solution accounted for 70.8% of the total variance in the items, in which the first and second component explained 57.3% and 13.5%, respectively. The items attention, command, orientation, appearance, motor behavior, verbal behavior and urinary incontinence loaded fairly high (0.6105) to high (0.9411) on the first component (Table 6 ). Vital function and oxygen saturation showed relatively high correlations (> 0.7080) with the second component. Urinary incontinence had a crossloading of 0.1560 on component 2 and oxygen saturation had a crossloading of 0.1872 on component 1, both indicating some minor association with that respective dimension. The communalities, meaning the proportion of the variance of that variable explained by both components, ranged for most variables between 0.6228 and 0.8892. In other words, these variables had relatively high to high relation with the 2 components. Communalities for oxygenation and vital function were rather low, but still above commonly accepted lower limits (0.3971 and 0.5364, respectively). Orthogonal and oblique axis rotation did not improve the initial unrotated solution. Table 6 NEECHAM Confusion Scale Component Loadings and Communalities (n = 54 patients, 194 test occasions) Loadings NEECHAM item Component I Component II Communalities Attention 0.9411 -0.0590 0.7991 Command 0.9246 -0.0430 0.8892 Orientation 0.9155 -0.0185 0.8385 Appearance 0.8886 -0.0967 0.6228 Motor 0.8682 0.0298 0.7546 Verbal 0.7891 0.0042 0.8568 Vital -0.0715 0.8205 0.5364 Oxygen 0.1872 0.7081 0.3971 Continence 0.6105 0.1560 0.6794 Concurrent validity To test concurrent validity of the NEECHAM Confusion Scale, paired observations of total NEECHAM scores of the nurses were correlated with total MMSE scores and total CAM scores of the researchers, respectively. A strong positive correlation coefficient (r = 0.75) was found with the MMSE, indicating that higher MMSE scores (better neurocognitive functioning) were related to higher NEECHAM scores (less severe confusion and/or delirium). Also, a strong negative correlation coefficient (r = -0.73) was found with the CAM, indicating that higher total CAM scores (more severe delirium) were related to lower total NEECHAM scores (more severe confusion and/or delirium). Diagnostic values A sensitivity analysis was performed by using ROC curve (Figure 1 ) and exploring sensitivities and specificities for different cut-offs of the total NEECHAM scores to determine the most optimal cut-off (table 7 ). ROC showed an area under the curve of 0.733 (Confidence Interval = 0.578–0.888). Based on ROC and diagnostic value of the different cut-offs, the most optimal cut-off point was found to be 27. This cut off showed a fairly good sensitivity of 76.9%, an acceptable specificity (64.6%) and a high negative predictive value (97.5%). However, the positive predictive value was low (13.5%), though the accuracy was acceptable (65.5%). Table 7 Sensitivities and specificities for a subset of threshold values of the NEECHAM total score (n = 54 patients, 194 test occasions) CAM algorithm NEECHAM total score Delirious N Not delirious n Total N ≤ 24 7 27 34 ≥ 25 6 154 160 Total 13 181 194 Sensitivity [(7/13) × 100] = 53.8%; Specificity [(154/181) × 100] = 85.1%; Predictive value of a positive test [(7/34 × 100] = 20.6%; Predictive value of a negative test [(154/160) × 100] = 96.3%; Accuracy [(7+154/7+27+6+154) × 100] = 83.0% ≤ 25 8 31 39 ≥ 26 5 150 155 Total 13 181 194 Sensitivity = 61.5%; Specificity = 82.9%; Predictive value of a positive test = 20.5%; Predictive value of a negative test = 96.8%; Accuracy = 81.4% ≤ 26 9 50 59 ≥ 27 4 131 135 Total 13 181 194 Sensitivity = 69.2%; Specificity = 72.4%; Predictive value of a positive test = 15.2%; Predictive value of a negative test = 97.0%; Accuracy = 72.2% ≤ 27 10 64 74 ≥ 28 3 117 120 Total 13 181 194 Sensitivity = 76.9%; Specificity = 64.6%; Predictive value of a positive test = 13.5%; Predictive value of a negative test = 97.5%; Accuracy = 65.5% ≤ 28 10 84 94 ≥ 29 3 97 100 Total 13 181 194 Sensitivity = 76.9%; Specificity = 53.6%; Predictive value of a positive test = 10.6%; Predictive value of a negative test = 97.0%; Accuracy = 55.2% ≤ 29 11 118 129 30 2 63 65 Total 13 181 194 Sensitivity = 84.6%; Specificity = 34.8%; Predictive value of a positive test = 8.5%; Predictive value of a negative test = 96.9%; Accuracy = 38.1% Figure 1 Receiver Operating Characteristic curve (ROC; Eng [41]). Discussion The purpose of this study was to determine the validity and diagnostic values of the Flemish translation of the NEECHAM Confusion Scale. In line with previous evidence, the findings of this study support the validity of the NEECHAM Confusion Scale (Table 1 ). Diagnostic values however were somewhat different than evidence from previous studies. Reliability analyses showed a high internal consistency (alpha coefficient = 0.88). Only Neelon et al. [ 22 ], reported a higher internal consistency (0.90), with the same physiological items (vital function and oxygen saturation) showing low item-total and inter-item correlations. As a matter of fact, item-total correlation for vital function in this study was extremely low and even negative (-0.0430 compared to 0.30 and 0.32 in the study of Neelon et al. [ 22 ]). In comparison with our study, Neelon et al. [ 22 ] tested the scale in two samples (n= 168 and 258, respectively) and data were not based on repeated measurements. Based on the low item-total and inter-item correlations of vital function and oxygen saturation, it could be suggested to delete these two items from the NEECHAM Scale. When we deleted the items 'vital' and 'oxygen' in our study, internal consistency of the NEECHAM increased as expected. Further evidence for this approach was our factor analyses which confirmed the findings of Neelon et al. [ 22 ] and Johansson et al. [ 28 ] showing that the vital function and oxygen items do not load on the general factor that includes the seven other items. The inclusion of such specific physiologic data seems to artificially constrain the scale's use [ 19 ] but needs to be further evaluated in future studies before excluding these items from the NEECHAM Scale. In line with Neelon et al. [ 22 ] and Johansson et al. [ 28 ], the continence item correlated with all items of the original subscale 1 (processing) and 2 (behavior). The continence item also loaded fairly high on the first component in the factor analysis. These findings suggest that continence rather seems to be an important indicator for delirium than belonging to a 3 rd subscale (e.g. physiological changes). Concurrent validity was good. The high correlations between the NEECHAM and the MMSE and CAM instruments (r = 0.75 and -0.73, respectively) – together with the variation observed in NEECHAM total scores and item distribution – indicate that the NEECHAM is a valuable instrument for rating severity of both cognitive dysfunctioning and delirium, respectively. Although values for sensitivity (76.9%) and specificity (64.6%) in our study are lower than those reported by Neelon et al. [ 25 ] (Table 1 ), these values are still within acceptable ranges. The area under the ROC curve, indicating how well the test separates the group being tested into those with and without delirium, was found to be fairly good (= 0.773). Positive predictive value was however low (13.5%) indicating the need for further studies to optimize this aspect of the scale. It has however to be mentioned that the scale was successfully used by resource nurses in an intervention study as demonstrated by Milisen et al. [ 29 ]. We found an optimal cut-point of 27 (see table 7 ) for detection of delirium and this in contrast with Neelon et al. [ 22 ] who found an optimal cut-point of 24 (see table 1 ). However, whether our cut-point or Neelon's [ 22 ] cut-point is ideal for different hospital settings is not known. Therefore, future studies should include a sensitivity analysis to explore which cut-off point is most valuable to detect delirium for different in-hospital patient populations. Neelon's study focused on older patients hospitalized for acute medical illness whereas our study included older post surgical hip fracture patients. Further, rather than merely comparing single scores with a threshold value, identifying a patient at risk for delirium may also be addressed by examining an acute decrease in total NEECHAM scores over time (e.g. 2 or more points), as recently shown by Matsushita et al. [ 31 ]. Another aspect that deserves further consideration is the reference criteria used. Neelon et al. [ 25 ] included at least two of the following clinical indicators: chart report of mental status problem, low MMSE score and presence of DSM-III symptoms for delirium versus use of the CAM-criteria in the current study. When however compared to other cognitive screening instruments or criteria published so far, the CAM appears to have more favorable sensitivity and specificity for detection of delirium [ 38 ]. Limitations of our study are as follows. Generalization of the findings may be hampered by the sampling methods (secondary analysis of data collected for the experimental group of a non-randomized trial) and the sample (restriction to older patients with hip fracture). Therefore, further studies including patients presenting a broad range of ages and clinical conditions (e.g. surgical and non-surgical patients) would benefit the generalization of findings. Our analyses were based on 194 observations in 54 older patients. Admittedly, these observations were not independent which could have potentially influenced the results. However, the main objective of this study was descriptive, not inferential, and this is not expected to be seriously affected by non-independence [ 39 , 40 ]. Furthermore, our findings concur with previous published evidence on reliability, validity and diagnostics value of the scale. It also needs to be mentioned that low prevalence of delirium (13/194 = 6.7%) could be an exploratory factor for the low positive predictive value. The higher the disease prevalence the greater the probability that a positive result will be 'correct'. Studies in samples with various prevalences of delirium are therefore indicated to test robustness of the findings. Conclusion This study contributes to the validation process of the (Flemish version of the) NEECHAM Confusion Scale. In line with previous evidence, this instrument seems to be a promising tool for clinical practice to screen for delirium in older hospitalized patients and to monitor for the course of cognitive dysfunction and severity of delirium. The minimal response burden on patients, even when used repeatedly, is certainly also an asset in clinical settings. Further validation studies (preferably not using added observations) in diverse clinical populations are however needed. Competing interests The author(s) declare that they have no competing interests. Authors' contributions KM, MDF, IAL, PLOB proposed the original idea for the study and designed the study protocol. KM and JG contributed to the translation part of the study. KM and AH oversaw field activity and executed statistical analyses. KM, MDF, AH, JG, IAL, PLOB, and SDG were involved in the interpretation of data. KM drafted the manuscript. MDF and SDG revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079888

Two cases of "cannabis acute psychosis" following the administration of oral cannabis

Background Cannabis is the most commonly used illegal drug and its therapeutic aspects have a growing interest. Short-term psychotic reactions have been described but not clearly with synthetic oral THC, especially in occasional users. Case presentations We report two cases of healthy subjects who were occasional but regular cannabis users without psychiatric history who developed transient psychotic symptoms (depersonalization, paranoid feelings and derealisation) following oral administration of cannabis. In contrast to most other case reports where circumstances and blood concentrations are unknown, the two cases reported here happened under experimental conditions with all subjects negative for cannabis, opiates, amphetamines, cocaine, benzodiazepines and alcohol, and therefore the ingested dose, the time-events of effects on behavior and performance as well as the cannabinoid blood levels were documented. Conclusion While the oral route of administration achieves only limited blood concentrations, significant psychotic reactions may occur.

Background As several countries in Europe have taken policies to decrease the penalties for cannabis possession, many people especially young persons have interpreted this move as giving support to consider cannabis as a benign drug [ 1 ]. However as stated by several reports cannabis is not a harmless substance and requires urgent attention considering public health issues such as car driving for example [ 2 ]. The relationship between Cannabis and acute psychosis is another important issue. In Pakistan and also India, Bhang, a beverage made from an infusion of cannabis leaves, and flowering tops combined with milk and nuts is reported to frequently induce psychotic manifestations among consumers [ 3 ]. Presenting symptoms include grandiosity, excitement, hostility, uncooperativeness, disorientation, hallucinatory behaviour and unusual thought content [ 3 ]. Recently five large-scale longitudinal studies and a systematic review have shown that cannabis use in adolescence is associated with a two-to threefold increase in the relative risk of later developing schizophrenia [ 4 ]. Furthermore short-term psychotic reactions, particularly in naive users have also been reported. Thomas [ 5 ] describes that one in seven people reported psychotic-like symptoms. Such reactions are usually acute, transient, self-limited however very unpleasant ("hearing voices, becoming convinced that someone is trying to harm you or that you are persecuted") [ 6 ]. But cannabinoids are considered able to trigger long-lasting psychotic decompensations in predisposed individuals, which may in part account for the epidemiological association described between cannabis consumption and psychotic disorders [ 7 , 8 ]. The therapeutic aspects of cannabis represent additional issues, as they are in constant development since several years. Synthetic THC (dronabinol) is available for restricted medical use in the USA since 1985. Nabilone, a synthetic THC analogue, is licensed in UK for the treatment of nausea and vomiting caused by chemotherapy unresponsive to usual anti-emetics. Clinical applications actually include nausea and vomiting, muscle spasticity in demyelinating diseases, loss of appetite in cancer and AIDS, pain, insomnia, asthma as well as other applications [ 9 ]. As these oral medications are becoming increasingly available, we think it is useful to report two cases of severe psychological sides effects, especially considering the lack of data in the literature on psychotic symptoms associated with oral synthetic or natural THC. Case reports We report two cases out of 8 healthy male volunteers who were included in a double blind crossover clinical study, approved by the ethics committee of the Department of Internal Medicine of the University of Lausanne. All subjects had to be occasional but regular cannabis users. Their urines were controlled to be negative for any drug of abuse (cannabis, opiates, amphetamines, cocaine, benzodiazepines) before each study period. The presence of ethanol was checked using a breathalyzer. All of them provided their written informed consent. This study was carried out to assess the effects of delta-9-tetrahydrocannabinol (THC) on psychomotor function and driving performance. It compared a medication containing 20 mg dronabinol (Marinol R ), and 2 hemp milk decoction containing either a medium (15.8 mg average dose determination) or a high dose of THC (45.7 mg) with matched placebos. The hemp plant fragments containing 1.5 % THC and 4.4 % THC-A were provided by Hiscia institute in Arlesheim, Switzerland. After administration, blood was sampled at regular intervals for cannabinoids determination by gas chromatography coupled with mass spectrometry (GC-MS-NCI). Clinical observations and 2 psychometric tests (roadsign recognition speed and accuracy on a tracking task) were also carried out. Furthermore, the subjects were asked to report their willingness to drive and the subjective effects on a VAS scale extending from 0 to 10 cm. The effects were assessed against placebo. These 2 cases were withdrawn from the study because of adverse events. We consider them worth reporting for the following reasons: in contrast to most other case reports where circumstances and blood concentrations are not known, our two cases reported here happened under defined clinical setting: the ingested dose, the time-events of effects on behavior and performance as well as the cannabinoid blood levels are fully documented. In addition, the consumption of other psychotropic major drugs could be ruled out. Case 1 The first subject was a 22-year-old medical student (weight: 65.3 kg, height: 1.82 m) and occasional cannabis smoker (about once per week). One hour after the administration of 20 mg of dronabinol, he started to laugh a lot and after 90 minutes, he manifested a severe anxiety with symptoms of derealisation and depersonalization. He reported "watching himself lying on the bed" and repeated several times the same questions at just a few minutes interval. Starting 2.5 hours after ingestion of dronabinol, and at the 4 hours and 5.5 hours post-ingestion series of tests, he was unable to perform the psychomotor tasks, despite reporting of weakening of symptoms approximately 165 minutes after their initiation. Before going to sleep (more than 10 hours after ingestion), he again felt a transient feeling of irrational anxiety and loosing the perception of his body. The next day he was well but a bit tired. Figure 1 shows the evolution of his blood concentrations of cannabinoids after ingestion of 20 mg dronabinol. At the time of strong adverse effects, the blood levels of THC and 11-OH-THC reached a concentration of 1.8 and 5.2 ng/mL, respectively. The subject reported a strong feeling of intoxication (figure 2 ). He also evaluated that his driving capability was strongly impaired (figure 2 ). Figure 1 Whole blood concentrations of THC, 11-OH-THC (actives metabolites) and THC-COOH (inactive metabolite) after oral intake of 20 mg dronabinol and of a hemp milk decoction containing traces of cannabinoids (placebo). Figure 2 Subjective effects (feeling of intoxication or driving capability) after oral intake of 20 mg dronabinol. The subject reported no feeling of intoxication or of driving impairment after ingestion of the placebo. Case 2 A 22-year-old student, also an occasional cannabis smoker (about twice a month), felt paranoid delusions with severe anxiety one hour after the administration of 16.5 mg of a THC decoction, and became suspicious during the experiment. He thought the investigators were concealing some problems. He was unable to perform the psychometric tests (roadsign recognition speed and accuracy on a tracking task) at the 1 hour and 2.5 hours post-ingestion series of tests. These effects persisted up to 4 hours after ingestion and weakened over the next 3 hours. The feeling was very unpleasant in comparison with that experienced after his usual smoking cannabis consumption. On the next day, he was well, with no recurrence. The time-concentration curves for the major cannabinoids were similar to those observed after ingestion of 20 mg dronabinol. One hour after drinking the hemp decoction, the THC and 11-OH-THC blood levels were of 6.2 and 3.9 ng/mL, respectively. Similarly to the other volunteer, he also indicated a strong feeling of intoxication and a very important decrease in his self-reported capacity to drive (results not shown). Conclusion A temporary form of drug-induced psychotic reaction after administration of oral cannabis has occurred in these two cases. Cannabis psychosis is the term proposed in the literature [ 10 ]. In 1958, Ames [ 11 ] reported in an experimental design with 10 subjects psychological symptoms such as severe anxiety, panic attacks, paranoid delusions and depersonalization. Talbott [ 12 ] in 1969 described 12 soldiers in Vietnam who had disorientation and hallucinations after their first use of cannabis. In Germany, 19 cases of toxic psychosis were reported after hashish use [ 13 ] and in Calcutta, Chopra [ 14 ] described retrospectively 200 patients hospitalized after the ingestion of large dose of cannabis between 1963 and 1968. Other reports in different countries showed similar features after bhang ingestion [ 3 , 15 ]. It usually results from taking large amount of the drug, generally in food or drink. The symptoms have some similarity with paranoid schizophrenia, which could raise the hypothesis that " symptoms of schizophrenic illness might be caused by an abnormal over-activity of endogenous cannabinoid mechanism in the brain" (Iversen [ 10 ] citing Emrich [ 16 ]). However because of the poor quality of information on previous cannabis experience, cannabis dose intake, other drug consumption and previous psychiatric comorbidity, some commentators have criticized these case series [ 12 , 17 , 18 ]. Case-control studies have been conducted comparing people with cannabis psychosis with persons suffering from schizophrenia [ 19 - 21 ]. However the results were inconsistent due in part to the small sample size of these studies. The originality of our two cases is that they were observed in an experimental setting, and therefore adds more evidence for the ability of oral cannabis to produce psychotic symptoms. In both our subjects, the effects appeared 1 hour to 1.5 hours after oral drug intake and lasted for 3 to 4 hours. Dronabinol (synthetic THC) is reported to have an onset of action at approximately 0.5 to 1 hour and peak effects between 2 and 4 hours. Psychoactive effects last 4 to 6 hours but the appetite stimulant effect may continue for 24 hours [ 22 ]. The issue of THC dose level is very important in terms of public health. A traditional cigarette of herbal cannabis in the 1960s and 1970s contained 1–3% THC: for a joint made of 750 mg of cannabis plant, the corresponding THC amount was 7 to 20 mg. However, the actual amount of cannabis taken up (i.e. the percent delivery to the respiratory tree) strongly depends on the smoking technique; it has been reported to reach approximately 50% [ 23 ]. Modern cigarettes (joint) based on intensive cannabis selection and improvement in plant cultivation contain 6 to 30% THC. Therefore, an average joint would correspond to 75 mg to 225 mg of THC! [ 22 ]. Through smoking, a 3.5% marijuana cigarette with about 900 mg plant materials can achieve plasma concentration in the range of 50 to 100 ng/ml. The maximum psychotropic effect or "high" occurs faster after smoking than by the oral route. Smoking is therefore the preferred route of cannabis administration for young users. Psychomotor function is considered to be obviously impaired above 10 ng/ml plasma THC for smoking cannabis. However in our two cases, the oral administration of cannabis produced circulating THC concentrations much lower than 10 ng/ml. We suggest several explanations for these differences. Firstly, the oral administration produces more active metabolite (11-OH-THC), which could more efficiently reach the effect site than THC. Secondly, as suggested by Chaudry [ 3 ], consuming oral cannabis may produce more potent, yet unknown psychotomimetic metabolites of THC. Thirdly, the slow absorption kinetics produces sustained plateau levels in the blood, which could influence the body and brain distribution. In a cocaine fatality, Giroud [ 24 ] found that THC and OH-THC were in higher concentration in brain than in blood. Finally, Leweke [ 25 ] in a study including 17 healthy volunteers found also one case that suffered a two-hour episode of paranoid psychotic state following the administration of dronabinol with a lower dose than our study. Furthermore D'Souza [ 26 ] administrating intravenous THC to 22 healthy subjects in a double blind randomised clinical trial found a range of transient symptoms resembling those seen in endogenous psychosis. At last, it is important to differentiate these transient psychotic states with spontaneous resolution from the type of psychosis that persist beyond the persistence of drug in the brain, therefore probably indicating a worsening of an underlying pathologic problem. In conclusion, doctors and users should be aware of the increasing availability of oral cannabis in "special" drinks or food as well as in medications under development. While the oral route of administration achieves only limited blood concentrations, significant psychotic reactions may occur. An increased incidence of psychotic episodes might be induced by this new trend and requires attention regarding this phenomenon in a public health perspective. Competing interests The author(s) declare that they have no competing interests. Pre-publication history The pre-publication history for this paper can be accessed here:

D:\keerthana\PMC001xxxxxx\PMC1079888.xml

1079889

A comprehensive update of the sequence and structure classification of kinases

Background A comprehensive update of the classification of all available kinases was carried out. This survey presents a complete global picture of this large functional class of proteins and confirms the soundness of our initial kinase classification scheme. Results The new survey found the total number of kinase sequences in the protein database has increased more than three-fold (from 17,310 to 59,402), and the number of determined kinase structures increased two-fold (from 359 to 702) in the past three years. However, the framework of the original two-tier classification scheme (in families and fold groups) remains sufficient to describe all available kinases. Overall, the kinase sequences were classified into 25 families of homologous proteins, wherein 22 families (~98.8% of all sequences) for which three-dimensional structures are known fall into 10 fold groups. These fold groups not only include some of the most widely spread proteins folds, such as the Rossmann-like fold, ferredoxin-like fold, TIM-barrel fold, and antiparallel β-barrel fold, but also all major classes (all α, all β, α+β, α/β) of protein structures. Fold predictions are made for remaining kinase families without a close homolog with solved structure. We also highlight two novel kinase structural folds, riboflavin kinase and dihydroxyacetone kinase, which have recently been characterized. Two protein families previously annotated as kinases are removed from the classification based on new experimental data. Conclusion Structural annotations of all kinase families are now revealed, including fold descriptions for all globular kinases, making this the first large functional class of proteins with a comprehensive structural annotation. Potential uses for this classification include deduction of protein function, structural fold, or enzymatic mechanism of poorly studied or newly discovered kinases based on proteins in the same family.

Background We restrict the definition of "kinase" to enzymes that catalyze the transfer of the terminal phosphate group from ATP (with a few exceptions, such GTP, as discussed below) to a substrate containing an alcohol, nitrogenous, carboxyl, or phosphate group as the phosphoryl acceptor. The substrate of a kinase can be a small molecule, lipid, or protein. Kinases play indispensable roles in numerous cellular metabolic and signalling pathways, and they are among the best-studied enzymes at the structural, biochemical, and cellular levels. Despite that all kinases use the same phosphate donor (in most cases, ATP) and catalyze apparently the same phosphoryl transfer reaction, they display remarkable diversity in their structural folds and substrate recognition mechanisms. This is probably due largely to the extraordinarily diverse nature of the structures and properties of their substrates. In order to investigate the relationship between structural fold and functional specificities in kinases, we carried out a comprehensive analysis of all available kinase structures and sequences [ 1 ] three years ago. This analysis surveyed more than 17,000 kinase sequences, which were classified into 30 families of homologous proteins. Furthermore, we found that 98% of these sequences fell into seven general fold groups with known structures. We were subsequently able to use this kinase classification scheme to analyze various aspects of kinase function and evolution, such as the shared catalytic and nucleotide substrate binding mechanisms across different kinase families and folds. Such protein classification has been shown to be in demand by biologists because it is a useful tool for analyzing various aspects of sequence/structure/function relationships in proteins, such as structure prediction or identification of functionally important residues. Given the rapid increase in the sizes of sequence and structure databases, it is also essential that a protein classification scheme remain stable over time. Ideally, the backbone of a classification scheme should not require fundamental revisions with the inclusion of additional information. The nearly three years that have passed since the completion of the initial kinase survey have seen a large influx of sequence and structural data due to large-scale projects such as genome sequencing and structural genomics initiatives, as well as functional studies by numerous individual research laboratories. There appear to be sufficient new developments in the field that warrant an update of the initial kinase survey and an evaluation of whether the families and fold groups identified in the original classification scheme still provide a comprehensive framework for all available kinase sequences. Here, we present an updated version of this kinase classification. This update serves two important purposes: to validate the robustness of the initial kinase classification scheme and to present, for the first time, a complete structural annotation of this large functional class of proteins. The updated kinase survey now includes over 59,000 sequences that belong to 25 families of homologous proteins. Despite that the total number of kinase sequences has increased more than three-fold, the framework of the original classification remains sufficient for describing all available kinase sequences. The initial survey also included large-scale structure predictions for kinases with unknown structure. The structures of several of these kinases have now been solved and the original fold group/family placements are shown to be correct. The two new kinase folds that have been characterized recently are discussed. Fold predictions were performed for those kinase families still lacking a homolog with solved structure. Results and discussion Overall, 59,402 sequences are classified into 25 families of homologous kinases. These kinase families are further assembled into 12 fold groups based on similarity of structural fold. 22 of the 25 families belong to 10 fold groups for which the structural fold is known. One additional family (polyphosphate kinase) is now associated with a predicted structural fold and presently forms a distinct fold group. The two remaining families are both integral membrane kinases and comprise the final fold group. Within a fold group, the core of the nucleotide-binding domain of each family has the same architecture, and the topology of the protein core is either identical or related by circular permutation. Homology between families within a fold group is not implied. The updated kinase classification is summarized in Tables 1 and 2 . For each fold group and family therein, the Pfam/COG members are listed as well as the specific kinase activities and a corresponding representative PDB or gi (NCBI gene identification) accession number. The total number of sequences in each family and fold group is also specified. The EC (Enzyme Commission) activities in bold have solved structures. Asterisks indicate that the first structure associated with that kinase activity was solved after the initial kinase classification was completed. Underlined entries are new kinase activities that were not included in the previous classification, although activities added due to re-organization or updating of the EC database are not underlined. It should be noted that the activity lists are not exhaustive, as they include only those kinase activities that have been annotated so far. Of the 190 kinases listed in the EC system over our chosen range (EC2.7.1.- through EC2.7.4.-), 126 activities can be placed in our kinase families. Enzymes that do not use ATP as the phosphate donor are intentionally excluded, except in cases where such kinases fall under existing families (for example, 2.7.1.90: diphosphate – fructose-6-phosphate 1-phosphotransferase in the phosphofructokinase-like family or 2.7.1.147: ADP-dependent glucokinase in the ribokinase-like family). Sequences for the remaining kinase activities have not been identified and thus are not included in our analysis, although it is possible that some of the unannotated kinases may be among the sequences with only general kinase function annotation, such as "similar to such-and-such kinase". Table 1 Classification of kinase activities by family and fold group, part 1. Group Family and PFAM/COG members Kinase Activity (E.C.) Representative PDB or gi Group 1: protein S/T-Y kinase/ atypical protein kinase/ lipid kinase/ ATP-grasp 23124 sequences protein S/T-Y kinase/ atypical protein kinase: COG0478, COG2112, PF00069, PF00454, PF01163, PF01633 22074 sequences 2.7.1.32 Choline kinase (*) PDB: 1cdk 2.7.1.37 Protein kinase 2.7.1.38 Phosphorylase kinase 2.7.1.39 Homoserine kinase 2.7.1.67 1-phosphatidylinositol 4-kinase 2.7.1.72 Streptomycin 6-kinase 2.7.1.82 Ethanolamine kinase 2.7.1.87 Streptomycin 3"-kinase 2.7.1.95 Kanamycin kinase 2.7.1.100 5-methylthioribose kinase 2.7.1.103 Viomycin kinase 2.7.1.109 [Hydroxymethylglutaryl-CoA reductase (NADPH 2 )] kinase 2.7.1.112 Protein-tyrosine kinase 2.7.1.116 [Isocitrate dehydrogenase (NADP+)] kinase 2.7.1.117 [Myosin light-chain] kinase 2.7.1.119 Hygromycin-B kinase 2.7.1.123 Calcium/calmodulin-dependent protein kinase 2.7.1.125 Rhodopsin kinase 2.7.1.126 [Beta-adrenergic-receptor] kinase (*) 2.7.1.129 [Myosin heavy-chain] kinase 2.7.1.135 [Tau protein] kinase (*) 2.7.1.136 Macrolide 2'-kinase 2.7.1.137 1-phosphatidylinositol 3-kinase 2.7.1.141 [RNA-polymerase]-subunit kinase 2.7.1.153 Phosphatidylinositol-4,5-bisphosphate 3-kinase 2.7.1.154 Phosphatidylinositol-4-phosphate 3-kinase lipid kinase: PF01504 321 sequences 2.7.1.68 1-phosphatidylinositol-4-phosphate 5-kinase PDB: 1bo1 2.7.1.127 1D-myo-inositol-trisphosphate 3-kinase (*) 2.7.1.140 Inositol-tetrakisphosphate 5-kinase 2.7.1.149 1-phosphatidylinositol 5-phosphate 4-kinase 2.7.1.150 1-phosphatidylinositol 3-phosphate 5-kinase 2.7.1.151 Inositol-polyphosphate multikinase 2.7.4.21 Inositol-hexakisphosphate kinase ATP-grasp: PF01326 729 sequences 2.7.1.134 Inositol-tetrakisphosphate 1-kinase PDB: 1dik 2.7.9.1 Pyruvate, phosphate dikinase 2.7.9.2 Pyruvate, water dikinase Group 2: Rossmann-like 17071 sequences P-loop kinases: COG0645, COG1618, COG1663, COG1936, COG2019, PF00265, PF00406, PF00485, PF00625, PF00693, PF01121, PF01202, PF01583, PF01591, PF01712, PF02223, PF02224, PF02283 7732 sequences 2.7.1.12 Gluconokinase (*) PDB: 1qf9 2.7.1.19 Phosphoribulokinase 2.7.1.21 Thymidine kinase 2.7.1.22 Ribosylnicotinamide kinase 2.7.1.24 Dephospho-CoA kinase 2.7.1.25 Adenylylsulfate kinase 2.7.1.33 Pantothenate kinase 2.7.1.37 Protein kinase (bacterial) 2.7.1.48 Uridine kinase (*) 2.7.1.71 Shikimate kinase 2.7.1.74 Deoxycytidine kinase (*) 2.7.1.76 Deoxyadenosine kinase 2.7.1.78 Polynucleotide 5'-hydroxyl-kinase (*) 2.7.1.105 6-phosphofructo-2-kinase 2.7.1.113 Deoxyguanosine kinase 2.7.1.130 Tetraacyldisaccharide 4'-kinase 2.7.1.145 Deoxynucleoside kinase (*) 2.7.1.156 Adenosylcobinamide kinase 2.7.4.1 Polyphosphate kinase 2.7.4.2 Phosphomevalonate kinase 2.7.4.3 Adenylate kinase 2.7.4.4 Nucleoside-phosphate kinase 2.7.4.8 Guanylate kinase 2.7.4.9 Thymidylate kinase 2.7.4.10 Nucleoside-triphosphate – adenylate kinase 2.7.4.13 (Deoxy)nucleoside-phosphate kinase 2.7.4.14 Cytidylate kinase 2.7.4.- Uridylate kinase phosphoenolpyruvate carboxykinase: COG1493, PF01293, PF00821 815 sequences 2.7.1.37 Protein kinase (HPr kinase/phosphatase) PDB: 1aq2 4.1.1.32 Phosphoenolpyruvate carboxykinase (GTP) 4.1.1.49 Phosphoenolpyruvate carboxykinase (ATP) phosphoglycerate kinase: PF00162 1351 sequences 2.7.2.3 Phosphoglycerate kinase PDB: 13pk 2.7.2.10 Phosphoglycerate kinase (GTP) aspartokinase: PF00696 2171 sequences 2.7.2.2 Carbamate kinase PDB: 1b7b 2.7.2.4 Aspartate kinase 2.7.2.8 Acetylglutamate kinase (*) 2.7.2.11 Glutamate 5-kinase 2.7.4.- Uridylate kinase phosphofructokinase-like: PF00365, PF00781, PF01219, PF01513 1998 sequences 2.7.1.11 6-phosphofructokinase PDB: 4pfk 2.7.1.23 NAD(+) kinase (*) 2.7.1.56 1-phosphofructokinase 2.7.1.90 Diphosphate – fructose-6-phosphate 1- phosphotransferase (*) 2.7.1.91 Sphinganine kinase 2.7.1.107 Diacylglycerol kinase 2.7.1.138 Ceramide kinase ribokinase-like: PF00294, PF01256, PF02110 2722 sequences 2.7.1.2 Glucokinase PDB: 1rkd 2.7.1.3 Ketohexokinase 2.7.1.4 Fructokinase 2.7.1.11 6-phosphofructokinase 2.7.1.15 Ribokinase 2.7.1.20 Adenosine kinase 2.7.1.35 Pyridoxal kinase (*) 2.7.1.45 2-dehydro-3-deoxygluconokinase 2.7.1.49 Hydroxymethylpyrimidine kinase 2.7.1.50 Hydroxyethylthiazole kinase 2.7.1.56 1-phosphofructokinase 2.7.1.73 Inosine kinase 2.7.1.92 5-dehydro-2-deoxygluconokinase 2.7.1.144 Tagatose-6-phosphate kinase 2.7.1.146 ADP-dependent phosphofructokinase 2.7.1.147 ADP-dependent glucokinase 2.7.4.7 Phosphomethylpyrimidine kinase (*) thiamin pyrophosphokinase 175 sequences 2.7.6.2 Thiamin pyrophosphokinase PDB: 1ig0 glycerate kinase (previously Group 15) 107 sequences 2.7.1.31 Glycerate kinase (*) PDB: 1to6 Table 2 Classification of kinase activities by family and fold group, part 2. Group Family and PFAM/COG members Kinase Activity (E.C.) Representative PDB or gi Group 3: ferredoxin-like fold kinases 10973 sequences nucleoside-diphosphate kinase: PF00334 923 sequences 2.7.4.6 Nucleoside-diphosphate kinase PDB: 2bef HPPK: PF01288 609 sequences 2.7.6.3 2-amino-4-hydroxy-6- hydroxymethyldihydropteridine pyrophosphokinase PDB: 1eqo guanido kinases: PF00217 324 sequences 2.7.3.1 Guanidoacetate kinase PDB: 1bg0 2.7.3.2 Creatine kinase 2.7.3.3 Arginine kinase 2.7.3.5 Lombricine kinase histidine kinase: PF00512, COG2172 9117 sequences 2.7.1.37 Protein kinase (Histidine kinase) PDB: 1i59 2.7.1.99 [Pyruvate dehydrogenase(lipoamide)] kinase 2.7.1.115 [3-methyl-2-oxobutanoate dehydrogenase (lipoamide)] kinase Group 4: ribonuclease H-like 2768 sequences COG0837, PF00349, PF00370, PF00871 2.7.1.1 Hexokinase PDB: 1dgk 2.7.1.2 Glucokinase 2.7.1.4 Fructokinase 2.7.1.5 Rhamnulokinase 2.7.1.7 Mannokinase 2.7.1.12 Gluconokinase 2.7.1.16 L-ribulokinase 2.7.1.17 Xylulokinase 2.7.1.27 Erythritol kinase 2.7.1.30 Glycerol kinase 2.7.1.33 Pantothenate kinase 2.7.1.47 D-ribulokinase 2.7.1.51 L-fuculokinase 2.7.1.53 L-xylulokinase 2.7.1.55 Allose kinase 2.7.1.58 2-dehydro-3-deoxygalactonokinase 2.7.1.59 N-acetylglucosamine kinase 2.7.1.60 N-acylmannosamine kinase 2.7.1.63 Polyphosphate-glucose phosphotransferase 2.7.1.85 Beta-glucoside kinase 2.7.2.1 Acetate kinase 2.7.2.7 Butyrate kinase 2.7.2.14 Branched-chain-fatty-acid kinase 2.7.2.- Propionate kinase Group 5: TIM β/α-barrel kinase 1119 sequences PF00224 2.7.1.40 Pyruvate kinase PDB: 1a49 Group 6: GHMP kinase 885 sequences COG1685, COG1907, PF00288, PF01971 2.7.1.6 Galactokinase (*) PDB: 1h72 2.7.1.36 Mevalonate kinase 2.7.1.39 Homoserine kinase 2.7.1.46 L-arabinokinase 2.7.1.52 Fucokinase 2.7.1.71 Shikimate kinase 2.7.1.148 4-(cytidine 5'-diphospho)-2- C- methyl-D- erythritol kinase (*) 2.7.4.2 Phosphomevalonate kinase Group 7: AIR synthetase-like 1843 sequences PF00586 2.7.4.16 Thiamine-phosphate kinase PDB: 1cli 2.7.9.3 Selenide, water dikinase Group 8: riboflavin kinase (previously Group 10) 565 sequences PF01687 2.7.1.26 Riboflavin kinase (*) PDB: 1nb9 Group 9: dihydroxyacetone kinase (previously Group 17) 197 sequences 2.7.1.29 Glycerone kinase (*) PDB: 1un9 Group 10: putative glycerate kinase (previously Group 16) 148 sequences COG2379 2.7.1.31 Glycerate kinase (*) PDB: 1o0u Group 11: polyphosphate kinase (previously Group 9) 446 sequences PF02503 2.7.4.1 Polyphosphate kinase gi|7465499 [48..730] Group 12: integral membrane kinases (previously Group 8) 263 sequences dolichol kinase: PF01879 127 sequences 2.7.1.108 Dolichol kinase gi|6323655 [349..513] undecaprenol kinase 136 sequences 2.7.1.66 Undecaprenol kinase gi|1705428 Occurrences of the same kinase activity in more than one family reflect cases of convergent evolution to the same kinase activity from different ancestors. For example, homoserine kinase entries are found in the protein kinase-like family (Group 1) and the GHMP kinase family (Group 6). These proteins each carry out the same biochemical reaction and therefore have identical EC specifications, but they belong to two unrelated protein families. Currently, an experimental structure is available only for the homoserine kinase from the GHMP kinase family. Framework of the classification remains unchanged The updated classification includes 42,092 additional sequences, 343 additional kinase structures, and 12 additional kinase specificities compared to the original classification. Although the total number of kinase sequences included in the classification has an impressive increase of more than three-fold (from 17,310 to 59,402), all new kinase sequences were found to be homologous to the previously established families, and thus are contained in the existing family and fold group classification. Furthermore, 343 additional kinase structures have been solved since the initial classification was completed. The majority of these structures correspond to kinases for which at least one representative structure was already known. For example, dozens of additional eukaryotic protein serine-threonine/tyrosine kinase structures were solved. Structures of 20 kinases with previously uncharacterized structures were also published (indicated by asterisks in Tables 1 and 2 ). The structural folds for 15 of these 20 kinases were predicted by our initial kinase classification based on their homology to proteins with known structures. All 15 of these predicted folds were shown be to correct by the experimentally determined structures. For example, choline kinase was expected to have a protein kinase-like fold similar to the other members of Family 1a (protein S/T-Y kinases and atypical protein kinases). The crystal structure of choline kinase [ 2 ] shows that this protein does indeed adopt a eukaryotic protein kinase-like fold. Likewise, pyridoxal kinase was shown to have a ribokinase-like fold [ 3 ], as was predicted in the initial kinase classification. Thus, the placements of these kinases in the classification scheme remain unchanged. The five remaining kinases with recently solved structures belong to families for which the fold was previously unknown. Two of these kinases, riboflavin kinase and dihydroxyacetone kinase, represent two new unique kinase folds. One glycerate kinase family, which was previously listed as an independent fold group, is now placed as an additional family in the Rossmann-like fold group due to similarities in architecture and topology of the predicted nucleotide-binding domain. As the nucleotide-binding domain cannot be confidently predicted for a second distinct glycerate kinase family, these sequences tentatively remain as a separate fold group. Lastly, inositol 1,4,5-trisphosphate 3-kinase is now known to be a member of the lipid kinase-like family (Family 2b). The total numbers of sequences, structures, families, and fold groups in the initial and updated classifications are summarized in Table 3 . Table 3 Comparison of initial and updated kinase surveys. Sequences Structures Families Families with Known Structure Fold Groups Fold Groups with Known Structure Initial Survey 17310 359 30 19 17 7 Updated Survey 59402 702 25 22 12 10 Two new kinase folds are characterized During the last two years the representative structures of Group 10 (riboflavin kinase) and Group 17 (dihydroxyacetone kinase) in the original kinase classification have been characterized, adding two new folds in the kinase fold repertoire. Riboflavin kinase (RFK) is an essential enzyme in the flavin cofactor biosynthetic pathway in both prokaryotes and eukaryotes [ 4 , 5 ]. The core of the riboflavin kinase structure [ 6 , 7 ] contains a 6-stranded β barrel with Greek key topology (Figure 1a ). This is the only kinase currently known to belong to the all-β class of proteins. The bound nucleotide is situated at one end of the β-barrel between two loop regions (L1 and L2), one of which contains a short 3 10 helix. The solvent-exposed L1 loop and 3 10 helix form an arch, under which the ADP phosphate tail and requisite Mg 2+ cation bind. The adenine ring is positioned by the Pro33 and Phe97 side chains, while the ADP β-phosphate interacts with the hydroxyl group of Tyr98 and the amino group of Asn36. However, the majority of contacts with the nucleotide are made by main chain amide and carbonyl groups from the two loop regions and the 3 10 helix (shown in magenta in Figure 1a ). The tightly bound Mg 2+ ion is coordinated directly to the side chains of Thr34 and Asn36, to the α-and β-phosphates of ADP, and presumably to the γ-phosphate of ATP as well [ 7 , 8 ]. Thr34 and Asn36 are part of the unique signature PTAN motif of riboflavin kinases which is located on a short β-strand following loop L1 and the 3 10 helix. Drastic conformational changes induced by binding of either nucleotide or flavin ligand have been demonstrated [ 7 , 8 ]. Because the fold of riboflavin kinase is not similar to any other known kinase structure, it remains as a separate fold group (now Group 8) in our kinase classification. Figure 1 Two new kinase folds . a) Riboflavin kinase (PDB|1q9s [8]). Loops L1 and L2 are shown in magenta. Residues 1 (Pro33) and 2 (Phe97) interact with the adenine ring of the nucleotide. Residues 3 (Thr34) and 4 (Asn36) coordinate the Mg 2+ cation. Residues 4 (Asn36) and 5 (Tyr98) interact with the phosphate tail of the nucleotide. In this and all other structure figures, the ATP analog is colored orange, substrate molecules are purple, and Mg 2+ cations are green balls. Ribbon diagrams were made using the MOLSCRIPT [57] program. b) Dihydroxyacetone kinase nucleotide-binding domain (PDB|1un9 [9]). Residues 1 (Leu435), 2 (Thr476), and 3 (Met477) pack around the adenine ring of the nucleotide. Residues 4 (Ser431) and 5 (Ser432) interact with the phosphate tail of the nucleotide. Residues 6 (Asp380), 7 (Asp385), and 8 (Asp387) are involved in coordinating the two Mg 2+ cations. Dashed lines indicate disordered regions in the structure. The second new kinase fold was revealed by the structure of the ATP-dependent dihydroxyacetone kinase from Citrobacter freundii [ 9 ]. Dihydroxyacetone kinase sequences are also widely distributed in organisms in all three kingdoms of life. This protein contains two regions separated by an extended linker. The N-terminal region (termed K-domain) is homologous to the non-ATP dependent DhaK protein in E. coli and other gram negative bacteria. It consists of two α/β domains and is responsible for dihydroxyacetone binding. The C-terminal region (termed L-domain, homologous to the DhaL protein in E. coli ) is the nucleotide-binding domain and is comprised of 8 antiparallel α-helices that form a closed barrel (Figure 1b ). The α-helices are all slightly tilted away from the axis of the barrel, forming a pocket in which a phospholipid is bound. The bound ATP analog is found to be located at the top of the α-barrel. The N-terminus of one helix (H4) is pointed toward the γ-phosphate of ATP and, together with a glycine-rich loop between helices H3 and H4, forms the primary binding site for the ATP phosphates. Ser432 interacts with the ATP α-phosphate, while Ser431 interacts with the ATP β-and γ-phosphates. Two Mg 2+ ions are coordinated by all three phosphates of ATP and by the three highly conserved aspartates (Asp380, Asp385 and Asp387) located on a loop between helices H1 and H2. Additionally, the adenine ring is packed against several hydrophobic side chains (Leu435, Thr476, and Met477). Dihydroxyacetone kinase is the only kinase known to have an all-α nucleotide-binding domain. It represents another new fold group (now Group 10) in our kinase classification scheme as its fold is unlike any other kinase with known structure. Two glycerate kinase families now with solved structures The initial kinase survey included two protein families with putative glycerate kinase activity. These proteins fall into two separate fold groups since no significant sequence similarity was detected between the two families despite their presumably identical biochemical activities. One family (previously Group 15) contains glycerate kinases from bacterial species, primarily of the firmicutes group and of the gamma subdivision of the proteobacterial group. The second family (previously Group 16) consists of proteins from eukaryotes and archaea in addition to several different bacterial species. Representative structures from each of these two families have recently been solved. Glycerate kinase from Neisseria meningitidis (PDB|1to6) (Rajashankar, K.R., Kniewel, R., Solorzano, V., and Lima, C.D. Glycerate Kinase from Neisseria meningitidis (Serogroup A). To be published .) is a member of the first glycerate kinase family (previously Group 15). The fold of this protein consists of two non-similar α/β domains (Figure 2a ). The N-terminal domain contains a central 5-stranded parallel β-sheet (strand order 21345) and several surrounding helices with Rossmann-like topology. The C-terminal domain contains a central 6-stranded mixed β-sheet (strand order 123456), with strand 2 antiparallel to the rest of the β-sheet. In this structure, the C-terminal domain is inserted between strands 2 and 3 of the N-terminal domain. The active site is likely to be in the cleft between the two α/β domains. Although this structure does not include a bound nucleotide or substrate, the two sulfate groups observed in the presumed active site may indicate the locations of the nucleotide and glycerate binding sites. In this structure, eight highly conserved polar or charged residues have side chains pointing into the presumed active site (Figure 2a ). Six of these eight residues, including two lysines, are contributed by the Rossmann-like domain. Furthermore, the crevice in which the sulfate group is located is significantly larger in the Rossmann-like domain that the corresponding crevice in the C-terminal domain, suggesting that the Rossmann-like domain may accommodate a larger molecule such as ATP. Based on the presence of these conserved lysine residues, which are common features of nucleotide binding sites in kinases, and the large crevice that may serve as the ATP-binding site, the Rossmann-like domain is predicted to perform the nucleotide binding role in this kinase. Thus, this family of glycerate kinases is added to the Rossmann-like fold group as a distinct family. Figure 2 Glycerate kinase structures . a) Neisseria meningitides glycerate kinase (PDB|1to6), a representative of first glycerate kinase family (previously Group 15). Highly conserved amino acids with side chains pointing into the presumed active site include 6 residues from the Rossmann-like domain ( 1 – Asp8, 2 – Lys11, 3 – Asp43, 6 – Glu286, 7 – Asp290, and 8 – Lys297) and 2 residues from the inserted domain ( 4 – Asp191 and 5 – Gln209). Glycine rich loops are shown in magenta. Sulfates are shown in ball-and-stick representation. b) Thermotoga maritima putative glycerate kinase (PDB|1o0u), a member of the second glycerate kinase family (previously Group 16, now Group 10). Highly conserved amino acids with side chains pointing into the presumed active site include 2 residues from the Rossmann-like domain ( 1 – Lys47 and 2 – Asp189) and 4 residues from the C-terminal domain ( 3 – Glu312, 4 – Arg325, 5 – Asp351, and 6 – Asn407). The glycine rich loop is shown in magenta. Putative glycerate kinase from Thermotoga maritima (PDB|1o0u) (Joint Center for Structural Genomics. Crystal Structure of Glycerate Kinase (TM1585) from Thermotoga maritima at 2.95 A Resolution. To be published .) is a member of the second glycerate kinase family (previously Group 16). The fold of this protein also consists of two non-similar α/β domains (Figure 2b ). The N-terminal α/β domain has Rossmann-like topology with the central 6-stranded β-sheet in the order of 654123. The C-terminal domain contains a 6-stranded mixed β-sheet with strand order 126345 and several helices packed on both sides of the β-sheet. The active site is likely to be in the cleft between the two α/β domains. In this structure, six highly conserved polar or charged residues are found with side chains pointing into the presumed active site (Figure 2b ). The C-terminal domain contributes four of these highly conserved residues, while the Rossmann-like domain contributes the remaining two residues in addition to a glycine-rich loop. Each of these domains contains one highly conserved basic residue that could potentially interact with the triphosphate tail of the bound ATP: Lys47 in the Rossmann-like domain and Arg325 in the C-terminal domain. Based on the available information, it is not possible to confidently predict which domain is responsible for nucleotide binding in this putative glycerate kinase. Therefore, this family is kept as a separate fold group until its active site is characterized. The annotation for these putative glycerate kinases is based on a gene found in a 5-kb fragment that is apparently responsible for complementation in Methylbacterium extorquens AM1 mutants lacking glycerate kinase activity [ 10 ]. However, other family members are annotated as putative glycerate dehydrogenases/hydroxypyruvate reductases based genetic analysis of the tartrate utilization pathway in Agrobacterium vitis [ 11 ]. Glycerate kinase and glycerate dehydrogenase/hydroxypyruvate reductase catalyze successive steps in the serine metabolism pathway. Therefore, the exact biochemical function of this enzyme family remains to be resolved. Inositol 1,4,5-trisphosphate 3-kinase is a member of the lipid kinase-like family Inositol 1,4,5-trisphosphate 3-kinase (I3P3K) catalyzes the first of several phosphotransfer reactions that convert inositol 1,4,5-trisphosphate, a ubiquitous second messenger in eukaryotes, to inositol pentakisphosphates, inositol hexakisphosphates, and inositol pyrophosphates. I3P3K is a known homolog of other inositol polyphosphate kinases, including inositol 1,4,5-trisphosphate 6-kinase and inositol hexakisphosphate kinase, but does not share significant sequence similarity to any kinase family with a previously solved structure. The solved structures of human I3P3K (PDB|1w2c [ 12 ]) and rat I3P3K (PDB|1tzd [ 13 ]) reveal that the catalytic core of this kinase adopts a protein kinase-like fold and is comprised of two domains with the active site cleft in between (Figure 3a ). The overall structure of I3P3K is most similar to the lipid kinases (PDB|1bo1 [ 14 ]), which belong to the same fold group as eukaryotic protein kinases in our kinase classification scheme. The shared structural core between lipid kinases and protein kinases includes all elements of the I3P3K N-terminal domain, and part of the β-sheet (strands 1, 4, and 5) and the three α-helices of the C-terminal domain (Figure 3a ). Figure 3 Structure of inositol polyphosphate kinases . a) Inositol 1,4,5-trisphosphate 3-kinase (I3P3K) (PDB|1w2c [12]) adopts a lipid kinase/protein kinase-like fold. Common core of lipid kinases, eukaryotic protein kinases, and I3P3K is shown in blue (α-helices) and yellow (β-strands); additional elements are grey. Residue 1 (Lys209) interacts with the nucleotide's phosphate tail, residue 2 (Glu215) stabilizes the orientation of residue 1 , residue 3 (Asp262) binds the sugar group of ATP, residue 4 (Lys264) likely interacts with the γ-phosphate during transfer, and residues 5 (Ser399) and 6 (Asp416) are both likely involved in coordinating two Mg 2+ cations. Mn 2+ is shown as a green ball and inositol 1,4,5-trisphosphate is shown in purple. b) Multiple sequence alignment of I3P3K (gi|10176869, PDB|1w2c, PDB|1tzd) and I5P2K (gi|6320521) with two related kinase families: lipid kinase representative PIPK (PDB|1bo1 [14]; Family 1b) and protein kinase representative twitchin kinase (PDB|1koa [58]; Family 1a). Italics denote α-helical regions for which the register of structure-based alignment cannot be obtained unequivocally due to significant structural divergence. Critical active site residues are indicated by white bold text highlighted in black/magenta, and are numbered the same as in panel (a). Magenta highlighting indicates residues that perform equivalent roles and are found in equivalent spatial locations, but do not align closely in sequence between the lipid kinase and protein kinase families. In this and other multiple alignments, sequences are labelled according to the NCBI gene identification (gi) number or PDB code and an abbreviation of the species name. Abbreviations in this alignment are as follows: Ag Anopheles gambiae , Am Apis mellifera , At Arabidopsis thaliana , Ce Caenorhabditis elegans , Dh Debaryomyces hansenii , Dm Drosophila melanogaster , Gg Gallus gallus , Gz Gibberella zeae , Hs Homo sapiens , Mg Mytilus galloprovincialis , Mm Mus musculus , Rn Rattus norvegicus , Sc Saccharomyces cerevisiae . First and last residue numbers are indicated before and after each sequence. Numbers of excluded residues are specified in square brackets. Residue conservation is denoted with the following scheme: mostly hydrophobic positions, highlighted yellow; mostly charged/polar positions, highlighted grey; small residues, red bold text. Locations of predicted (gi|6320521, gi|10176869) and observed (PDB|1w2c_A, PDB|1tzd_B, PDB|1bo1_A, PDB|1koa) secondary structure elements (E, β-strand; H, α-helix) are marked above the sequences (with the exception of gi|10176869 which is shown below the sequence) in italics and normal font, respectively. The mode of nucleotide binding in I3P3K is also very similar to that of eukaryotic protein kinases, as each of the critical nucleotide binding and Mg 2+ binding residues in I3P3K plays the same role as a corresponding protein kinase residue. Lys209 (human I3P3K; hI3P3K) forms a hydrogen bond with the α-and β-phosphates of ATP and corresponds to the highly conserved Lys72 in protein kinase A (PKA). This lysine residue is oriented by Glu215 in hI3P3K (corresponding to Glu91 in PKA). A second highly conserved lysine residue (Lys264 in hI3P3K) interacts with the 3-OH phosphate acceptor group of the inositol 1,4,5-trisphosphate substrate and likely stabilizes the γ-phosphate during transfer, similar to the role of Lys168 in PKA. Although Lys264 (hI3P3K) and Lys168 (PKA) are contributed by different structural elements in different regions of the protein sequence, these residues are found in equivalent spatial locations and likely play the same role in catalysis. A Mn 2+ ion is coordinated by Asp416, which corresponds to the conserved magnesium-binding residue Asp184 of the DFG motif in PKA. Ser399 is expected to coordinate a second divalent cation that is not modeled in the I3P3K structure, as this residue is found in the equivalent spatial location of the conserved magnesium-binding residue Asn171 in PKA. These active site similarities also extend to members of the lipid kinase family, such as phosphatidylinositol phosphate kinase IIβ (PIPK; PDB|1bo1) (Figure 3b ), although a representative structure with bound nucleotide has not yet been solved. Although I3P3K shares similarity of the overall fold as well as the active site with the related lipid kinase and protein kinase-like families, I3P3K is more closely related to the lipid kinase family. I3P3K and the lipid kinases share conserved motifs, including the substrate-binding/catalytic "DLK" motif (Asp262 to Lys264 in hI3P3K) and the magnesium-binding "SSLL" motif (Ser398 to Leu401 in hI3P3K), which are not found in protein kinases. Additionally, DALI [ 15 ] identifies lipid kinase representative PIPK (PDB|1bo1) as the closest structural neighbor of I3P3K [ 13 ]. Thus, based on the similarity of structural fold and the conservation of critical nucleotide-binding, magnesium-binding, and catalytic residues I3P3K can be assigned to the lipid kinase family in the kinase classification (Family 1b) despite the lack of significant sequence similarity. Predicted folds for remaining kinases with unknown structures Fold predictions (see Methods) were made for each remaining family of kinases without a solved structure, with the exception of the integral membrane kinases (dolichol kinase and undecaprenol kinase). These kinases include inositol 1,4,5-trisphosphate 3-kinase, inositol 1,3,4,5,6-pentakisphosphate 2-kinase, eukaryotic pantothenate kinase, and polyphosphate kinase. Inositol 1,4,5-trisphosphate 3-kinase (I3P3K; previously Group 11) and inositol 1,3,4,5,6-pentakisphosphate 2-kinase (I5P2K; previously Group 12) both catalyze phosphorylation reactions in the production of inositol polyphosphate (IP) second messengers. These kinases were placed in separate fold groups in the initial survey based on a lack of significant sequence similarity to each other or any other known kinase family. Before the crystal structures of I3P3K were reported during the preparation of this manuscript, fold predictions for both I3P3K and I5P2P were carried out. The results of fold predictions guided by 3D-Jury [ 16 ], secondary structure predictions, and observed presence of critical conserved sequence motifs indicated that both of these IP kinases would likely adopt a structural fold similar to lipid kinases and eukaryotic protein kinases, which are possibly related families that share a common ATP-binding site and structural core [ 17 ]. Furthermore, a multiple alignment of representative I3P3K, I5P2K, lipid kinase, and protein kinase sequences shows that the critical functional residues in these proteins are also conserved in the IP kinases (Figure 3b ). For example, I3P3K and I5P2K each have the conserved lysine/arginine residue that is important for orienting the α-and β-phosphates of nucleotide's triphosphate tail and the aspartate/glutamate that interacts with the ribose of ATP (residues 1 and 3 in Figure 3b , respectively). Additionally, the serine/threonine and the aspartate residues involved in coordinating the two requisite Mg 2+ cations are conserved in these kinases as well (residues 5 and 6 in Figure 3b , respectively). Both I3P3K and I5P2K also have a predicted glycine-rich loop in the N-terminal region of the protein. From the multiple sequence alignment, it becomes apparent that I3P3K and I5P2K are more closely related to the other lipid kinases than to protein kinases. In addition to phosphorylating similar substrates, the IP kinases and lipid kinase family members each have critical active site lysine residue involved in stabilizing the γ-phosphate of ATP during transfer (residue 4 in Figure 3b ) that has migrated in the sequence/structure relative to the protein kinase-like family. The solved structures of I3P3K from human [ 12 ] and rat [ 13 ], which were published during manuscript preparation and are discussed above, confirm this non-trivial fold assignment as well as the predicted functional roles played by the conserved active site residues. This further increased our confidence in the I5P2K prediction. Thus, I3P3K and I5P2K are now assigned as members of the lipid kinase-like family (Family 1b) in the kinase classification. The spatial structure of eukaryotic pantothenate kinase (previously Group 14) is unknown. The crystal structure of prokaryotic pantothenate kinase identifies this enzyme as a member of the P-loop kinase family [ 18 ]. However, due to the lack of sequence identity between the prokaryotic and eukaryotic versions of this protein [ 19 ] in conjunction with dissimilar predicted secondary structure patterns, eukaryotic pantothenate kinase is expected to adopt a fold distinct from its prokaryotic counterpart. Although standard sequence similarity search methods failed to obtain any reasonable structural assignment, several fold recognition servers strongly suggested that the eukaryotic pantothenate kinases adopt a ribonuclease H-like fold. The ribonuclease H-like fold is composed of three layers (α/β/α), including a 5-stranded mixed β-sheet with strand order 32145, where strand 2 antiparallel to the rest of the sheet. The topology of the core of this fold is βββαβαβα. A duplication of this fold is also seen in several other ribonuclease H-like kinases (Group 4), although the closest structural template identified by 3D-Jury is a non-kinase homolog of this family (2-hydroxyglutaryl-CoA dehydratase component A; PDB|1hux [ 20 ]). Importantly, the ribonuclease H-like kinase group contains the ASKHA (acetate and sugar kinase/hsp70/actin) superfamily. Nucleotide binding and divalent metal coordination are achieved by several motifs conserved within the ASKHA superfamily [ 21 ]. These conserved motifs include the ADENOSINE motif that interacts with the ribosyl and the α-phosphoryl group of ATP, the PHOSPHATE 1 motif that interacts with Mg 2+ through coordinated water molecules, and the PHOSPHATE 2 motif that interacts with the β-and γ-phosphoryl groups of ATP. The conservation of these motifs in the eukaryotic pantothenate kinases is noted in Figure 4 . Thus, based on the presence of these distinguishing motifs in addition to the similarity of secondary structure patterns (Figure 4 ), the eukaryotic pantothenate kinases are added to the ribonuclease H-like family. Figure 4 Eukaryotic pantothenate kinase is a ribonuclease H-like kinase . Multiple sequence alignment for representative sequences of the pantothenate kinase family and two related ribonuclease H-like families with known structure (2-hydroxyglutaryl-CoA dehydratase component A (PDB|1hux [20]) and hexokinase I (PDB|1dgk [59])) is shown. The PHOSPHATE 1, PHOSPHATE 2, and ADENOSINE motifs are indicated by dashed boxes. Abbreviations of species names are as follows: Af Acidaminococcus fermentans , At Arabidopsis thaliana , Bc Bacillus cereus , Ca Clostridium acetobutylicum , Ce Caenorhabditis elegans , Dm Drosophila melanogaster , En Emericella nidulans , Hs Homo sapiens , Mm Mus musculus , Mt Methanothermobacter thermautotrophicus , Pa Pichia angusta , Ta Thauera aromatica , Tt Thermoanaerobacter tengcongensis . Locations of predicted (gi|4191500) and observed (PDB|1hux_A, PDB|1dgk_N) secondary structure elements (E, β-strand; H, α-helix) are marked above the sequences in italics and normal font, respectively. Polyphosphate kinase (PPK) synthesizes inorganic polyphosphate (polyP) by catalyzing the transfer of the γ-phosphate of ATP to a linear polymer of tens or hundreds of orthophosphate residues. Additionally, this enzyme can catalyze the transfer of a phosphate group from polyP to ADP or GDP and can generate ppppG by transferring a pyrophosphate group from polyP to GDP as well [ 22 ]. PPK sequences have been identified in many bacterial species as well as in some archaea. Fold predictions indicate that PPK is comprised of three domains: two phospholipase D-like subdomains, which are clearly recognizable by standard sequence comparison methods such as RPS-BLAST, and an N-terminal Rossmann-like domain. The phospholipase D (PLD) fold consists of a 7-stranded mixed β-sheet (strand order 1765234) flanked by several α-helices. PPK contains two PLD-like subdomains, presumably arranged in the same manner as other PLD superfamily members such as tyrosyl-DNA phosphodiesterase (PDB|1jy1 [ 23 ]) and the homodimer of bacterial endonuclease Nuc from Salmonella typhimurium (PDB|1bys [ 24 ]), which is the closest structural template identified by 3D-Jury [ 16 ]. The active site of enzymes in the PLD superfamily is located between two PLD-like subdomains. This active site is highly symmetrical due to the five equivalent highly conserved residues that are contributed by each PLD-like subdomain. Among these are two histidine residues (one from each subdomain) proposed to perform critical catalytic roles. One histidine may act as a nucleophile by attacking the phosphodiester bond that is cleaved by known PLD-like enzymes and form a phospho-histidine intermediate, while the second conserved histidine could serve as a general acid by protonating the leaving group [ 24 ]. The conservation of these critical active site residues is shown in Figure 5 . Consistent with the proposed mechanism for PLD-like enzymes, PPK has been shown to form a phospho-histidine intermediate during the phosphotransfer reaction [ 25 , 26 ] (residue 1 in Figure 5 ). If nucleotide binding in the kinase reaction is carried out by the PLD-like domains, PPK will denote a new fold group in the kinase classification since the PLD-like topology is unlike any exiting kinase fold group. However, the possibility that N-terminal domain of PPK may be responsible for binding of ATP cannot be ruled out at this point. Fold prediction suggests that the PPK N-terminal region likely adopts a Rossmann-like fold, which is also seen in thousands of other kinase sequences (Group 2). We are currently unable to confidently predict whether the Rossmann-like or PLD-like region of PPK is responsible for nucleotide binding in the kinase reaction. Thus, PPK is presently considered a separate fold group in our kinase classification. Figure 5 Second and third domains of polyphosphate kinase are homologous to phospholipase D . Multiple sequence alignment for representative sequences of polyphosphate kinase (gi|7465499, Group 11) and the phospholipase D family (PDB|1bys [24]) is shown. Highly conserved active site residues are highlighted in black and shown in white bold text. Abbreviations of species names are as follows: Bh Bacillus halodurans , Bj Bradyrhizobium japonicum , Ca Clostridium acetobutylicum , Ch Cytophaga hutchinsonii , Dh Desulfitobacterium hafniense , Ec Escherichia coli , Pa Pseudomonas aeruginosa , Rp Rickettsia prowazekii , St Salmonella typhimurium , Wg Wigglesworthia glossinidia brevipalpis . Locations of predicted (gi|7465499) and observed (PDB|1bys_A) secondary structure elements (E, β-strand; H, α-helix) are marked above the sequences in italics and normal font, respectively. Comprehensive structural annotation of kinases Of the 25 kinase families, 22 currently have at least one homolog with a solved structure (Tables 1 and 2 ). The structural folds of each domain within one additional family (polyphosphate kinase) are predicted as discussed above. The two remaining families are integral membrane kinases. Although the tertiary structure of dolichol kinase and undecaprenol kinase are not yet determined, secondary structure predictions indicate that both of these families adopt all α-helical conformations. Thus, structural annotations of all kinase families are now revealed, including fold descriptions for all globular kinases, and the kinase fold groups listed in Tables 1 and 2 present the complete structural depiction of this entire functional class of proteins. The structural folds adopted by kinases include some of the most widely spread protein folds, including the Rossmann-like fold, ferredoxin-like fold, and TIM β/α-barrel fold. The kinase fold repertoire also includes representatives of all major classes (all α, all β, α+β, α/β) of protein structures, demonstrating that nature has found ways to utilize all varieties of secondary structure combinations to carry out the kinase reaction. Additional kinase activities in the classification The updated classification also includes 12 additional kinase activities. However, 7 of these activities reflect changes within the EC database rather than newly characterized kinase sequences. For example, while the structure of adenosylcobinamide kinase was published in 1998, its EC number (EC 2.7.1.156) was only assigned in April 2004. The sequences and structures of this kinase were included in initial kinase survey (e.g. PDB|1cbu [ 27 ]) and were placed in the P-loop kinase family of the Rossmann-like fold group. The updated kinase classification does include 5 newly annotated or characterized kinases (indicated by underlining in Tables 1 and 2 ). The first sequences associated with each of these activities (2.7.1.52 Fucokinase, 2.7.1.92 5-dehydro-2-deoxygluconokinase, 2.7.1.138 Ceramide kinase, and 2.7.1.140 Inositol-tetrakisphosphate 5-kinase) were identified after the initial kinase survey was completed. Sequences with [Hydroxymethylglutaryl-CoA reductase (NADPH 2 )] kinase activity (2.7.1.109) were included in the initial classification, although only a general kinase activity ("AMP-activated protein kinase") was assigned at the time. Thus, the specific kinase activity of this enzyme is a new addition to the kinase classification as well. As can be seen from Tables 1 and 2 , all of these newly annotated kinases (underlined in the tables) belong to existing kinase families containing members that are well characterized both biochemically and structurally. The link between these kinases and members of the existing families can all be identified by BLAST [ 28 ] with E-values less than 1e -5 . Therefore, the catalytic mechanisms of these newly annotated kinases may be inferred from their closely related homologs. Two families previously annotated as kinases are removed from the classification Two kinase families were removed from the classification. In both cases, the sequences were annotated as kinases in the NCBI database, but further biochemical studies have indicated that they most likely do not have kinase activity. The case of the ThrH protein (previously Family 2g – L-2-Haloacid dehalogenase-like family) is an unusual one. Earlier genetic studies of the thrH gene of Pseudomonas aeruginosa have shown that over-expression of ThrH complements both homoserine kinase and phosphoserine phosphatase activities in vivo . The gene product of thrH was thus annotated as "bifunctional homoserine kinase/phosphoserine phosphatase isoenzyme" [ 29 ]. A more recent structural and biochemical study of ThrH has shown that this protein does not have ATP-dependent kinase activity. Instead it possesses phosphoserine phosphatase activity and is also able to transfer a phosphate group from phosphoserine to homoserine, presumably via a phospho-enzyme intermediate [ 30 ]. Thus, although ThrH is able to generate phosphohomoserine and complement homoserine kinase activity in vivo , it achieves this through a completely different chemical mechanism from that of true homoserine kinase. Thus, ThrH is in fact a phosphatase and phosphotransferase but not a kinase and is subsequently removed from the kinase classification. The putative tagatose 6-phosphate kinase (T6P kinase, previously Group 13) activity was initially suggested for the agaZ gene product based on the computational analysis and reconstruction of the putative N-acetylgalactosamine metabolic pathway in E. coli [ 31 ]. However, no tagatose 6-phosphate kinase activity for either AgaZ or its homolog GatZ can be detected experimentally, and genetic studies have suggested that gatZ is associated with tagatose-1,6-bisphosphate aldolase activity [ 32 , 33 ]. Results of transitive PSI-BLAST searches and fold predictions with 3D-Jury [ 16 ] also suggest similarity between AgaZ and tagatose- and fructose-bisphosphate aldolases with TIM β/α barrel fold. The alignment of the putative T6P kinases with the aldolase families revealed that several residues in the aldolases that are involved in substrate binding and catalysis are also conserved in the AgaZ/GatZ protein family (data not shown). These include two histidine residues involved in the coordination of the catalytic Zn 2+ and the aspartate residue that is proposed to protonate the substrate during the aldolase reaction [ 34 ]. Thus, based on both functional study and structural prediction, it is likely that these proteins carry out an aldolase reaction rather than a kinase reaction. Therefore, this family is removed in the updated kinase classification as well. More diversity of structural folds and nucleotide binding in kinases In the original kinase survey study, the substrate binding and phosphoryl transfer reaction mechanisms were analyzed across protein families and fold groups, and several distinct modes of nucleotide binding have emerged. One recurring theme observed was the bound nucleotide located at the C-terminus of β-strands and N-terminus of α-helices (i.e. on β-α loops). Signature motifs that interact with the nucleotide are also common. These motifs are often rich in glycines and found on both β-α and β-β loops. One such example is the so-called P-loop (phosphate-binding loop) formed by the Walker-A motif, which is found in a variety of different proteins that bind nucleotides [ 35 ]. The P-loop, which is located on a β-α loop, wraps around the triphosphate tail of the bound nucleotide. Together with the positive dipole of the α-helix and some positively charged side chains, a strong anion hole is created for the binding of the phosphate groups of the nucleotide. The newly characterized riboflavin kinase is the only known kinase with an all-β structural core. RFK contains a novel nucleotide-binding motif that encompasses an arched loop (L1 in Fig. 1a ), a 3 10 helix, and a reverse turn followed by a short β-strand (Figure 1a ). This short β-strand encompasses the highly conserved PTAN sequence motif. The threonine and asparagine in the motif are directly involved in the coordination of Mg 2+ and thus the binding of MgATP (Figure 1a ). The mechanism of the phosphotransfer reaction in RFK appears to be direct in-line transfer of the γ-phosphate of ATP to the 5' hydroxyl group of riboflavin, which may be activated by a glutamate residue (Glu86) [ 7 , 8 ]. The most unique features of RFK appear to be that the phosphate is transferred through a hole beneath the highly dynamic Loop L1, and the proper positioning of the catalytic residues depends on binding of the substrates. The second kinase with a novel fold is dihydroxyacetone kinase, which is the only known kinase with an all-α nucleotide-binding domain. The binding of MgATP is accomplished in part through interactions with a glycine-rich loop between helices H3 and H4 and the N-terminal positive dipole of Helix H4. Uniquely, two Mg 2+ ions were found to ligand to the ATP phosphates and a cluster of three highly conserved aspartate residues on a loop between helices H1 and H2. The mechanism of phosphotransfer in DhaK is not clear since the complex conformation of the crystal structure is influenced by the crystal packing and appears not in its active form. A reaction mechanism involving a phospho-enzyme intermediate cannot be ruled out at this point. Thus, the newly characterized riboflavin kinase and dihydroxyacetone kinase reveal spectacularly different structures compared to those previously known and have enriched the kinase fold repertoire, which now includes all major classes of protein structures with α/β, α+β, all-β, and all-α folds. Although the substrate recognition and catalytic mechanisms of the two newly characterized enzymes share certain features with other kinases, such as utilization of a helix dipole and backbone amide groups in a glycine-rich loop for nucleotide binding, as a whole they are distinctly unique. Supplementary material Supplementary material is available by anonymous ftp at , which includes lists of NCBI gene identification (gi) numbers for sequences from each kinase family and a table cataloging functional residues from kinase family representatives. Conclusion We have performed an updated comprehensive survey of all available kinase sequences and structures. All experimentally characterized kinase families, with the exception of the integral membrane kinases, are now associated with a known or predicted structural fold. Therefore, the kinases are the first large functional class with a comprehensive structural annotation for its known members. Additionally, we find that, despite a three-fold increase in the number of kinase sequences and two-fold increase in the number of kinase structures, the framework of our classification remains sufficient for describing all available kinases. Furthermore, we find that no fold predictions made in the initial kinase survey are now shown to be incorrect. Thus, the updated kinase survey serves to confirm the soundness of our classification scheme in addition to presenting the final global picture of this entire functional class. Potential uses for this classification include deduction of protein function, structural fold, or enzymatic mechanism of poorly studied or newly discovered kinases based on proteins in the same family. Methods Constructing updated families of homologous kinases sequences The updated version of the kinase classification scheme was assembled with the same strategy that was applied in the construction of our first kinase survey [ 1 ], with the previous classification used as a framework for this update. Briefly, the hmmsearch program of the HMMER2 package [ 36 ] was used to assign sequences from the NCBI non-redundant (nr) database (July 2, 2004; 2,911,742 sequences, including environmental sequences) to a set of 57 profiles describing catalytic kinase domains (E-value cutoff 0.1). This set of kinase profiles (from Pfam [ 37 ] version 5.4 and COG version 2 [ 38 ]) was constructed during the initial kinase survey. As these profiles had been assembled into families of homologous sequences in the initial classification scheme, the sequences assigned to these profiles by hmmsearch were then placed in the appropriate kinase families. Additionally, the GREFD program of the SEALS package [ 39 ] was used to extract from the nr all sequences for which the definition line contained the pattern "kinase". For any kinase sequence not already assigned to a kinase family (either by hmmsearch or in the previous classification), three iterations of PSI-BLAST [ 28 ] were carried out against the nr database (E-value cutoff 0.001). Any kinase producing a hit to a sequence already assigned (either by hmmsearch or in the previous classification) was subsequently placed in the corresponding kinase family. The appropriate placement of the remaining unassigned kinase sequences was determined by manual inspection of multiple alignments, secondary structure predictions, and distant PSI-BLAST hits. These proteins were placed into existing kinase families based on the presence of conserved catalytic residues and other distinguishing motifs as well as overall sequence similarity. Sequences that were fragments, non-kinase entries (e.g. kinase inhibitors), or non-catalytic entries (e.g. regulatory subunits) were removed. Such sequences were identified by their annotations in the non-redundant database and by their lengths being too short to cover the complete protein. In the case of non-kinase or non-catalytic entries, lack of kinase activity was confirmed based on either literature available concerning the sequences in question or on obvious homology to a protein with known non-kinase function. The lists of newly identified kinase sequences were appended to those for each of the kinase families included in the initial classification. The meaning of families and fold groups in the new version of the classification remain unaltered: the families contain homologous kinase sequences, while the fold groups imply similarity of structural fold but not homology. Fold group classification In the initial classification, fold groups were assembled based solely on similarity of structures. Families in the same fold group share structurally similar nucleotide-binding domains that are of the same architecture and topology (or related by circular permutation) for at least the core of the domain. Some of the recently solved kinase structures allowed for the merging of certain kinase families to previously established fold groups based on these same structural similarity guidelines. Fold predictions To provide fold assignments for the remaining structurally uncharacterized kinase families, initial analysis was performed with standard sequence similarity search methods such as transitive PSI-BLAST [ 28 ], RPS-BLAST [ 40 ], and profile HMMs from SMART [ 41 ]. All searches were initiated with the representative sequences (selected in the initial survey [ 1 ]) of the families. Transitive PSI-BLAST (E-value threshold 0.01) was run against the NCBI non-redundant protein sequence database until convergence. CDD (RPS-BLAST) [ 42 ] and SMART (profile HMMs) [ 43 ] web tools were used with default settings to detect distant homology to other conserved protein domains annotated in the SMART, PFAM [ 37 ] and COG [ 44 ] databases. In addition, RPS-BLAST was also exploited to compare query sequences directly to the PDB using the GRDB system [ 45 ]. Further analysis was carried out using Meta Server [ 46 ], which assembles various secondary structure prediction and fold recognition methods. Collected predictions were screened with 3D-Jury [ 16 ], the consensus method of fold recognition servers. The default servers used by the 3D-Jury system for consensus building include: ORFeus [ 47 ], Meta-BASIC [ 48 ]., FFAS03 [ 49 ], mGenTHREADER [ 50 ], INBGU [ 51 ], RAPTOR [ 52 ], FUGUE-2 [ 53 ], and 3D-PSSM [ 54 ]. Final fold/template selections were based on 3D-Jury reliability scores as well as those of individual servers, correctness of mapping of predicted and observed secondary structure elements, and conservation of functionally and/or structurally important residues. In the case of inositol 1,3,4,5,6-pentakisphosphate 2-kinase, initial fold assignment was based on functional analogy to 1-phosphatidylinositol-4-phoshate 5-kinase, which phosphorylates similar substrates. Sequence-to-structure alignments Multiple sequence alignments for considered protein families were prepared using PCMA [ 55 ] followed by manual adjustment. Sequence-to-structure alignments between analyzed kinase families and their distantly related template families were built using consensus alignment approach and 3D assessment [ 56 ] based mainly on 3D-Jury results for representative kinase sequences. Sequences of distantly related proteins of known structure were aligned first based on the superposition of their 3D structures. In the case of inositol 1,3,4,5,6-pentakisphosphate 2-kinase, sequence-to-structure alignment was prepared manually with respect to the results of secondary structure predictions and the preservation of functionally critical residues as well as the hydrophobic core of the protein. Alterations within the classification Although the framework of the classification remains essentially unchanged, the organization within the classification has been slightly modified. More specifically, the numbering of the fold groups has been adjusted so that all kinase families with unsolved structures are at the end. Furthermore, the EC (Enzyme Commission) numbers have been updated to reflect the current organization of the EC database. Therefore, the EC content of each family may differ somewhat between the initial and updated classifications, but these changes do not indicate new additions to the family unless otherwise indicated. Authors' contributions SC performed the update of the classification and participated in drafting the manuscript. KG performed fold predictions. HZ participated in drafting the manuscript. NVG conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.

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1079890

Crystal structure of THEP1 from the hyperthermophile Aquifex aeolicus: a variation of the RecA fold

Background aaTHEP1, the gene product of aq_1292 from Aquifex aeolicus , shows sequence homology to proteins from most thermophiles, hyperthermophiles, and higher organisms such as man, mouse, and fly. In contrast, there are almost no homologous proteins in mesophilic unicellular microorganisms. aaTHEP1 is a thermophilic enzyme exhibiting both ATPase and GTPase activity in vitro . Although annotated as a nucleotide kinase, such an activity could not be confirmed for aaTHEP1 experimentally and the in vivo function of aaTHEP1 is still unknown. Results Here we report the crystal structure of selenomethionine substituted nucleotide-free aaTHEP1 at 1.4 Å resolution using a multiple anomalous dispersion phasing protocol. The protein is composed of a single domain that belongs to the family of 3-layer (α/β/α)-structures consisting of nine central strands flanked by six helices. The closest structural homologue as determined by DALI is the RecA family. In contrast to the latter proteins, aaTHEP1 possesses an extension of the β-sheet consisting of four additional β-strands. Conclusion We conclude that the structure of aaTHEP1 represents a variation of the RecA fold. Although the catalytic function of aaTHEP1 remains unclear, structural details indicate that it does not belong to the group of GTPases, kinases or adenosyltransferases. A mainly positive electrostatic surface indicates that aaTHEP1 might be a DNA/RNA modifying enzyme. The resolved structure of aaTHEP1 can serve as paradigm for the complete THEP1 family.

Background Comparative genomics led to the definition of 4873 clusters of orthologous groups of proteins (COGs) by comparing protein sequences encoded in (currently 66) completely sequenced genomes [ 1 ]. Aimed at finding thermophile-specific proteins among bacteria, extended phylogenetic patterns searches based on the COG-database were performed. Using this strategy, COG1618 was detected as a cluster containing proteins from all thermophilic and hyperthermophilic but only one mesophilic organism [ 2 - 4 ]. Surprisingly, although also absent from unicellular eukaryotes, COG1618-homologs are present in many higher multicellular organism such as Homo sapiens , Mus musculus , Danio rerio , Rattus norvegicus , etc. Because of this unusual phylogenetic distribution, aaTHEP1, the gene product of aq_1292 from the hyperthermophilic bacterium Aquifex aeolicus , was characterised biochemically as the first member of COG1618 proteins [ 2 ]. The analysis revealed that aaTHEP1 is an NTPase catalyzing ATP and GTP hydrolysis at turnover rates of 5 × 10 -3 s -1 and 9 × 10 -3 s -1 , respectively, with a K m in the micromolar range and a temperature optimum between 70 and 80°C. Although COG1618 proteins are annotated as "predicted nucleotide kinases"such an activity could not be confirmed for aaTHEP1 experimentally and its in vivo function remains unknown. To further characterize the aaTHEP1 function, we resolved its three dimensional structure by X-ray crystallography. Results and discussion Overall structure, domain class and architecture Selenomethionine substituted aaTHEP1 was purified as described earlier [ 2 ] and eluted as a monomer from the final gel filtration column. Analysis of its nucleotide loading state using HPLC revealed that it was partially loaded with ADP (approx. 30%, data not shown). It was crystallized using PEG3350 as precipitant in the presence of KH 2 PO 4 (see Methods) and crystals diffracted up to 1.4 Å using synchrotron radiation (see Table 1 ). Initial phases were obtained using a MAD phasing protocol (see Methods) and a model was build and refined. The final model has an R cryst of 16.8% and an R free of 20.8% and contains one aaTHEP1 molecule in the asymmetric unit. 172 amino acid residues, 249 water molecules, one phosphate, one magnesium and two sodium ions were included in the model. No electron density was found for residues D38-K43 which are part of a disordered loop. aaTHEP1 consists of a single compact domain confirming the gel filtration experiments as well as the resistance of aaTHEP1 to limited proteolysis [ 2 ]. It is build up of nine strands and six helixes in the sequential order βαβββββαβααβαβα (Figures 1 , 2 , 3 ) which is in agreement with previously recorded CD-spectra showing an equal ratio of β-sheets and α-helices [ 2 ]. All nine strands form a single sheet in topological order 918723465 wherein a five-stranded parallel and a four-stranded antiparallel region can be distinguished (Figure 3 ). Whereas the parallel part of the sheet almost lies in a plane, its antiparallel region is curved defining a convex (outer) and a concave (inner) side of the beta-structure (Figure 2 ). Spatially restricted to the parallel region, two α-helixes are located outside of the sheet. In contrast, a set of four helixes is distributed over the whole bended sheet at its inner side. This set consists of three parallel large α-helixes in identical N- to C-orientation who are accompanied by a further perpendicularly arranged much smaller 3/10-helix located near their N-terminal sides. The edge of the antiparallel region of the sheet forms a small bended lid that covers this smaller 3/10-helix. In summary, the overall topology of aaTHEP1 is a central sheet with helical structures on each side. According to the CATH protein structure classification [ 5 ], aaTHEP1 is assigned to class 3.40.50.300 i. e. "P-loop containing nucleotide triphosphate hydrolases, homologous superfamilies with Rossmann fold topology" which are mixed alpha-beta proteins with 3-layer(α/β/α) sandwich architecture. Structural alignments and fold classification For comparison with other structures in the pdb-database, the DALI algorithm was employed [ 6 ]. The closest homologue of aaTHEP1 was found to be cob(I)alamin adenosyltransferase (pdb-code: 1G5R, Z-score = 9.9) that catalyzes the final step in the conversion of vitamin B(12) to coenzyme B(12) and has a RecA-like protein fold. A comparison between the topologies of aaTHEP1, cob(I)alamin adenosyltransferase and RecA clearly shows the structural similarity (Fig. 3 ) despite only 9% sequence identity in the aligned region. In contrast to cob(I)alamin adenosyltransferase and RecA, aaTHEP1 contains an extension of its β-sheet consisting of strands β3-β6. We conclude that the structure of aaTHEP1 represents a variation of the RecA protein fold. Topology of the P-loop Although being closest DALI-homologue, the structure of cob(I)alamin adenosyltransferase (CobA) differs significantly from aaTHEP1 within the P-loop (Figure 4 ). Whereas aaTHEP1 bears a P-loop typical for P-loop hydrolases, the P-loop of CobA is shorter by one amino acid which flattens its structure. This is an essential feature for the adenosyl transfer reaction [ 7 ]. Thus, we do not expect aaTHEP1 to catalyze an adenosyl transfer. A survey comparing sequences and structures of all P-loop-fold proteins led to the definition of two major divisions, the GK- and the ASCE-class of NTPases [ 8 ]. Whereas the GK-class includes all GTPases and kinases, the ASCE-class includes all further NTPases. Structurally, the GK-class enzymes contain adjacent P-loop and Walker B strands. In contrast, as it is the case for both aaTHEP1 and the RecA superfamily, the ASCE-proteins contain an additional strand between and a catalytic essential glutamate (E107 in aaTHEP1) within the Walker B motif, thus indicating that aaTHEP1 neither belongs to the group of GTPases nor to the kinase family. The catalytic centre No electron density for an ADP molecule was found indicating that only the nucleotide-free protein crystallized. However, we found electron density for a phosphate ion in the putative nucleotide binding site where the β-phosphate of the nucleotide is expected. This is a usual phenomenon, since negatively charged ions are often found in empty nucleotide binding sites ( e. g. [ 9 ]). In other ATPases and GTPases, the aspartate residue of the consensus site DxxG (D106 in aaTHEP1) is involved in positioning a water-bridged magnesium ion presumably important for nucleotide hydrolysis [ 10 , 11 ]. In the nucleotide free aaTHEP1, there is also a magnesium ion at the corresponding position which is octahedrally coordinated to the hydroxyl group of T14 of the P-loop, a phosphate oxygen and four water molecules. One of these water molecules (W24) makes a hydrogen bond to D106. Thus, the arrangement of the magnesium ion is similar as this found in the nucleotide-bound conformation of other ATPases and GTPases. To determine possible orientations of the nucleotide which was biochemically shown to undergo hydrolysis [ 2 ], we constructed a superposition of aaTHEP1 with RAS complexed with GppNHp (pdb-code: 5P21 [ 12 ]), and RecA complexed with ADP (pdb-code: 1MO3,[ 13 ]) by aligning the P-loop including the precedent β-strand for spatial orientation (Figure 5 ). We then analyzed the resulting position of the nucleotides (GppNHp from Ras and ADP from RecA) relative to the aaTHEP1 surface (Figure 5 ). In both cases, the nucleotide would be located in a cleft of the aaTHEP1 surface and would sterically not clash with residues of aaTHEP1. The position of the phosphates is rather similar whereas the orientation of the ribose and especially the position of the base is markedly different in the ADP and GppNHp although the base would be close to conserved residues in both orientations. We cannot exactly envisage the base orientation of the nucleotide bound to aaTHEP1, but it is very likely that the overall orientation of the nucleotide and the position of the phosphates is correctly predicted. Consequently, the large remaining cleft located adjacently to the predicted position of the γ-phosphate is unoccupied. The pocket itself is rather unpolar but it is lined by a highly conserved patch of basic residues (Figure 5 ) to which a negatively charged cosubstrate, e. g. DNA/RNA could bind. The protein surface The location of conserved residues in a protein structure often points to sites which are functionally important, e. g. the catalytic centre or conserved binding sites [ 14 ]. To detect putative binding sites of aaTHEP1, we colour coded the surface of aaTHEP1 with respect to the conservation of exposed amino acids. As can be seen in Figure 5 , there is only one highly conserved region located in and around a cleft of the protein surface which includes the Walker A motif (P-loop). We conclude that this particular region represents the functionally most important site, i. e. the nucleotide and cosubstrate binding site of aaTHEP1. Not even a single amino acid residue conserved in all species aligned in Figure 1 can be detected on the residual protein surface of aaTHEP1. For that reason, we conclude that binding of the physiological cosubstrate is restricted to the neighbourhood of the nucleotide binding pocket. Analysis of the electrostatic surface potential of aaTHEP1 strikingly revealed a number of positively charged clusters, whereas almost no negatively charged regions can be found (Figure 5 ). This is in agreement with the strong binding of aaTHEP1 to cation exchangers and its theoretical pI of 9.9. The largest positively charged spot is located in a conserved region close to the nucleotide binding cleft. Based on this observation and the similarity to the RecA protein we speculate that aaTHEP1 may be a DNA or RNA modifying enzyme. Gene functions can be predicted by searching for the conservation of operons and gene orders because genes found in gene strings, particularly in multiple genomes, can be assumed to be functionally linked [ 15 ]. For THEP1, we detected 4 genomes ( Aeropyrum pernix K1, Archaeoglobus fulgidus DSM 4304, Thermoplasma acidophilum DSM 1728 and Thermoplasma volcanium GSS1) where the THEP1-gene is immediately followed by a COG1867 protein on the same strand. In Pyrococcus furiosus , this protein is characterized as a N2, N2-dimethylguanosine tRNA methyltransferase [ 16 ]. Thus, aaTHEP1 may also play a role in tRNA modification. Furthermore, both COG1867 proteins and THEP1 proteins can be considered to belong to the group of PACE-proteins (proteins from Archaea without assigned function that are conserved in Eukarya) [ 17 ]. PACE proteins are described being involved in fundamental cellular functions and several of them are obviously related to RNA metabolism [ 18 ]. The human homologue The human homologue MGC13186 (hsTHEP1) shows 39% sequence identity to aaTHEP1 (Figure 1 ) and was first described in a study aiming at identifying full-length ORF for all human and mouse genes[ 19 ]. No function is yet described for this protein. However, gene profiling data from UniGene are available [ 20 ]. hsTHEP1 is widely expressed in most of the examined tissues including brain, heart, lymph node, skin and pancreas whereas no expression was found in blood, thymus, bladder and spleen. It is especially highly expressed in embryonic and various tumour tissues. From these data we conclude that hsTHEP1 has a general function in many human tissues. Conclusion The crystal structure of aaTHEP1 uncovered a modified RecA-like fold. Although the function of aaTHEP1 remains unclear, the structure led us to conclude that the enzyme does not belong to the group of GTPase, kinases or adenosyltransferases. Analysis of the electrostatic surface potential revealed several positively charged clusters indicating the presence of putative nucleic acid binding sites. Since aaTHEP1 has homologues in thermophilic bacteria and vertebrates it can serve as a model for the complete COG1618 protein family. Methods Nomenclature To aid a consistent nomenclature of the THEP1 protein family we propose to adopt the name THEP1 to all members across the species, e.g. hsTHEP1 for the human protein, mmTHEP1 for the mouse protein, etc.. Crystallization, data collection, processing, structure solution, refinement and validation Recombinant aaTHEP1 was purified from Escherichia coli as described earlier [ 2 ]. Bacteria were grown in minimal media without methionine containing 50 mg/l L-selenomethionine [ 21 ]. Crystals of the dimension 250 × 80 × 35 μm 3 were obtained by the hanging drop method after mixing equal volumes of 13 mg/ml aaTHEP1 with reservoir buffer containing 15 % PEG-3350 and 0.1 M potassiumdihydrogenphosphate. For cryo-protection, crystals were soaked for 10 sec in 30 % PEG-3350, 200 mM potassiumdihydrogenphosphate and flash-frozen in liquid nitrogen. The diffraction data were collected at the Swiss Light Source (SLS) from a single crystal. Data were processed and scaled using XDS [ 22 ] and XSCALE [ 22 ]. The positions of the three selenium sites in the asymmetric unit were determined using SHELXD [ 23 ]. Those positions were refined and the electron density of the protein calculated by SHARP [ 24 ]. Solvent flattening and histogram matching were done by SOLOMON [ 25 ] and DM [ 26 ]. ARP/WARP was used to automatically build 85% of the backbone and sidechains [ 27 ]. For further model interpretation XFIT XtalView [ 28 ] was used. Refinements were made with Refmac [ 29 ]. PROCHECK [ 30 ] and Whatcheck [ 31 ] were used to validate the structure. Secondary structures were calculated using DSSP [ 32 , 33 ]. DALI-searches [ 6 ] were carried out at [ 34 ], GRATH [ 35 ] at [ 36 ] and further structural comparisons using SSAP [ 37 ] were done at [ 38 ]. BLAST was performed at [ 39 ]. Figure 1 was prepared using GeneDoc available at [ 40 ]. All figures depicting structures were prepared using PyMol [ 41 ] or Swiss pdb-viewer [ 42 , 43 ]. The X-Ray coordinates and structure factors have been deposited in the PDB database under pdb-code 1YE8. Authors' contributions MR carried out the protein expression, purification and crystallization experiments and participated in structure determination and data analysis. OD participated in structure determination and data analysis. CK participated in data analysis. AW and MK conceived of the study, and participated in its design and coordination. MK drafted the manuscript. All authors read and approved the final manuscript.

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1079891

Internal fixation of femoral shaft fractures in children by intramedullary Kirschner wires (a prospective study): its significance for developing countries

Background To evaluate internal fixation by intramedullary Kirschner wires as a surgical technique in the treatment of femoral shaft fractures in children by a prospective study. Methods 17 femoral shaft fractures at various levels in 16 children aged 2–15 years were treated by closed intramedullary Kirschner wiring under image intensifier control between May 2000 and October 2003. No external splint was used. Results Fracture union was achieved in 6–14 weeks. Non-weight bearing crutch walking was started 2–3 days after surgery. Full weight bearing started 6–14 weeks. Average operative time was 40 min (range 20–72 min). Wires were removed after 8–22 weeks. There were no infections, no limb length disparity. One child had pin track ulceration. A big child of 14 years had angulation of the fracture. Conclusion Intramedullary nailing of femoral shaft fractures in children by stainless steel Kirschner wires is an effective method, which compares well with other studies. It is a simple procedure, which can be easily reproduced. Blood loss is minimal, and the operative time short. There is no need pre-bend the wires in a C or S curve. Stainless steel Kirschner wires are cheap, universally available, and can be manufactured locally. The cost of Image intensifiers is affordable in most of the cities of the developing countries. The hospital does not have to maintain a costly inventory. Provides early mobility, return to home and, school. Gives a predictable clinical pathway and reduces occupancy of hospital beds. The technique was successfully applied for internal fixation of other diaphyseal fractures in children and some selected diaphyseal fractures in adults. Based on my experience and a review of the literature, I recommend this technique as a modality for treatment of femoral shaft fractures in children aged 2 to 14 years.

Background This technique has been used successfully at King Khalid hospital, Najran, Saudi Arabia since 1995. This prospective study was prompted by a review of earlier reports by Shakeel [ 1 ], Salem Al Zahrani [ 8 ], and Ligier and Metazieau [ 6 ]. Fracture treatment in children relies on rapid healing and spontaneous correction of angulated fractures; therefore most of the diaphyseal fractures can be treated by plaster alone. Operative treatment of children's fractures is often looked at critically [ 2 ]. Conventional treatment of femoral shaft fractures in children is by traction followed by a hip spica or a Thomas' splint. Conservative treatment of femoral shaft fractures gives good results in children under 5 years of age. But above that age, all such fractures cannot be treated by conservative methods. There is a possibility of loss of reduction and malunion. Plaster immobilisation has its own complications like pressure sores, nerve palsies, soiling of the skin and the plaster, breakage of the plaster, joint stiffness. The child is immobilised and needs an attendant for personal care. Reeves et al [ 22 ] reported that the cost of non-operative treatment is 40 % higher than operative treatment. In the last few decades, the trend worldwide has been towards some form of fixation for children's fractures, especially the femoral shaft, and the indications for operative management have been widened. Rush [ 3 ] used the intramedullary rods of his design. Intramedullary nailing was made popular by Ender and Simon-Weidner in Europe [ 4 ], and by Pankovitch in the United States [ 5 ]. Beaty, Austin and Canale [ 12 ] studied the preliminary results and complications of interlocking intramedullary nailing of femoral shaft fractures in adolescents. Gonzalez and Herranz[ 13 ] recommend the avoidance of rigid intramedullary nails introduced through the piriformis fossa in children less than 13 years of age. Antegrade intramedullary nailing through the piriformis fossa may cause coxa valga, epiphyseodesis of greater trochanter, thinning of the femoral neck because of damage to the growth plate[ 13 ]. Saxer[ 14 ] advises the introduction of intramedullary Kuntscher nail through the sub-trochanteric zone or the use of plate and screw. External fixation has been advocated by Aronson and Tursky[ 9 ]. They reported angular deformity and shortening of more than 13 mm in proximal third femoral shaft fractures treated by conservative means. External fixation has its own complications; pin track infection and refracture[ 9 , 18 , 19 ]. Also, the child has to accommodate an external device. Compression plating was used by Van Neikerk [ 10 ], Ward [ 11 ] and Hansen [ 17 ]. The disadvantages are the risk of infection, large soft tissue dissection, delayed union, limb length disparity, another large exposure to remove the implants [ 10 , 11 ]. The other disadvantages are periosteal stripping, evacuation of fracture haematoma and blood loss. Tarek Mirdad [ 21 ] reported blood loss requiring blood transfusion in 41 % of children treated by compression plating. Also, a period of 3–4 weeks of protected weight bearing is recommended after removal of plate and screws. Ligier and Metaizeau have successfully treated 123 femoral shaft fractures in children by Elastic Stable Intramedullary Nailing (ESIN) [ 6 ]. Pradeep Kumar [ 7 ], Shakeel [ 1 ] and Zahrani [ 8 ] recommend the efficacy of Kirschner wires for flexible intramedullary nailing of femoral shaft fractures in children. Shakeel reports reduced psychological trauma on the child and the parents [ 1 ]. Methods Between May 2000 and October 2003, 17 femoral shaft fractures at various levels in 16 children aged 2–15 years were treated by internal fixation with intramedullary Kirschner wires at King Khalid hospital, Najran, Ministry of Health, Saudi Arabia. Inclusion criteria a. Displaced fractures, with or without comminution. b. Multiple fractures. c. Fractures found to be unstable on closed reduction. d. Fractures which displaced in traction. e. Fractures in patients with polytrauma and patients under intensive care to facilitate nursing. f. Irritable patients with brain injury. g. Associated vascular injury needing repair. Exclusion criteria Undisplaced fractures and fractures in a good position were treated by traction and hip spica. The goal To provide rapid healing of the fracture in a correct position, ease of nursing care, early mobilisation, and return to home and school. Operative technique Under general anaesthesia, the patient was placed supine on an orthopaedic table with the feet strapped to the footplates and longitudinal traction applied ensuring correct linear and rotational alignment clinically and radiologically using an image intensifier. 3 small children were operated upon a radiolucent table, traction being applied by an assistant. 2 stainless steel Kirschner wires of 30–45 cm length and 2.5–3.5 mm diameter (depending on the size of the medullary canal and the child) were prepared by bending them at an approximate angle of 45°, 2 cm from the tip and cutting off the sharp points to prevent inadvertent penetration of the cortex. The wires were not pre-bent in a 'C' or 'S' curve. The wires were loaded onto a 'T' handled introducer with a Jacob's chuck. Two small skin incisions were made proximal to the superior pole of the patella, one laterally and the other antero-medially. Entry portals were made into the distal femoral metaphysis proximal to the growth plate of the distal femur with a sharp bone awl laterally and antero-medially. The wires were introduced retrograde by hand or gentle hammering. The lateral wire was introduced first. The tips of the wire are placed just distal to the growth plates of the greater and lesser trochanters, the bends pointing towards the side of the entry portal [Figure 1 ]. Angular and rotational malalignment spontaneously corrects on passage of the wires across the fracture. Figure 1 Proximal femoral shaft fracture. Image intensifier screening in two planes perpendicular to each other confirmed proper placements of the wires. The tail portions of the wires were bent towards the fracture and cut 1 cm away from the entry portal in the cortex [Figure 2 ]. The skin wounds were sutured and dressed. Fracture stability, correct linear and rotational alignment was assessed on table. Figure 2 Comminuted proximal femoral shaft fracture. All patients had received a pre-operative bolus of intravenous antibiotic. Antegrade medullary Kirschner wiring was carried out in one small child with a single intramedullary Kirschner wire. The entry portal was on the lateral cortex distal to the growth plate of the greater trochanter. Post-operative rehabilitation No external splint was used except for 2 patients who had ipsilateral tibia and fibula fractures, which were being treated by plaster immobilisation in an above knee plaster cast. The children were started on non-weight bearing crutch walking and knee physiotherapy 2–3 days after the surgery and were ready for discharge 3–4 days after surgery. The children with polytrauma stayed on for a longer period in the hospital because of associated injuries. Sutures were removed on the 10 th day after surgery. The children were encouraged to attend school three weeks after surgery avoiding sports and physical training. The school authorities consented to this and allowed an attendant for the children. Full weight bearing was allowed after clinico-radiological fracture union. Union was defined clinically by the absence of bony tenderness and abnormal mobility at the fracture site, and no pain at the fracture site on weight bearing. Radiological fracture union was defined by the presence of callus bridging the fracture and partial obliteration of the fracture line in 2 views perpendicular to each other. The children were assessed for malunion both linear and rotational, and limb length disparity. The wires were removed under general anaesthesia with the help of pliers, which can be locked onto the wire and hammered on. Results The children were aged 2 years to 15 years. There were 7 left sided and 8 right-sided fractures, and 1 bilateral. 14 children had met with a road traffic accident and 2 had a fall from a height. All were closed fractures [Table 2 ]. Table 2 Details of patients Patient Age Sex Side Open/closed Level of # Operative time In min Open/closed Reduction Stay fixation In days Stay removal In days Weight bearing In weeks Time # union In weeks Time removal In weeks Discharge In weeks 1 6 M L C U3-M3 jn 47 C 5 2 6 6 13 17 2 7 M L C U3 35 C 19 3 6 14 20 24 3 12 F Bil. C M3 75 C 13 3 6 10 20 30 4 2 M L C U3 43 C 13 Lost To Follow up - 5 14 M R C M3 72 O 13 4 12 12 20 20 6 12 M R C U3 36 C 06 3 14 14 22 22 7 8 M R C U3-M3 jn 50 C 11 2 8 8 16 26 8 10 M L C M3 com 46 C 11 3 6 6 16 24 9 10 M L C M3 35 C 12 2 8 8 20 20 10 3 M L C U3 com 25 C 12 Lost To Follow up - 11 11 M R C M3-L3 jn 40 O 61 2 8 8 20 20 12 4 M R C M3 20 C 8 6 6 6 14 16 13 14 M R C M3 32 C 5 IL nail - - - - 14 3 1/2 M L C L3 46 O 5 2 4 6 8 10 15 4 1/2 F R C M3-L3 jn 45 C 6 2 6 6 10 12 16 15 M R C M3 26 C 7 2 8 10 12 14 There were 6 subtrochanteric, 8 midshaft and 3 distal fractures. One child had bilateral femoral shaft fractures [Figure 3 ]. 3 fractures were communited. 12 children had associated injuries and 10 children had polytrauma [Table 1 ]. Table 1 Associated injuries Associated injuries Patients Eye abrasion 1 Ipsilateral tibia and fibula fractures 2 Humerus fracture 1 Blunt abdomen injury 5 Blunt chest injury 1 Brain injury 7 Clavicle fracture 1 Facial injury 1 Ipsilateral femoral artery tear 1 Figure 3 Bilateral femoral shaft fracture. The average operative time was 40 min (range 20–72 minutes). Open reduction had to be carried out in 3 fractures, one for an associated femoral artery tear, which needed repair. The other two were for muscle interposition. A percutaneous bone lever was introduced into the fracture site to reduce another fracture. Open reduction needed a minimum exposure with minimum blood loss, minimum handling of the periosteum and, no increase in the morbidity or alteration in the post-operative rehabilitation. Hospital stay for fracture fixation was between 5–13 days, children with polytrauma stayed longer for treatment of associated injuries. Hospital stay for wire removal was between 2–5 days. Clinico-radiological fracture union was achieved between 6–14 weeks at which time the children started full weight bearing. The wires were removed between 8–22 weeks [Table 2 ]. Complications Pin track ulceration in 1 child. Pins were trimmed under local anaesthesia and the wounds healed in a week's time. Exuberant callus formed in 2 children with brain injury, but this did not affect the final outcome. The fracture angulated in a big child of 14 years. The wires were removed and a closed interlocked medullary nailing was carried out. The fracture went on to union without any further complications. No infections were encountered. All the children had a complete range of movement of the hip and the knee joints. No shortening. Lengthening cannot be commented on, as this was a short-term study. Criteria for malunion [ 16 ] More than 15° angulation in coronal plane, 20° in sagittal plane, and 10° in rotational malalignment is unacceptable. This is variable with age with as much as 45° angulation in sagittal plane being acceptable in infants due to the remodelling potential. Discussion Long years of experience and study have established the place of surgical management of femoral shaft fractures in children. Of the various modalities, ESIN is proving to be the best surgical option. The aim is to encourage the formation of bridging periosteal callus. The wires, the bone and the muscles provide the stability. The muscles act as guy ropes, so even in an irritable child or the hyperactive child, the muscle activity just complements the fixation. Muscle action also causes spontaneous correction of any angular deformities. Micromotion allowed by the elasticity of the fixation promotes external bridging callus. The periosteum is not disturbed and being a closed procedure there is no evacuation of the fracture haematoma or risk of infection. Callus formation is twice as fast as with conventional methods [ 6 ]. In this study the wires were not pre-bent to a 'C' or 'S' curve. Kiely [ 20 ] studied the construct of C, S and straight intramedullary wires in a model simulating a fractured femur of a 6 years old child. He concluded that the principle of flexible nailing does not apply to children's femoral shaft fractures. The factor may be the small diameter of the model used in the study. He also concluded that any of the described nail combinations could be used to stabilise a small diameter bone. This provides the surgeon with a greater range of options when managing fractures. The present study agrees with these findings. In this study, retrograde wiring was used for 2 distal fractures and a single ante grade wire for another distal fracture achieving good results. Other researchers have not reported operating on a child as young as 2 years. In this study, children were allowed to continue normal activities after removal of the wires. This short-term study compares well with other studies on treatment of femoral shaft fractures in children by both conservative and operative methods. It agrees with other studies on elastic nailing with titanium nails and stainless steel Kirschner wires. Staheli [ 15 ] defined the ideal treatment of femoral shaft fractures in children as one that controls alignment and length, does not compress or elevate the extremity excessively, is comfortable for the child and convenient for the family, and causes the least negative psychological impact possible. This technique comes close to achieving this ideal. Conclusion Intramedullary nailing of femoral shaft fractures in children by stainless steel Kirschner wires is an effective method, which compares well with other studies. It is a simple procedure, which can be easily reproduced. Blood loss is minimal, and the operative time short. There is no need pre-bend the wires in a C or S curve. Stainless steel Kirschner wires are cheap, universally available, and can be manufactured locally. The cost of Image intensifiers is affordable in most of the cities of the developing countries. The hospital does not have to maintain a costly inventory. Provides early mobility, return to home and, school. Gives a predictable clinical pathway and reduces occupancy of hospital beds. The technique was successfully applied for internal fixation of other diaphyseal fractures in children and some selected diaphyseal fractures in adults. Based on my experience and a review of the literature, I recommend this technique as a modality for treatment of femoral shaft fractures in children aged 2 to 14 years. Competing interests The author(s) declare that they have no competing interests. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079892

Uroflowmetry nomogram in Iranian children aged 7 to 14 years

Background As the voiding habits of Iranian children differs from other children because of some cultural and religious considerations, we aimed to establish normal reference values of urinary flow rates in Iranian children between 7 to 14 years of age. Methods Eight hundred and two uroflowmetry studies were performed on children with no history of a renal, urological, psychological or neurological disorder, between the ages 7 and 14. Five hundred twenty five studies from 192 girls and 335 boys were considered in this study excluding the staccato/interrupted voiding pattern or voided volume less than 20 ml. The voiding volume, the maximum and average urinary flow rates were extensively analyzed. Results The maximal and average urine flow rate nomograms were plotted for both girls and boys. Mean maximum urine flow rate was 19.9 (ml/sec) for boys and 23.5 (ml/sec) for girls with a mean voided volume of 142 (ml) for boys and 147 (ml) for girls. Flow rates showed a close association with voiding volume in both sexes. The maximum and average flow rates were higher in girls than in boys, and they showed a significant increase in flow rates with increasing age, where boys did not. The mean maximum urine flow rates (19.9 ml/sec for boys and 23.5 ml/sec for girls) were found to be higher in this study than other studies. Conclusion Nomograms of maximal and average flow rates of girls and boys are presented in centile form, which can help the physician to evaluate the response to medical or surgical treatment and be useful for the screening of lower urinary tract disturbances in children, for a wide range of voided volumes.

Background Uroflowmetry is the most commonly used form of urodynamic testing. Simply stated, Uroflowmetry is the measurement of the rate of urine flow over time. It is easy to perform and it is a non-invasive test. Uroflowmetry alone is rarely able to determine the cause of a voiding dysfunction; however, it is extremely useful in selecting patients for more complex urodynamic testing [ 1 ]. It also provides an objective way of determining response to treatment of certain diseases [ 2 ]. Measured flow rates in children were first published by Kaufman [ 3 ] in 1957. He estimated normal flow rates at between 13 and 26 ml/sec with a lowest voiding volume of 100 ml. Scott and Mcllhaney [ 4 ] measured maximum flow rate of 8–29 ml/sec at a voiding volume of over 100 ml. The lower limits of normal maximum and average urine flow rates are still unclear in both sexes. Recommended values for the lower limits of normal for the maximum urine flow rate in men range between 15 (ml/sec) [ 5 ] and 20 (ml/sec) [ 3 ]; in women, the values range between 12 [ 6 ] and 20 (ml/sec) [ 7 ]. Siroky et al. [ 8 ] constructed nomograms using data from a relatively small number of younger men. The nomogram of Jorgensen et al. [ 9 ] was restricted to older woman. All of the above mentioned nomograms were made for adults; one of the famous nomograms for children is Miskole's nomogram that was based on 433 micturitions. At maximum bladder fullness, the overall average flow rates in this study were 16 (ml/sec) in girls and 14 (ml/sec) in boys. The children were divided into three groups according to their body surface [ 10 ]. The aim of the present study was to establish the normal reference values in children of both sexes for maximum and average flow rates for a wide range of voiding volumes in Iran. Besides, there is no pediatric uroflowmetry nomogram for Iranian children and most uroflowmetry software use adult nomograms. Methods This cross-sectional study was performed from April 2000 to February 2001 among 7–14 years old children. The study was approved by the Institutional Board Review and the Medical Ethics Committee of Tehran University of Medical Sciences. The samplings were done in 15 different schools throughout the city of Tehran. We had many discussion sessions for children's parents as well as their teachers and other school staff, explaining the program and its harmlessness for the children. Children who wanted to take part in the program voluntarily and their parents filled in an informed consent were entered in this study. Participants (n = 802) were told by their teacher to come to school with a full bladder in the morning. The mictiograph consisted of an electronic transducer installed in a specific toilet, with the recording equipment in a separate place. Testing was performed in a completely private toilet, lockable from inside. A nurse, skilled in clinical pediatric urology, helped us with the sampling, especially in girls' schools. Children started voiding when the operator told them. All children voided at maximum sensation of bladder fullness. No positional restriction during testing was applied and children were told to void in their habitual positions rather than specifying them to sit or stand. Uroflowmetry was performed using a uroflowmeter model Micromedics System/H, Model no. DPU- 441, Type 1001H 1995, England. Uroflowmetry was performed on 802 children with no history of renal, urological, psychological or neurological disorder, 275 of the studies were excluded from the study because their voided volumes were less than 20 ml [ 11 ] or their flow curves were not bell shape (staccato or interrupted). For each subject data regarding maximum flow rate (Q-max), average flow rate (Q-ave), voided volume (vv), time to maximum flow, flow time and voiding time, as well as age, sex, height and weight was recorded. Statistical analysis was performed using SPSS software for Windows version 11(SPSS Inc., Chicago, IL). A P value of 0.05 or less was considered as statistically significant. Several transformations of data were assessed and the goodness-of-fit tested to determine whether a linear, hyperbolic, parabolic or logarithmic function best described the relation between the maximum or average flow rates and the voided volume [ 12 ]. The quintile regression method was used to establish the percentile levels (5, 10, 25, 50, 75, 90, and 95). For presentation, the nomograms have been expressed in centile form and prepared for boys and girls separately. Differences were assessed for significance using Student's t -test. Results We included Uroflowmetry studies of 527 children, 335 boys (63.6%) and 192 girls (36.4%) 7 to 14 years old. The mean age was 10.44 ± 2.25 years for boys and 9.73 ± 2.17 for girls. The mean voided volumes were 142 ± 97.5 ml in boys and 147 ± 89.74 ml in girls. The mean maximum flow rate was 19.95 ± 10 (ml/sec) in boys and 23.52 ± 8.7 (ml/sec) in girls. The mean average urine flow rates were 8.1 ± 4.4 (ml/sec) for boys and 8.8 ± 4.2 (ml/sec) for girls. The minimum acceptable flow rates for boys and girls regarding age are shown in Table 1 . Flow curves were bell shaped in 87% of all voids, staccato in 11% (32 boys and 26 girls) and intermittent in 2% (7 boys and 2 girls). Table 1 Minimum acceptable flow rates for boys and girls between 7 to 14 years old (according to uroflowmetry parameters of healthy children who voided 100 ml or more). Age Boys Girls Q-max Q-Ave Q-max Q-Ave 7–9 10 3.8 11 3.8 10–11 9.8 4 11.6 4.6 12–14 9.7 4.2 13.8 5.4 Maximum and average flow rates showed no significant increase with age in boys (Pearson's correlations: -0.11, P = 0.099 for Q-max and 0.09, P = 0.835 for Q-ave) but showed a statistically significant increase with age in girls (Pearson's correlations: 0.22 for Q-max, P = 0.001 and 0.27 for Q-ave, P = 0.002). So, in our study girls showed statistically significant increases in urine flow rates with age but boys did not. This increase was approximately 0.92 (ml/sec/year) for maximum urine flow rate and 0.51 (ml/sec/year) for average urine flow rate in girls between the ages 7 and 14. Both sexes showed increases in maximum and average urine flow rates with increasing voided volume. The data sets for the study in both sexes allowed estimation of centiles for flow rates in the range of 20 to 350 ml. In boys, equations for the nomograms related flow rates with voided volume. These became: (a) Square root (Q-max) = 2.05 + 0.2 × Square root (VV) Root mean square error = 0.44 (H 0 : slope = 0, P < 0.001) (b) Square root (Q-ave) = 1.36 + 0.12 × Square root (VV) Root mean square error = 0.38 (H 0 : slope = 0, P < 0.001) In girls, the association between voided volume and flow rate was best fitted by logarithmic function. The final equations for the nomogram graphs were then: (a) Ln (Q-max) = 1.06 + 0.42 × Ln (VV) Root mean square error = 0.36 (H 0 : slope = 0, P < 0.001) (b) Square root (Q-ave) = - 0.67 + 0.74 × Ln(VV) Root mean square error = 0.39 (H 0 : slope = 0, P < 0.001) The equations for each centile are given in Table 2 . Nomograms for the maximum and average urine flow rates for boys are shown in Figures 1 and 2 , respectively. Figures 3 and 4 show the nomograms for the maximum and average urine flow rates for girls. Table 2 The equations for nomograms The fitted parameters (5, 10, 25, 50, 75, 90, 95 percentiles) for the nomograms of maximum and average flow rates against voided volume for boys and girls. The parameter values substitute into the general equation: Sqrt (Q-max or Q-ave) = A × log (Sqrt (vv)) + B for Boys Log (Q-max) = A × log (vv) + B for Girls Sqrt (Q-ave) = A × log (vv) + B for Girls Percentiles Boys Girls Q-max Q-Ave Q-max Q-Ave 5 th A +0.155 +0.087 +0.55 +0.51 B +1.22 +0.82 -0.21 +0.48 10 th A +0.164 +0.093 +0.63 +0.58 B +1.39 +0.92 -0.36 -0.65 25 th A +0.189 +0.124 +0.54 +0.67 B +1.59 +0.99 +0.27 -0.69 50 th A +0.229 +0.131 +0.38 +0.82 B +1.67 +1.25 +1.23 -1.09 75 th A +0.245 +0.138 +0.28 +0.85 B 2.11 +1.53 +1.90 -0.89 90 th A +0.207 +0.14 +0.33 +0.86 B +3.06 +1.87 +1.87 -0.65 95 th A +0.179 +0.134 +0.24 +0.73 B +3.77 +2.29 +2.39 +0.33 Figure 1 Uroflowmetry nomogram for maximum urine flow rates in Boys 7–14 in Iran Figure 2 Uroflowmetry nomogram for average urine flow rates in Boys 7–14 in Iran Figure 3 Uroflowmetry nomogram for maximum urine flow rates in Girls 7–14 in Iran Figure 4 Uroflowmetry nomogram for average urine flow rates in Girls 7–14 in Iran Discussion These nomograms were constructed to provide normal reference values in both sexes for urinary flow rate, covering a wide range of voided volumes and in centile form. Our nomogram is the only uroflowmetry nomogram for children in Iran. The use of nomograms avoids the dangers of referencing flow rates to any one voided volume. A maximum urine flow rate of 12 (ml/s) in men or 15 (ml/s) in women might fall just inside the fifth centile curve at 200 (ml) voided volume, although well outside the same curve at 400 (ml). The normality of these flow rates may then be interpreted quite differently at these 2 voiding volumes. Siroky and colleagues were among the first to develop a nomogram that allowed uroflow to be corrected for voided volume. They also applied their nomogram to evaluation of men with bladder outlet obstruction. The authors stated that Q-max was a more sensitive indicator of outlet obstruction than Q-ave [ 8 ]. The Liverpool [ 13 ] nomogram was created from the micturitions of 331 normal adult men and 249 normal women. Men showed a significant decline in urinary flow rates with age, but women didn't show statistically significant variation in flow rates in this study. The authors believed that the voiding difficulties can be excluded in women by uroflowmetry, based on the 10th centile ranking that differentiates between women who are at low or high risk of having voiding dysfunction. Haylen and colleagues also believed that uroflowmetry could identify a subgroup of symptomatic men with high Q-max who are likely to have detrusor instability [ 13 ]. The use of statistical transformation by Gierup [ 14 ] and Jensen et al. [ 15 ] overcomes the problems created by inaccuracy when untransformed standard deviations were used. Churchill et al. [ 16 ] created nomograms for maximum urine flow rates for boys using regression analysis to fit functions to the data. Di Scipio [ 17 ] found only low correlations of age, height, weight and body surface area to average and maximum flow rates. Abele and Krepler [ 18 ] reported that the flow rates of girls and boys were similar. Miskole [ 10 ] nomogram noted that the curves of 50% of the average and maximum flow rates were the same in both sexes when the voided volume was less than100 ml. But when the voided volume is more than 100 ml, the curves for the girls were higher. Gutierrez Segura's report [ 19 ] confirmed that the maximum and average flow increased with volume and age. Mean values were higher in girls than boys, and these differences were greater for maximum flow with larger volumes and older age except in the 12–14 year old group. He reported a normal flow pattern in 90% of healthy children, which is comparable to 87% Bell shape flow pattern in our study. The mean maximum and average urine flow rates found higher in girls than in boys. This is interpretable by the girl's shorter urethra. The mean maximum urine flow rates (19.9 ml/sec for boys and 23.5 ml/sec for girls) were found to be lower in this study than those reported in western studies (Miskole [ 10 ]: Mean maximum urine flow rate 14 ml/sec for boys and 16 ml/sec for girls). This may be explained to some extent by the fact that because of religious considerations children have very early toilet training and parents force them to void every 1–2 hours in the toilet-training period to avoid wetting. These may cause a learned early sensation to void resulting in full sensation at lower bladder volumes. Low voided volume in healthy children is also confirmed in both sexes during 3 days voiding diary in this country (unpublished data). Conclusion Nomograms in centile form are very useful for diagnosing urinary flow disturbances over a wide range of voided volumes. The present study provides reference values of maximal and average flow rates of normal boys and girls. Uroflowmetry is become a screening test for urinary problems in children. Competing interests The author(s) declare that they have no competing interests. Authors' contributions AMK conceived the study, participated in its design and coordination and finalized the manuscript. CAY and OR participated in design, collected the necessary data, reviewed the literature, and wrote a first draft of the manuscript. PT have reviewed the literature, edited the draft, revised the statistical analyses and nomograms and submitted the manuscript. PM participated in the design of the study. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079893

Exogenous glycosaminoglycans coat damaged bladder surfaces in experimentally damaged mouse bladder

Background Interstital cystitis is often treated with exogenous glycosaminoglycans such as heparin, chondroitin sulphate (Uracyst), hyaluronate (Cystistat) or the semi-synthetic pentosan polysulphate (Elmiron). The mechanism of action is presumed to be due to a coating of the bladder surface to replace the normally present chondroitin sulphate and heparan sulphate lost as a result of the disease. This study used fluorescent labelled chondroitin sulphate to track the distribution of glycosaminoglycans administered intravesically to mouse bladder that had been damaged on the surface. Methods The surfaces of mouse bladders were damaged by 3 mechanisms – trypsin, 10 mM HCl, and protamine sulphate. Texas Red-labeled chondroitin sulphate was instilled into the bladders of animals with damaged bladders and controls instilled only with saline. Bladders were harvested, frozen, and sectioned for examination by fluorescence. Results The normal mouse bladder bound a very thin layer of the labelled chondroitin sulphate on the luminal surface. Trypsin- and HCl-damaged bladders bound the labelled chondroitin sulphate extensively on the surface with little penetration into the bladder muscle. Protamine produced less overt damage, and much less labelling was seen, presumably due to loss of the label as it complexed with the protamine intercalated into the bladder surface. Conclusion Glycosaminoglycan administered intravesically does bind to damaged bladder. Given that the changes seen following bladder damage resemble those seen naturally in interstitial cystitis, the mechanisms proposed for the action of these agents is consistent with a coating of damaged bladder.

Background Interstitial cystitis (IC) is being re-examined within a broader context of chronic pelvic pain and pelvic floor dysfunction with the result that it is being recognized that the prevalence of IC is much higher than previously estimated [ 1 , 2 ]. It has long been evident that, whatever the cause, IC is a disease of the urothelium and the symptoms are triggered by urine [ 3 ]. In general, the bladder exhibits thinning and denudation of urothelium and distinctive changes in the muscle [ 4 ], and there is very strong evidence that the urothelium fails to differentiate properly in IC [ 5 , 6 ] One of the main therapeutic approaches is so-called "glycosaminoglycan (GAG) replacement therapy." This is partly based upon earlier observation that the so-called "GAG Layer" is lost or damaged in the interstitial cystitis bladder [ 6 , 7 ]. The "GAG Layer" is a complex set of proteoglycans and glycoproteins on the bladder surface has been isolated and characterized from cow and human bladder [ 8 - 11 ]. Whether or not glycosaminoglycans actually restore impermeability of the bladder in interstitial cystitis is controversial [ 12 - 15 ]. Nonetheless, many patients respond to intravesical instillations of pentosan polysulphate (Elmiron) [ 16 ], hyaluronate (Cystistat) [ 17 ], chondroitin sulphate (Uracyst) [ 18 ]or heparin [ 19 ]. In this paper, we use three different models of bladder damage to determine whether GAG instilled intravesically actually binds preferentially to damaged bladder. We prepared a fluorescent-labeled chondroitin sulphate molecule by covalently attaching Texas Red to the reducing terminus of the GAG chain and then observing bladder sections by fluorescence to determine where the labelled GAG was found. Methods Texas Red-labelled chondroitin sulphate was prepared by mixing 10 μL Texas Red Hydrazide (Molecular Probes, Eugene, OR), 10 mM in dimethyl formamide, 40 μL of 1 mM chondroitin sulphate (Stellar Healthcare, London, Ontario), and 5 μL of 50% acetic acid, The chondroitin sulphate was assumed to be 20,000 molecular weight, yielding a concentration of 20 mg/ml as being equivalent to 1 mM. The reaction was allowed to occur overnight at room temperature. The next morning, the bond was reduced and made permanent by adding 10 μL of 150 mM sodium cyanoborohydride for 2 h. The labelled ChS was separated from unincorporated label by gel filtration on Biogel P-4. Mice (C57BL/6NHsd, 9–10 weeks old) were anesthetized by intraperitoneal injection of ketamine 40 mg/kg and xylazine 5–10 mg/kg and catheterized with a 24 Ga × 3/4" intravenous catheter (Terumo Medical, Elkton, MD). Bladder damage was produced by 3 methods. (a) Protamine, 1 mg/ml, was instilled into the bladder until fullness and allowed to remain for 10 minutes. The protamine was then washed out with 3 fillings of buffered saline. (b) Dilute HCl, 10 mM was prepared by dilution in saline and was administered in the same way as described for the protamine except that a 0.1 M sodium bicarbonate wash was used as the first rinse;. (c) Trypsin, 1 mg/ml, was dissolved in saline and instilled into the mouse bladder where it was allowed to remain for 30 min. The trypsin was washed out with three fillings of buffered saline. Control animals were treated with buffered saline only. Three animals were in each group. The mouse bladders were then filled with Texas Red-labeled chondroitin sulphate, which was allowed to remain for 1 hr. The bladder was gently rinsed as described above. The animals were then euthanized by cervical dislocation with death being confirmed by opening the chest. The bladders were removed, flash frozen in OCT, and sections were obtained for fluorescence. The bladders were sectioned (2 μm thick) with a cryostat and then examined first by ordinary fluorescence (2 sections per animal) and then by confocal microscopy using a Leica TCS NT confocal system (1 section). A representative field was selected for confocal microscopy. Texas Red emission was stimulated with excitation at 568 nm and the fluorescence was collected using a 630 nm bandpass filter. The nuclei were visualized by labelling with 20 μM Hoechst 33259. Sections were also stained with hematoxylin and eosin (H&E) and a low-pH Alcian Blue counterstained with Nuclear Fast Red. For analysis of the fluorescence intensities to assess relative binding, areas of urothelium in the image were outlined with the "lasso" tool of PhotoShop. Total pixels within the area were counted. The red colour was then detected within that area and the average brightness and number of pixels was calculated. The integrated intensity was calculated as the average pixel intensity × number of red pixels/total area in pixels. For the controls, the unenhanced image was used so that it would be comparable to the other images. Images were interpreted without knowledge of the treatment. Results Figure 1 shows that each of the models of bladder damage produces a different pattern of damage. This figure shows both a conventional H&E stain (A) and an Alcian Blue stain (B) with the fluorescent images shown in (C). The controls showed an even urothelium with a well-formed endogenous "GAG layer" that appeared as a thin, blue line on the luminal surface of the urothelium. The luminal surface was even and showed the characteristic umbrella cells. The separation of the entire urothelium from the stroma seen in all the damaged bladders probably represents actual damage induced by, for example, edema resulting from loss of permeability rather than an artefact of sectioning. Trypsin removed most of the entire umbrella cell layer along with the "GAG layer." Protamine treatment produced little overt urothelial damage, but did roughen the luminal surface. The dilute HCl treatment also stripped off the umbrella cells and weakened the connection between urothelium and the stroma. Figure 1 Damage produced by each model of bladder damage and binding of Texas Red-labelled chondroitin sulphate to mouse bladder . Images in the first two columns are transmitted light images of bladder sections stained with H&E or Acid Alcian Blue to demonstrate bladder damage. Acid Alcian Blue binds to glycosaminoglycans and therefore stains the connective tissue and the "GAG Layer," which is indicated by arrows in the control image. The fluorescence images are digitally combined images of Hoechst 33258 fluorescence (blue) to show nuclei and Texas Red-labelled chondroitin sulphate (red). Arrows have been added to the fluorescence image of the protamine-treated bladder to show the location of the urothelium. From top to bottom, the rows show the controls, bladders treated with 1 mg/ml trypsin (30 min), 1 mg/ml protamine (10 min) and 10 mM HCl (10 min.) respectively. The fluorescence image of the control was digitally enhanced to demonstrate the small amount of binding that occurred, but which was undetectable at the settings used for the other images (Table 1). As shown in the fluorescence images, in control animals which experienced only saline instillation into the bladder, the label was found only on a narrow band corresponding to the luminal cell layer. The amount of TR-labeled chondroitin sulphate that actually bound to the controls was minimal, and the image shown in the figure was digitally enhanced to visualize the small amount that did bind. The dilute HCL damage model and the trypsin-damage model showed increased binding of the label to the bladder surface, but the binding still was confined mainly to the damaged urothelium. In the protamine damage model, the amount of binding was reduced. In no case, however, was significant penetration of the bladder by the label seen. Table 1 shows the quantitative image densitometry measurement of the amount of red fluorescence found at the urothelial surface of each of the images. Binding to the normal bladder surface was undetectable, but could be revealed by digital enhancement of the image. The trypsin- and HCl-damaged bladders bound the most labelled chondroitin sulphate, whereas the protamine damaged bladder bound about 1/5 th the amount bound by the other two models. These values are consistent with the level of damage observed. Table 1 Comparison of binding of TR-labeled Chondroitin Sulphate to bladder urothelium in experimental bladder damage. Control Trypsin Protamine Dil. HCl Mean pixel intensity 0 139 ± 17 167 ± 24 156 ± 28 Red Area (pixels) 0 5,952 2,058 4,814 Urothelial Area (pixels) 366,025 81,879 79,623 64,528 Normalized Red 0 0.0727 0.0133 0.0746 Integrated Red 0 10.1 ± 1.3 2.2 ± 0.3 11.6 ± 2.1 Discussion Although large placebo responses have clouded clinical trials of replacement GAG therapy, thousands of patients have benefited from therapy with one of the agents used for allegedly replacing the bladder "GAG Layer." The mechanism of action could arise from physically coating the bladder surface and restoring impermeability as Parsons has claimed [ 19 ], or could be due to a more complex mechanism [ 20 ]. The normal urothelium has minimal affinity for chondroitin sulphate and binds it only on the luminal surface of the umbrella cells in miniscule quantities. However, the chondroitin sulphate did not penetrate into the deeper muscle layers of the bladder when the urothelium was damaged or destroyed, but bound extensively to the damaged bladder surface. Two of the bladder models yielded virtually identical results, but the pattern seen with protamine damage was different, and less chondroitin sulphate was bound than was observed in the trypsin and dilute HCl damage models. Lewis and co-workers [ 21 ] observed that protamine was inserted into the apical cell membrane, destroying the permeability barrier, but that glycosaminoglycans extracted the protamine, thereby restoring the bladder impermeability. Our findings are not inconsistent with this model, in which case both the extracted protamine and its complexed chondroitin sulphate would be removed with the fluid. The net result could be decreased binding of the label. However, the protamine instillation produced less overt damage as well. The observed pattern of binding to damaged bladder suggests that its main action is on the urothelial surface and is not due to a pharmacologic effect. Both heparin and pentosanpolysulphate have pharmacologic effects on the coagulation system [ 22 ], whereas chondroitin sulphate, a normal constituent of the bladder surface, and hyaluronate do not [ 23 ]. All of these glycosaminoglycan molecules have effects on mast cells and other cells within the bladder at high concentrations in vitro [ 24 - 26 ]. The fact that all of these glycosaminoglycan molecules alleviate symptoms in some patients in spite of not penetrating into the bladder and remaining on the surface, even in the face of extensive damage, suggests a physical effect is responsible for clinical efficacy at least of chondroitin sulphate. We suggest that one possible mechanism for the symptom-relieving effect of this class of compounds is they may exclude urinary proteases rather than salts. Theoretical calculations based on actual measurements of the amount of glycosaminoglycan found normally on the human bladder surface showed the presence of a dense layer of glycosaminoglycan will yield a bound water layer that reduces the concentration of salts at the bladder surface [ 20 ]. Lewis and Clausen earlier had demonstrated that urinary proteases degrade bladder ion channels [ 27 ], and Negrete and co-workers demonstrated the impermeability of rabbit bladder lay in the apical membrane [ 15 ]. Because the highly charged GAG layer with its attendant bound water layer would be a far more significant barrier for macromolecules than small, inorganic ions, we speculate that the GAG layer may normally function to exclude macromolecules rather than salts. Therefore the therapeutic benefit of exogenous glycosaminoglycans may result from exclusion of proteases or other macromolecules from the bladder surface where they could produce additional damage. Conclusion Chondroitin sulphate instilled intravesically into three animal models of bladder damage coated the damaged bladder surface and failed to penetrate the bladder. This suggests they act at the bladder surface rather than in deeper layers of the bladder. Competing interests This work was supported by a grant from Stellar Healthcare, Inc., a company that markets Uracyst for the treatment of Interstitial Cystitis. Authors' contributions Jean Coffman obtained frozen sections and provided histologic interpretations. Kimberly Kyker performed the confocal microscopy and provided interpretations. Robert Hurst was responsible for the overall design of the experiments and their interpretation and for writing the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

D:\keerthana\PMC001xxxxxx\PMC1079893.xml

1079894

Casodex treatment induces hypoxia-related gene expression in the LNCaP prostate cancer progression model

Background The changes in gene expression profile as prostate cancer progresses from an androgen-dependent disease to an androgen-independent disease are still largely unknown. Methods We examined the gene expression profile in the LNCaP prostate cancer progression model during chronic treatment with Casodex using cDNA microarrays consisting of 2305 randomly chosen genes. Results Our studies revealed a representative collection of genes whose expression was differentially regulated in LNCaP cells upon treatment with Casodex. A set of 15 genes were shown to be highly expressed in Casodex-treated LNCaP cells compared to the reference sample. This set of highly expressed genes represents a signature collection unique to prostate cancer since their expression was significantly greater than that of the collective pool of ten cancer cell lines of the reference sample. The highly expressed signature collection included the hypoxia-related genes membrane metallo-endopeptidase (MME), cyclin G2, and Bcl2/adenovirus E1B 19 kDa (BNIP3). Given the roles of these genes in angiogenesis, cell cycle regulation, and apoptosis, we further analyzed their expression and concluded that these genes may be involved in the molecular changes that lead to androgen-independence in prostate cancer. Conclusion Our data indicate that one of the mechanisms of Casodex action in prostate cancer cells is induction of hypoxic gene expression.

Background Prostate cancer remains the second leading cause of cancer death in men. The progression of prostate cancer from an androgen-dependent, treatable stage to an androgen-independent, non-curable stage is still the primary cause of mortality. Despite screening for early diagnosis, it is still not possible to determine which patients will progress to the androgen-independent stage [ 1 ]. Androgen-dependent prostate cancer is responsive to androgen-ablation therapy and the cancer cells die by apoptosis [ 2 ]. Androgen ablation or blockage of androgen action through the androgen receptor has been the cornerstone of treatment of advanced prostate cancer [ 3 ]. Casodex is a non-steroidal anti-androgen commonly used in the treatment of prostate cancer. Treatment of patients with Casodex has been shown to decrease the progression of early stage prostate cancer to the advanced stages [ 4 ]. One of the issues in androgen-blockade therapy is the occurrence of androgen-independent cells which are thought to be the predominant cell type after relapse from therapy. These androgen-independent cells often express mutations in the androgen receptor which accounts for their lack of response to anti-androgens. Recently, it was shown that treatment of patients with Casodex did not result in the occurrence of cells expressing androgen receptor mutations [ 5 ]. In our studies, Casodex was chosen since this anti-androgen was shown to act as an androgen receptor antagonist in LNCaP cells. LNCaP cells express a mutation in the hormone binding domain which causes these cells to be stimulated by other anti-androgens [ 6 ]. Other studies have shown that Casodex can act as an agonist in prostate cancer [ 7 ]. cDNA microarray analysis has become a powerful tool to measure global gene expression changes in both patient biopsy samples and cell culture models. The ability to quantitatively measure differential gene expression provides an opportunity to compare differences in samples which identify potential pathways and modulators of disease processes [ 8 ]. A recent study measured differences in gene expression in patient samples and found a clear difference between prostate cancer and benign prostatic hyperplasia [ 9 ]. Another study examined differences in gene expression in prostate cancer cell lines and found a unique set of genes whose expression was up regulated [ 10 ]. Yet another study showed expression of the enhancer of zeste homologue 2 (EZH2), a polycomb group protein, was overexpressed in androgen-independent prostate cancer and may be a potential modulator of progression [ 11 ]. An unusual high tolerance of cancer tissues to low oxygen conditions was one of the first observations regarding cancer cells [ 12 ]. The significance of hypoxia in cancer has only recently become a focus of many studies. Recently, it was suggested that expression of hypoxic genes may be responsible for the progression to malignancy in prostate cancer [ 13 ]. It was also shown that the more hypoxic a prostate tumor in a patient, the more likely the patient was to experience therapy failure [ 14 ]. It has also been suggested that the mechanism of prostate cell death upon androgen withdrawal is due to apoptosis induced by ischemia and hypoxia [ 15 ]. In this study we utilized the previously described LNCaP prostate cancer progression model [ 16 , 17 ] to address changes in gene expression that accompany the progression to androgen-independence. Our previous studies showed that chronic treatment of LNCaP-R and LNCaP-UR cells with Casodex selected for cells resistant to apoptosis induction [ 18 ]. In the present study, we utilized cDNA microarray analysis to measure the changes in gene expression that had occurred in the LNCaP model after treatment with Casodex to identify potential modulators of prostate cancer progression. We used low passage, androgen-dependent, LNCaP-R cells and high passage, androgen-independent, LNCaP-UR cells and treated cells with Casodex for five weeks to mimic chronic treatment in patients. Our results indicate a set of highly expressed genes that may serve as a signature for prostate cancer progression. A subset of these highly expressed genes were the hypoxia-related genes membrane metallo-endopeptidase (MME), cyclin G2, and Bcl2/adenovirus E1B 19 kD ( B -cell lymphoma n ineteen kDa i nteracting p rotein, BNIP3). This study identifies for the first time the novel pathway of hypoxic gene expression that may modulate the progression of prostate cancer to the androgen-independent state and demonstrate the induction of hypoxia as a mechanism of Casodex action. Methods Chemicals and reagents Bicalutamide (Casodex) was provided by AstraZeneca (UK) and was dissolved in fresh dimethylsulfoxide (DMSO) to a concentration of 50 mM, divided into aliquots, and stored at -20°C until further use. Normal human prostate epithelial cell RNA (PrEC) was purchased from Clonetics (Rockland, ME). RPMI 1640, Dulbecco's Modified Essential Media (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (PS), TRIzol, and Hank's Balanced Salt Solution (HBSS) were purchased from Life Technologies (San Diego, CA). Total human reference RNA was purchased from Stratagene (La Jolla, CA). Cell culture LNCaP cells were maintained in RPMI 1640 supplemented with 7% FBS and 1% PS. DU145 and PC3 cells were grown in DMEM supplemented with 5% FBS and 1% PS. Treatment of LNCaP cells with Casodex was performed as follows: Cells were seeded into T75 flasks in RPMI 1640 with 7% FBS and 1% PS and allowed to grow to 70% confluence. The media was then removed and replaced with RPMI 1640 with 7% FBS and 1% PS media containing 1 μM Casodex (vehicle 0.01%). Control cells were fed with media and passaged as needed. Cells grown in media containing Casodex were fed once every three days but did not require passaging during the five-week treatment. Isolation of RNA RNA was isolated from cells using TRIzol according to manufacturer's instructions. Briefly, media were removed and cells were rinsed once in HBSS. A volume of 3 ml of TRIzol was added to each T75 flask and cells were scraped and placed into tubes for chloroform phase separation. RNA was precipitated using isopropanol, rinsed with 70% ethanol in diethylpyrocarbonate water (DEPC), and resuspended in RNase free water. The quality and quantity of RNA was determined using RNA 6000 Nano chips on the Agilent 2100 Bioanalyzer (Agilent Technologies). Array fabrication The slides used in this study were generated as follows: cDNA inserts from Research Genetics Sequence Verified Human cDNA library were amplified using standard procedures in a 96-well format, each well containing 100 μl of PCR reaction mix and 1 μl of bacterial culture as the template. The reaction buffer contained 1.5 mM MgCl 2 , 1× PCR buffer, 0.2 mM dNTPs, 0.25 mM primers, 2.5 units Taq DNA polymerase, and 0.2 % Tween 20. Following an initial denaturation step of 94°C for 60 seconds, 33 cycles of 94°C for 45 seconds, 55°C for 45 seconds, and 72°C for 180 seconds was performed. Verification of the amplified PCR products was performed by agarose gel electrophoresis. Individual PCR products were precipitated overnight at -20°C with sodium acetate (pH 5.2) and ethanol. Following precipitation, the DNA pellets were allowed to dry at room temperature and were resuspended by addition of 30 μl of 3× SSC and 0.1% sarcosyl solution. This procedure resulted in a concentration range of 100 to 499 ng/μl of DNA per sample. The PCR products were spotted 2,305 spots at 375 μm spacing / 2.2 cm 2 / per slide onto poly-L-lysine-coated microscope slides using an Affymetrix GMS 417 arrayer per manufacturer's suggestions at 50% humidity and 25°C. The slides were allowed to dry overnight before the DNA was cross-linked to the slides by UV irradiation. Probe synthesis and hybridization A total of 20 μg RNA from each control and treated samples were reverse transcribed in the presence of amino-allyl modified dUTP [5-(3-aminoallyl)-2'-deoxyuridine 5 triphosphate] and indirectly labeled with cyanine 3 (cy 3) or cyanine 5 (cy 5) dye using the FairPlay microarray labeling kit (Stratagene) according to manufacturers instruction. The absorbance of the samples was measured at 260 nm (for cDNA), 550 nm (for cy 3), 650 nm (for cy 5) to give an indication of reverse transcription and dye labeling efficiency. Cy3 and cy5 labeled samples were mixed with 20 μg human Cot 1 DNA (Stratagene) and 20 μg poly-adenine (Sigma) and the samples were dried in a speed vacuum on medium heat for 2 hours. Dried samples were resuspended in 18 μl of hybridization buffer containing 50% formamide, 5× sodium chloride and sodium citrate (SSC), and 0.1% sodium dodecyl sulfate (SDS). The samples were denatured by incubation at 95°C for 3 minutes then placed on ice for 30 seconds. Slides were prehybridized by incubation for 1 hour at 42°C in prehybridization buffer containing 1.0% bovine serum albumin (BSA), 0.1% SDS, and 5× SSC. The slides were rinsed three times in milli Q water, dipped into 99% isopropanol, and air-dried. Labeled probes were hybridized for 16 hours in a 42°C water bath. Following hybridization, slides were sequentially washed for 5 minutes each in buffers containing; 1× SSC and 0.2% SDS, 0.1× SSC and 0.2% SDS, and 0.1% SSC. Slides were immediately scanned using a GenePix 4000A scanner (Axon Instruments, Union City, CA). Data extraction and analysis Multi image tiff files generated using the GenePix 4000A scanner were imported in GenePix Pro version 4 software (Axon Instruments) for data extraction. Extraction of signal intensity data from each spot on the images was performed and visually checked for accuracy. Raw fluorescent intensities at 532 and 635 nm were measured and local area background was subtracted from each spot using GenePix Pro. Files generated from the extraction were imported in Microsoft Excel for normalization. Trimmed global median normalization was performed on the ratio of medians. For data tables, candidate genes were chosen according to fluorescent intensity readings at 635 nm above 400 units. Normalized ratios of the medians from each of the three hybridizations were averaged and the standard deviation was reported. Normalized, averaged data sets were then analyzed using Cluster analysis and visualized using Treeview [ 19 ]. RT-PCR RNA transcripts were characterized by RT-PCR using SuperScript II RNase H - Reverse Transcriptase (Invitrogen, Life Technologies) according to manufacturer's instructions. Total RNA (5 μg) isolated from cells using TRIzol was reverse transcribed to cDNA using oligo-dT primers. The PCR product for hypoxia-inducible factor-1α (HIF-1α) was amplified using the following primers: F: 5'-CCTTCGATCAGTTGTCACCA-3' and R: 5'-TGGGTAGGAGATGGAGATGC and gave a 211 bp product. The PCR product for membrane metalloendopeptidase (MME, also referred to as CD10, CALLA, and NEP) was amplified as described by Xiao et. al. [ 20 ] and gave a 268 bp product. For a control, β-actin was amplified in parallel using primers; F: 5'-GAAGAGCTACGAGCTGCC-3', and R: 5'-TGATCCACATCTGCTGGA-3' and gave a 368 bp product. Cyclin G2 primers were, F: 5'-AGCCATCAAATGGGGTAGTG-3', and R: 5'-CTTGGGGCAATAGGAATGAA-3' and gave a product of 502 base pairs. BNIP3 primers were; F: 5'-GCTCCTGGGTAGAACTGCAC-3', and R: 5'-TCTTCATGACGCTCGTGTTC-3' and gave a 411 base pair product. The following RT-PCR cycling parameters were used; 42°C for 2 minutes, 42°C for 50 minutes, 70°C for 15 minutes, and hold at 4°C indefinitely. A volume of 5.0 μl of the RT-PCR reaction product was added to each PCR reaction with 1.0 μl each of forward and reverse primers. The PCR cycling parameters used for Cyclin G2 and BNIP3 were; Step 1, 95°C for 3.0 min; Step 2, 94°C for 1 min; Step 3, 60°C for 1 min; Step 4, 72°C for 45 sec; Step 5, repeat steps 2 through 4 thirty-five times; Step 6, 72°C for 3 min; and Step 7, hold at 15°C until further use. The PCR product of VEGF was amplified as described by Munaut et al [ 21 ], Glut-1 was amplified as described by Ito et al [ 22 ], and Carbonic anhydrase-IX and XII were amplified as described by Nishimori et al [ 23 ]. Real time PCR analysis Quantitative Real Time PCR reactions were performed using QuantiTect SYBR Green RT-PCR kit (Qiagen Inc., Valencia, CA) as per manufacturer's instructions. Each reaction was set up with 1/10 th the volume of cDNA generated by RT-PCR as described above. The PCR cycling parameters used for HIF-1α are initial incubation of 95°C for 15 minutes, 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, 72°C for 30 seconds, were performed. Reactions, in duplicate, were performed on a BioRad iCycler thermalcycler using iCycler software version 2.3v. The comparative CT equation denotes the expression level of the gene in a given sample, normalized with in the sample to an endogenous reference gene and relative to the expression level of the same gene in another sample can be represented as ΔΔCT= [ΔCT (sampleX) ] - [ΔCT (calibratorsample) ] and ΔC T = [CT (targetgene) ] - [CT (referencegene) ] [ 24 ]. Western blot analysis Protein extracts were prepared from cells using a protein lysis buffer containing 50 mM Tris-HCl, pH 7.5, 2.0 mM phenylmethylsulfonyl fluoride (PMSF), 5.0 mM iodoacetamide, 5.0 mM ethylene diamine tetraacetic acid (EDTA), 150 mM NaCl, 0.5% nonylphenoxy polyethoxy ethanol (NP-40), and 0.5% nonanoyl-N-methylglucamide (Mega-9). Protease inhibitors leupeptin (2 μg/ml) and pepstatin (1 μg/ml) (Roche, Mannheim, Germany) were added just prior to the addition of lysis buffer to the cells. Protein concentrations in the extracts were quantitated using bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). From each extract, 10 to 50 μg of total protein was separated on a 12% SDS-PAGE with a 5% stacking gel. After electrophoresis, proteins were transferred to 0.2 μm PVDF membranes (BioRad, Hercules, CA) using transfer buffer that contained 25 mM Tris-HCl and 700 mM glycine. Membranes were blocked in 7% powdered milk dissolved in 1× TTBS (14 mM Tris, 154 mM NaCl, and 0.1% Tween-20, pH adjusted to 7.5 with HCl) overnight at 4°C. Primary antibodies were incubated for 2 hours at room temperature in 5% powdered milk dissolved in 1× Tween-20 in TTBS. The antibody for the mitochondrial import receptor protein (TOM20) was provided by Dr. Masataka Mori, Kumamoto University School of Medicine, Kumamoto, Japan. Primary antibodies used were: polyclonal rabbit anti-human TOM20 at 1:1,500 dilution, polyclonal rabbit anti-human BNIP3 (BD Pharmingen, San Diego, CA) at 1:8,000 dilution; and polyclonal goat anti-human Cyclin G2 (Santa Cruz Biotechnology) at 1:1,500 dilution. The antigen-antibody complexes were detected using the appropriate secondary antibodies conjugated to horseradish peroxidase (Promega, Madison, WI) dissolved in 1× TTBS with 5% powdered milk and incubated with membranes for 1 hour at room temperature. Membranes were developed using ECL+ (Amersham Pharmacia Biotech, Arlington Heights, IL) and films exposed for appropriate times to detect signal. Cell fractionation The cytosolic and heavy membrane fractions of cells were separated using the ApoAlert Cell Fractionation kit (Clontech Laboratories, Palo Alto, CA) according to manufacturer's instructions. Briefly, cells were mechanically dislodged and collected by centrifugation at 600 × g for 5 minutes at 4°C. The pellets were washed with 1 ml ice-cold wash buffer, and resuspended in 800 μl of ice-cold fractionation buffer. After incubation on ice for 10 minutes, cells were homogenized by 50 strokes using a Dounce homogenizer (pestle B). The homogenates were centrifuged at 700 × g for 10 minutes at 4°C. The supernatant was collected and further centrifuged at 10,000 × g for 25 minutes at 4°C. The resulting supernatant was designated as the cytosol fraction and the pellet (heavy membrane fraction) was resuspended in 100 μl of fractionation buffer mix. Protein concentrations in the extracts were quantitated using bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). Results Genes highly expressed upon Casodex treatment To analyze the expression of genes that accompany the progression of prostate cancer to the androgen-independent phenotype, we performed three separate cDNA microarray experiments, on androgen-dependent LNCaP-R and androgen-independent LNCaP-UR cells cultured with or without treatment for five weeks in 1 μM Casodex. Hybridizations were performed against a common reference RNA pool so that comparisons across samples and experiments could be made. The commercially available common reference RNA pool contains RNA from ten different cancer cell lines. Of the 2305 genes examined, we found 160 genes whose expression was altered at least 2-fold in the treated cells. Table 1 shows the list of genes whose expression was significantly increased compared to the reference RNA and Table 2 shows the fold change of these genes in response to treatment with Casodex. These genes were overexpressed in all three hybridizations. After normalization and filtering, a total of 160 genes from the samples were analyzed using hierarchical clustering to show the similarities of expression. The dendogram separated the samples according to overall similarity and shows that control and treated LNCaP-R samples reside on the same branch and control and treated LNCaP-UR samples reside on a parallel branch (Fig. 1 ). Of the 160 genes clustered were MME, cyclin G2, and BNIP3 all of which showed differential regulation between the LNCaP-R and UR cells and all of which clustered in a same node (Fig. 1 ). The correlation coefficient of this node was 0.85 which suggests that the expression pattern of these genes was 85% similar. Table 1 Highly expressed Casodex responsive genes in the LNCaP model. Data show fold-increase in gene expression over reference samples. Each datum point represents mean ± SE from 3 independent experiments. LNCaP-R LNCaP-UR GenBank # Name Control ± SE Treated ± SE Control ± SE Treated ± SE AA663884 synaptosomal-associated protein 15.18 ± 3.35 20.67 ± 9.33 20.92 ± 1.07 11.68 ± 0.61 AA700604 sorbitol dehydrogenase 13.73 ± 3.23 19.60 ± 6.50 21.08 ± 1.58 11.08 ± 1.39 T73556 fatty-acid-Coenzyme A ligase 5.11 ± .34 2.45 ± 0.74 3.94 ± 0.46 1.07 ± 0.25 AA028987 EST 5.38 ± 6.95 8.43 ± 5.68 6.22 ± 2.05 11.00 ± 3.74 R98851 membrane metallo-endopeptidase 10.28 ± 5.90 55.90 ± 30.2 79.29 ± 16.62 153.50 ± 56.07 N68465 UDP-N-acteylglucosamine pyrophosphorylase 4.37 ± 3.73 3.99 ± 1.44 12.47 ± 2.03 9.03 ± 2.41 H84113 retinal outer segment membrane 1 8.03 ± 2.06 14.73 ± 4.93 9.79 ± 1.39 7.09 ± 0.65 AA041499 cell division cycle 4-like 11.66 ± 7.07 10.67 ± 1.84 3.47 ± 0.44 3.64 ± 1.14 AA456695 H2B histone family, member Q 4.67 ± 3.02 4.23 ± 1.14 2.56 ± 0.27 4.67 ± 1.13 R82299 S-adenosylmethionine decarboxylase 1 8.35 ± 2.23 14.84 ± 4.06 11.11 ± 2.19 6.98 ± 0.51 AA063521 BCL2/adenovirus E1B 19 kD 3.59 ± 3.34 13.14 ± 3.94 6.76 ± 1.79 12.46 ± 2.32 AA419164 retinoic acid receptor, beta 9.90 ± 3.40 11.14 ± 2.23 7.44 ± 0.98 5.01 ± 0.46 AA459039 serine protease inhibitor, Kunitz type, 2.89 ± 2.90 10.44 ± 1.46 6.28 ± 0.99 9.94 ± 1.44 AA489752 cyclin G2 2.86 ± 2.67 10.97 ± 1.10 6.92 ± 1.62 15.14 ± 4.80 AA412053 CD9 antigen (p24) 4.80 ± 1.05 8.78 ± 2.72 5.44 ± 0.96 6.12 ± 0.84 Table 2 Gene expression fold change in the LNCaP model treated with Casodex. Fold Change GenBank# Name LNCaP-R Control vs. Treated LNCaP-UR Control vs. Treated T73556 fatty-acid-Coenzyme A ligase - 2.09 - 3.68 R98851 membrane metallo-endopeptidase + 5.44 + 1.94 AA063521 BCL2/adenovirus E1B 19 kD + 3.66 + 1.84 AA459039 serine protease inhibitor, Kunitz type, + 3.61 + 1.58 AA489752 cyclin G2 + 3.84 + 2.19 (+, fold up regulated and -, fold downregulated) Figure 1 Cluster diagram of LNCaP cells treated with Casodex . Clustering was performed as described in Materials and Methods. LNCaP-R C, control. LNCaP-R T, treated 5 weeks in 1 μM Casodex. LNCaP-UR C, control. LNCaP-UR T, treated 5 weeks in 1 μM Casodex. Columns , individual samples and rows , individual genes. Cells represent the expression level of the indicated gene where green is downregulated, red is up regulated, black is zero, and gray is missing. The correlation in this node is 0.85. Hypoxia regulated gene expression in the LNCaP model treated with Casodex HIF-1 is a transcriptional activator involved in oxygen homeostasis in cells [ 25 ]. In tumors where hypoxic conditions are common, HIF-1 levels are increased and stimulate gene expression that promotes angiogenesis [ 26 ]. HIF-1α is the inducibly expressed subunit that forms a heterodimer with the constitutively expressed HIF-1β. HIF-1α has been implicated in androgen-independent prostate cancer [ 13 ] and is thought to mediate angiogenesis in tumors by transcriptional up regulation of vascular endothelial growth factor [ 27 ]. BNIP3 was shown to be regulated by HIF-1α [ 28 , 29 ] which led us to examine whether the high levels of BNIP3 detected in our hybridizations were accompanied by expression of HIF-1α. Figure 2 (panel A) shows the results of an RT-PCR and reveals the expression of HIF-1α in normal human prostate epithelial cells, PC3 and DU145 cells, and the LNCaP model. Using RT-PCR, we did not observe significant differences in the intensity of the PCR band between different cell lines and upon treatment with Casodex. To more quantitatively assess the message level, we performed a quantitative Real Time PCR analysis of HIF-1α along with β-actin as an internal control. The data presented in Figure 2 (panel B) shows a clear up regulation of HIF-1α expression in the androgen-independent PC3 and DU145 cells when compared to the low levels expressed in normal prostate epithelial cells (PrEC). In the LNCaP model, low levels of HIF-1α are expressed in the untreated LNCaP-R and LNCaP-UR cells. However, upon Casodex treatment, both LNCaP-R and UR cells show increased expression of HIF-1α which more closely resembles the expression pattern observed in the androgen-independent PC-3 and DU-145 cells. Figure 2 A. RT-PCR of HIF-1α . RT-PCR was performed as described in Materials and Methods and shows the presence of HIF-1α (upper panel) and β-actin as internal control (lower panel). Lane 1, normal prostate (PrEC). Lanes 2 and 3, androgen-independent prostate cell lines PC3 and DU145, respectively. Lane 4, LNCaP-R control. Lane 5, LNCaP-R treated 5 weeks in 1 μM Casodex. Lane 6, LNCaP-UR control. Lane 7, LNCaP-UR treated 5 weeks in 1 μM Casodex. B. Quantitative real time PCR for HIF-1α expression was perfomed as described in materials and methods. CT values was determined using the formula ΔΔCT = [ΔCT (sampleX) ] - [ΔCT (calibratorsample) ] and ΔC T = [CT (targetgene) ] - [CT (referencegene) ]. The graph was drawn using Graph Pad Prism 3.0. Verification of MME, Cyclin G2, and BNIP3 message in the LNCaP model MME is a neutral endopeptidase expressed on the cell surface and catalyzes the degradation of biologically active neuropeptides [ 30 ]. Prostate epithelial cells express MME, including the LNCaP prostate cancer cell line, and there is evidence that the presence of MME on the surface of prostate cancer cells promotes metastasis [ 31 , 32 ]. In contrast, other reports suggest that MME can inhibit prostate cancer cell growth [ 33 ] and can inhibit prostate cancer cell migration [ 34 ] and loss of MME expression can contribute to the progression to androgen-independence [ 35 ]. MME was shown to be up regulated at the transcriptional level by androgen in LNCaP prostate cancer cells [ 36 ]. RT-PCR was performed to verify message expression of MME. Figure 3 shows the results of RT-PCR of MME in normal human prostate epithelial cells, PC3 and DU145 cells, and the LNCaP model control and treated with Casodex. The data show a single product of expected size (268 bp) in all samples tested. As a normalization control, we included β-actin in parallel experiments (Figure 3 , panel B). MME protein was reported to be undetectable in PC3 and DU145 cells [ 32 ]. However, our results show that MME message was expressed in PC3, DU145 and LNCaP cells. Figure 3 RT-PCR of MME . RT-PCR was performed as described in Materials and Methods and confirms the presence of MME. A single band at 268 bp (black arrow) indicates MME message expression. Lane 1, normal prostate (PrEC). Lanes 2 and 3, androgen-independent prostate cell lines PC3 and DU145, respectively. Lane 4, LNCaP-R control. Lane 5, LNCaP-R treated 5 weeks in 1 μM Casodex. Lane 6, LNCaP-UR control. Lane 7, LNCaP-UR treated 5 weeks in 1 μM Casodex. β-actin (bottom panel) was included as a control. Cyclin G2 is a cell cycle inhibitor [ 37 ] whose expression is induced during apoptosis. Cyclin G2 is expressed at varying levels through the cell cycle and was shown to be present in cerebellum, thymus, spleen, kidney and prostate tissue [ 38 ]. Interestingly, cyclin G2 was recently shown to be induced by hypoxia [ 39 ]. Figure 4 verifies cyclin G2 message expression (product at 502 bp) in the LNCaP cells treated or untreated with Casodex. RT-PCR confirmed the presence of cyclin G2 in control and treated LNCaP cells. However, western blot analysis showed that Casodex-treated cells overexpressed cyclin G2 protein (Figure 5 ). Figure 5 shows protein levels of cyclin G2 in LNCaP cells treated or untreated with Casodex. Cyclin G2 protein levels appear to be up regulated upon treatment with Casodex in both the LNCaP-R (lane 2) and LNCaP-UR (lane 4) cells. These results are consistent with cyclin G2 message up regulation indicated in microarray analysis (Table 2 ). Figure 4 RT-PCR of Cyclin G2 . RT-PCR was performed as described in Materials and Methods and confirms the presence of Cyclin G2 message expression in LNCaP cells control or treated with 1 μM Casodex for 5 weeks. Cyclin G2 message is indicated (black arrow) at 502 bp. Lane 1, LNCaP-R control. Lane 2, LNCaP-R treated 5 weeks in 1 μM Casodex. Lane 3, LNCaP-UR control. Lane 4, LNCaP-UR treated 5 weeks in 1 μM Casodex. Figure 5 Western blot of Cyclin G2 . Western blot was performed as described in Materials and Methods and shows the presence of Cyclin G2 (CCNG2) protein in LNCaP cells control or treated with 1 μM Casodex for 5 weeks. Lane 1, LNCaP-R control. Lane 2, LNCaP-R treated 5 weeks in 1 μM Casodex. Lane 3, LNCaP-UR control. Lane 4, LNCaP-UR treated 5 weeks in 1 μM Casodex. BNIP3 is a proapoptotic Bcl-2 family member localized to the mitochondrial membrane and was shown to interact with antiapoptotic family members to promote apoptosis [ 40 ]. The transmembrane region of BNIP3 localizes this protein to the mitochondrial membrane and it was shown that deletion mutants lacking the transmembrane region were unable to induce apoptosis [ 40 ]. BNIP3 was shown induce apoptosis in hypoxic cardiac cells. [ 41 - 43 ] and is itself induced by hypoxia in tumors [ 29 ]. Figure 6 verifies by RT-PCR BNIP3 message expression (411 bp) in the LNCaP model control and treated with Casodex. RT-PCR's were repeated for each MME, cyclin G2, and BNIP3 biological replicates and similar results were observed (data not shown). Figure 6 RT-PCR of BNIP3 . RT-PCR was performed as described in Materials and Methods and confirms the presence of BNIP3 message expression in LNCaP cells control or treated with 1 μM Casodex for 5 weeks. BNIP3 message expression is indicated (black arrow) at 411 bp. Lane 1, LNCaP-R control. Lane 2, LNCaP-R treated 5 weeks in 1 μM Casodex. Lane 3, LNCaP-UR control. Lane 4, LNCaP-UR treated 5 weeks in 1 μM Casodex. BNIP3 in the LNCaP model treated with Casodex In order to assess the significance of BNIP3 message up regulation in the LNCaP model, we performed western blots to determine the subcellular localization of the protein. Cytosolic and mitochondrial fractions were subjected to western blot analysis and TOM20, an integral mitochondrial membrane protein [ 44 ], was included as an internal control. Figure 7 shows that BNIP3 was detected in the mitochondrial fraction of both LNCaP-R and UR cells (lanes 2, 4, 6, and 8), and BNIP3 is overexpressed in the Casodex-treated LNCaP-R and LNCaP-UR cells. Figure 7 Cell fractionation and western blot of LNCaP cells treated with Casodex . Cell fractionation of cytosolic (Cyto) and mitochondrial (Mito) fractions and western blot was performed as described in Materials and Methods. LNCaP cells either treated with 1 μM Casodex for 5 weeks or control were fractionated and subjected to western blot and show that LNCaP-UR cells overexpress BNIP3 which is predominately localized in the mitochondrial fraction. Lane 1, LNCaP-R control cytosolic fraction. Lane 2, LNCaP-R control mitochondrial fraction. Lane 3, LNCaP-R treated with 1 μM Casodex for 5 weeks, cytosolic fraction. Lane 4, LNCaP-R treated with 1 μM Casodex for 5 weeks, mitochondrial fraction. Lane 5, LNCaP-UR control cytosolic fraction. Lane 6, LNCaP-UR control mitochondrial fraction. Lane 7, LNCaP-UR treated with 1 μM Casodex for 5 weeks, cytosolic fraction. Lane 8, LNCaP-UR treated with 1 μM Casodex for 5 weeks, mitochondrial fraction. TOM20, a mitochondrial integral membrane protein, (bottom panel) was included as a control. Expression of other hypoxia-related genes In order to determine if other hypoxia-related genes were being regulated in the LNCaP model treated with Casodex, we measured mRNA expression levels of vascular endothelial growth factor (VEGF), glucose transporter-1 (Glut-1), and carbonic anhydrases 9 and 12 (CAIX and CAXII). VEGF isoforms 189, 165, and 121 are shown in Fig. 8 , panel A. We did not detect a band representative of VEGF isoform 145. Isoform 121 showed relatively equal expression in all cells tested however, isoform 165 appeared upregulated in LNCaP-R cells after five weeks treatment with Casodex (Fig. 8 panel A. lane 5). Isoform 189 appeared upregulated in DU145 cells (lane 2) compared to PC3 cells (lane 1) and increased in LNCaP-R and UR cells after treatment with Casodex (lanes 5 and 7, respectively). Increased glucose uptake, mediated by Glut-1, is thought to protect cells from apoptosis in hypoxic conditions [ 45 ]. Expression levels of Glut-1 are shown in Fig. 8 panel B and shows basal levels of Glut-1 in PC3 and DU145 cells (lanes 1 and 3). Treatment of LNCaP-R and UR cells with Casodex resulted in an upregulation of Glut-1 message (lanes 5 and 7, respectively). CAIX and XII are inducible by HIF-1α [ 39 ] and were shown to be expressed at very low levels in PC3 cells and negative for expression in DU145 cells [ 46 ]. Fig. 8 panel C shows CAIX expression is barely detectable in PC3, DU145, LNCaP-R control and treated with Casodex (lanes 2 through 5). LNCaP-UR cells showed an approximate two-fold increase in CAIX expression after treatment with Casodex (lanes 6 and 7). CAXII expression was detected in both PC3 and DU145 cells (Fig. 8 , panel D, lanes 1 and 2), however, in LNCaP-R cells treated with Casodex, the levels appeared to decrease approximately two-fold (lanes 4 and 5). LNCaP-UR cells showed approximate equal expression of CAXII after treatment with Casodex (lanes 6 and 7). Figure 8 RT-PCR of HIF-1 induced genes . LNCaP cells were treated with 1μM Casodex for 5 weeks. RNA was isolated from both treated and untreated cells as described in Materials and Methods. Expression of VEGF isoforms (panel A), Glut-1 (panel B), CA-IX (panel C), CA-XII (panel D) and β-Actin (panel E) were examined by RT-PCR. In each panel, the cell lines shown are PC-3 (lane 2), DU-145 (lane 3) LNCaP-RC (lane 4), LNCaP-RT (lane 5), LNCaP-URC (lane 6) and LNCaP-URT (lane 7). The 100 bp DNA ladder (lane1) is included as a size marker. PCR amplification was performed as described under Materials and Methods. Discussion In this study, cDNA microarray analysis was performed to identify genes involved in prostate cancer cell response to chronic treatment with Casodex. The LNCaP prostate cancer progression model was used due to its ability to progress from an androgen-dependent to an androgen-independent phenotype [ 17 ]. With this model, it was possible to measure changes in gene expression that accompanied progression from androgen-dependence to androgen-independence in a similar genetic background in response to chronic treatment with Casodex. Since patients with both androgen-dependent and androgen-independent prostate cancer are treated with androgen-ablation therapy, it is important to understand what changes in gene expression occur in both cases in order to identify potential therapeutic targets for more specific intervention independent of androgen-signaling status. The treatment time course of LNCaP in Casodex containing media was based on pilot studies which showed no change in the antiapoptotic protein Bcl-2 after growth of the cells for three weeks. After five weeks in Casodex containing media, there was an apparent decrease in Bcl-2 protein and we measured a decrease in apoptosis [ 18 ]. We surmised from these studies that the changes occurring in LNCaP cells resulted after extended exposure to Casodex and therefore, set the time course of five weeks treatment. The three independent microarray analyses consistently showed a set of highly expressed genes (Table 1 ). Given that the reference RNA contained a set of 10-pooled cancer cell lines, we termed these highly expressed genes as "prostate cancer signature genes". We chose to further examine expression of MME, cyclin G2, and BNIP3 since these genes were significantly up regulated in the LNCaP model upon treatment with Casodex (Table 2 ) and these genes are each involved in hypoxia-response. Several genes showed downregulated expression in response to Casodex treatment in LNCaP-UR cells (Table 1 ) however, only fatty-acid-Coenzyme A ligase showed downregulated expression in both LNCaP-R and UR cells (Table 2 ). Although we did not further investigate this downregulation, it is possible that hypoxic conditions resulted in fatty-acid metabolism alterations which caused the downregulation of this ligase. Clustering of the 160 genes showed that MME, cyclin G2, and BNIP3 were similarly expressed (Fig. 1 ) and clustered in a similar node (correlation = 0.85) which supports the notion that a hypoxic response is a predominant phenomena in LNCaP cells response to treatment with Casodex. The dendogram in Figure 1 revealed the similarities and differences between representative cell lines in our LNCaP model. Control and treated LNCaP-R samples clustered in the same branch and in parallel, control and treated LNCaP-UR samples clustered in the same branch (Fig.1). Since LNCaP-R and LNCaP-UR cells differ in terms of growth, morphology, response to androgen, and sensitivity to apoptosis [ 16 ], it was expected the two would cluster in separate branches. These data demonstrate the distinct genetic expression profiles largely due to the androgen-dependent and androgen-independent difference between LNCaP-R and LNCaP-UR cells. We probed for HIF-1α expression by RT-PCR. The results showed the presence of HIF-1α at the expected molecular weight in all cells. Quantitative PCR analysis indicated up regulation of HIF-1α in Casodex treated LNCaP-R and LNCaP-UR cells (Figure 2 , panel B). This result is consistent with up regulation of hypoxia-related genes upon treatment with Casodex (Tables IA and IB ). Further, up regulation of HIF-1α in the Casodex-treated cells is similar to the high levels of HIF-1α in the androgen-independent PC3 and DU145 cells, indicating that Casodex treatment may drive the LNCaP cells towards androgen-independence, consistent with our previous observations [ 18 ]. We verified the identity and expression of some of the genes identified by microarray analysis and relevant to hypoxia. MME, cyclin G2, and BNIP3 message identity and expression were confirmed by RT-PCR analysis (Figs. 3 , 4 , and 6 , respectively). MME has been implicated in prostate cancer and though the data are conflicting on whether this gene promotes or protects against the progression, our data suggest a role for this gene in prostate cancer progression to androgen-independence. Though others report that MME was not detectable in PC3 and DU145 cells [ 29 ]. This disparity may be due to subtle differences in culturing techniques or PCR conditions between our laboratory and others working with these cells. In the LNCaP-R cells, MME expression was up regulated over 5-fold and in LNCaP-UR cells MME was up regulated nearly 2-fold in response to Casodex (Table 2 ). In context, MME was up regulated over 150-fold in the LNCaP-UR cells compared to the reference RNA (Table 1 ) which strongly suggests this gene plays a role in androgen-independent prostate cancer. Precisely what function MME plays in the LNCaP model is currently under investigation. Cyclin G2 was shown to inhibit cell cycle progression and promote apoptosis [ 37 ]. Our data showed an increase in cyclin G2 message (Table 2 ) and protein expression (Fig. 5 , lanes 2 and 4) in the LNCaP model upon treatment with Casodex. Aside from the known induction of cyclin G2 by HIF-1α [ 39 ] the increased expression of cyclin G2 in our Casodex treated samples could also be due to the long duration of time the cells spend in the culture flask. The experiment warrants that Casodex treatment begins after the cells have come to approximately 70% confluence. At this density, there is a short time period after the addition of media containing Casodex of sustained logarithmic growth after which the cells appear to cease growth before the 5 weeks treatment has ended (visual inspection, data not shown). It is possible that cyclin G2 is involved in the growth arrest observed in the LNCaP model during treatment with Casodex. BNIP3 is a proapoptotic Bcl-2 family member and as such, its up regulation and overexpression would predict induction of apoptosis. We have previously shown that in LNCaP cells treated with 1 μM Casodex for 5 weeks then treated with 5 nM docetaxel, the cells pretreated for 5 weeks in Casodex exhibited an increased resistance to the induction of apoptosis by docetaxel when compared to cells not pretreated with Casodex but treated with docetaxel [ 18 ]. We addressed the possibility that BNIP3 protein may be missorted or mutated. It was shown that C-terminal deletion mutants of BNIP3 lost the ability to induce apoptosis [ 40 ]. We did detect the presence of BNIP3 in the mitochondrial membrane fraction of cells suggesting that BNIP3 is localized correctly to the mitochondrial membrane presumably via its C-terminal transmembrane region. Determining why the increased levels of BNIP3 do not induce apoptosis in the LNCaP model is currently under investigation. Expression of VEGF isoforms 165 and 121 showed similar levels in LNCaP-R and UR cells treated with Casodex. However, the 189 isoform appeared to be upregulated upon treatment with Casodex. The 189 isoform may be preferentially induced in response to Casodex in the LNCaP model. The absence of VEGF isoform 145 in all cells tested suggest either that it is not a preferred expressed isoform in prostate cancer cells or that our PCR cycling conditions did not favor its expression though this latter possibility is unlikely since the primers used were designed to amplify all four splice variants. Glut-1 expression levels showed upregulation in the LNCaP model after treatment with Casodex suggesting that the cells were responding to Casodex treatment by presumably increasing glucose uptake to protect against hypoxia-induced damage. CAIX and XII showed variable expression (Fig. 8 . panels C and D) with CAIX detected only in the LNCaP-UR cells. CAXII showed variable expression in all cells and was downregulated in LNCaP-R cells treated with Casodex (Fig. 8 panel D, lane 5) but appeared unchanged in LNCaP-UR cells after treatment (lanes 6 and 7). These data suggests that CAIX and XII may not be major contributors in LNCaP cells response to Casodex treatment. It was recently shown, using the LNCaP prostate cancer cell line, that one of the mechanisms of Casodex in prostate cancer was the assembly of an inactive transcriptional complex on androgen response elements in the DNA suggesting that altered coactivator and/or corepressor genes may be responsible for androgen-independent progression [ 47 ]. Our data showing differential expression of hypoxia related genes up regulated upon treatment with Casodex are supported by this finding. Conclusion We conclude from these studies that hypoxia plays a critical role in the progression of prostate cancer to the androgen-independent phenotype and furthermore, identify induction of a hypoxic response as one of the mechanisms of Casodex in prostate cancer. Competing interests The author(s) declare that they have no competing interests. Authors' contributions CAR and VKG contributed equally to the work. CAR carried out the cell culture, treatment and microarray studies. VKG performed the immunoblot analyses. JDE performed the microarray analysis and assisted in data analysis. JKV conceived of the study, participated in its design and analysis, and direct the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

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1079895

Glycogen synthesis correlates with androgen-dependent growth arrest in prostate cancer

Background Androgen withdrawal in normal prostate or androgen-dependent prostate cancer is associated with the downregulation of several glycolytic enzymes and with reduced glucose uptake. Although glycogen metabolism is known to regulate the intracellular glucose level its involvement in androgen response has not been studied. Methods We investigated the effects of androgen on glycogen phosphorylase (GP), glycogen synthase (GS) and on glycogen accumulation in the androgen-receptor (AR) reconstituted PC3 cell line containing either an empty vector (PC3-AR-V) or vector with HPV-E7 (PC3-AR-E7) and the LNCaP cell line. Results Androgen addition in PC3 cells expressing the AR mimics androgen ablation in androgen-dependent prostate cells. Incubation of PC3-AR-V or PC3-AR-E7 cells with the androgen R1881 induced G1 cell cycle arrest within 24 hours and resulted in a gradual cell number reduction over 5 days thereafter, which was accompanied by a 2 to 5 fold increase in glycogen content. 24 hours after androgen-treatment the level of Glucose-6-P (G-6-P) had increased threefold and after 48 hours the GS and GP activities increased twofold. Under this condition inhibition of glycogenolysis with the selective GP inhibitor CP-91149 enhanced the increase in glycogen content and further reduced the cell number. The androgen-dependent LNCaP cells that endogenously express AR responded to androgen withdrawal with growth arrest and increased glycogen content. CP-91149 further increased glycogen content and caused a reduction of cell number. Conclusion Increased glycogenesis is part of the androgen receptor-mediated cellular response and blockage of glycogenolysis by the GP inhibitor CP-91149 further increased glycogenesis. The combined use of a GP inhibitor with hormone therapy may increase the efficacy of hormone treatment by decreasing the survival of prostate cancer cells and thereby reducing the chance of cancer recurrence.

Background Androgen withdrawal leads to apoptosis of normal prostate cells and is the principal therapy to treat advanced prostate cancer [for a review, [ 1 ]]. Metabolic events known to be associated with androgen withdrawal are reduction in glucose uptake, downregulation of several glycolytic enzymes and of some key enzymes of the pentose-phosphate shunt [ 2 - 5 ]. Androgen withdrawal led to transcriptional downregulation of the pyruvate dehydrogenase E1 alpha (PDH E1α) gene in rat ventral prostate and in PC3 prostate cancer cells transiently transfected with the androgen receptor. Reduced transcription of PDH E1α is associated with a reduction of the glucose oxidative pathway [ 6 ]. In contrast, androgen stimulated CO 2 production derived from glucose [ 2 ]. These results suggest that glucose transporters and several catabolic enzymes are regulated in an androgen-dependent manner. Glycogen metabolism is regulated by intermediates of glycolysis, by covalent modification and by glycogen and purines. The two major enzymes GS and GP are controlled by phosphorylation and allosterically by effector molecules [ 7 - 9 ]. Glycogen synthase (GS) in its phosphorylated form is inactive but can be activated allosterically by G-6-P. This can facilitate the dephosphorylation by a glycogen-bound PP1-type phosphatase to the active form [ 10 , 11 ]. Active GS is inactivated by phosphorylation by several important protein kinases: casein kinase II, calmodulin-dependent kinases, protein kinase A (PKA), protein kinase C (PKC) [ 12 , 13 ]. Glycogen synthase kinase 3 (GSK-3), a major kinase inactivating GS, phosphorylates several sites on GS but only when GS has been phosphorylated at other sites [ 14 ]. Partial dephosphorylation of a specific N- or C-terminal residue increases the sensitivity of GS to activation by G-6-P [ 15 ]. Glycogen phosphorylase (GP) also exists in two forms, the active phosphorylated a-form (GP-a) and the inactive b-form (GP-b). cAMP and calcium stimulate the activation of GP through PKA and phosphorylase (PHOS) kinase, which seems to be the only kinase phosphorylating GP [ 16 ]. Muscle GP is allosterically activated by the binding of AMP, whereas G-6-P and glucose are allosteric inhibitors [ 9 ]. We have recently shown that the cyclin-dependent kinase inhibitor flavopiridol, which is in clinical trials as an anticancer agent, is also a potent GP inhibitor and binds to the purine-nucleotide inhibitor-binding site of GP [ 17 , 18 ]. Inhibition of glycogen degradation by the specific GP inhibitor CP-91149 also growth inhibited cells that expressed high levels of brain GP but not cells expressing low levels of brain GP [ 19 ]. CP-91149 binds at a site located at the subunit interface in the region of the central cavity of the dimeric structure and stabilizes the inactive form of GP [ 20 - 23 ], These observations raised the possibility that glycogen metabolism, and in particular brain GP, may be a potential target for anticancer therapy. Therefore, to understand the regulation and role of glycogen metabolism in prostate cancer in response to androgen we measured intracellular glycogen stores, the activities of GS and GP and G-6-P in prostate cancer cell lines. Our results indicate that glycogen accumulation and reduction in cell growth are associated with the androgen response of prostate cancer cells and can be further enhanced by GP inhibition with the GP inhibitor CP-91149. Thus androgen-dependent growth arrest and cell death can be further enhanced by GP inhibition. Methods Cell lines and cell culture The construction and characterization of PC3 cells reconstituted with the androgen receptor (AR) has been reported [ 24 ]. For these experiments, PC3-AR cells were stably transfected with vector pZ16E67 BN containing the human papilloma virus E7 protein cDNA (PC3-AR-E72 and E73) or vector pZipNeoSV(X)1 alone (PC3-AR-V1 and V2)[ 25 ]. LNCaP cells were obtained from ATCC and experiments were performed with cells around passage 23. All cells were grown in RPMI 1640 lacking phenol red supplemented with charcoal-stripped 5% fetal bovine serum and containing penicillin (100 units/ml), and streptomycin (100 μg/ml). Stably transfected PC3-AR-E7 or V cells were maintained with 100 μg/ml hygromycin and 500 μg/ml G418. Cultures of LNCaP cells were supplemented with 4 nM of the stable testosterone derivative R1881. Androgen response was induced by either adding 4 nM R1881 (PC3-AR-E7 or V) or by omitting androgen from the culture medium (LNCaP). PC3-AR-E7 or V cells were plated in a density of 5 × 10 5 cells per 100 mm dish and LNCaP in a density of 3 × 10 5 cells per 100 mm dish. Cells were kept under 95% air and 5% CO 2 at 37°C in a humidified incubator. Glycogen synthase (GS), phosphorylase (GP) activity assays, glycogen and G-6-P determination GS assays were performed as described with some modifications [ 26 ]. For the assay cells from one 100 mm plate were harvested, washed in PBS and lysed in lysis buffer (50 mM Tris/HCl (pH 7.5), 250 mM NaCl, 0.1% NP40, 5 mM EDTA, 50 mM NaF, 1 mM for phenyl-methyl-sulfonyl-fluoride, protease inhibitor cocktail (Boehringer-Mannheim)). Cell lysates were diluted equally with GS dilution buffer. The reaction was started by adding 25 μl assay buffer to 25 μl of the lysate. The assay buffer contained 5 mM UDP-glucose and 1 μCi/ml of [ U -14 C ]UDP-glucose. 10 mM G-6-P was added to determine the activity of allosterically activated GS. The reaction was performed at 30°C for 15 min and stopped by pipetting the mixture on a p81 Whatman filter. The filters were immersed in 66% EtOH, washed several times in EtOH, once in acetone, air-dried and counted in a liquid scintillation counter (LKB Wallac). GP activity and glycogen content were determined as described with modifications [ 17 , 27 , 28 ]. The G-6-P concentration was determined in the following way. Nine 100 mm petri dishes were prepared for the isolation of G-6-P and cells collected, sedimented by centrifugation and 0.75 ml 6% perchloric acid added to the cell pellet. Samples were homogenized, iced for 10 min, vortexed and centrifuged at 6000 rpm for 15 min in the Sorvall at 4°C. 0.6 ml of supernatant was neutralized by adding 134 ul of 4 M KOH/0.8 M imidazole. A pH around 7 was confirmed with pH paper. Samples were then centrifuged for 20 min to precipitate the salts. The supernatant was stored at -80°C until assayed in a Hitachi Fluorometer. The reaction was started by adding G-6-P dehydrogenase from Leuconostroc mesenteroides and incubated at room temperature for 25 min. Samples were measured before the reaction and immediately afterwards. The excitation wavelength was 365 nm and the emission wavelength 435 nm. Flow cytometric analysis Cells were fixed with 70% ethanol, DNA was stained with propidium iodide. The intensity of fluorescence was measured using a Becton Dickenson flow cytometer at 488 nm for excitation and at 650 nm for emission [ 29 ]. The cell cycle profile was analyzed using Modifit's Sync Wizard (Verity Software Inc.). Results Growth, cell cycle arrest and glycogen content in PC3-AR-E7 and PC3-AR-V cells treated with R1881 The effects of androgen on glycogen metabolism in prostate cancer were investigated using the PC3-AR model [ 30 , 31 ]. PC3 cells do not express the androgen receptor (AR) and are, therefore, androgen-independent. Paradoxically, when PC3 cells are reconstituted with AR (PC3-AR), they become sensitive to the addition of androgen by exiting from the cell cycle and undergoing apoptosis [ 24 ]. This phenomenon is until now unexplained. Therefore, to induce an androgen response, the androgen R1881 was added to PC3-AR (PC3-AR-V) cells. Glycogen content normalized to cell count was determined after different incubation times (Fig. 1A ). Glycogen content doubled when cells were incubated with R1881 for 24 hours and further increased upon longer incubation times with R1881. This effect was not observed in PC3 cells lacking AR and treated with R1881 for up to 72 hours. Figure 1 The effect of R1881 on viable cell number, cell cycle and glycogen content of PC3-AR-V and PC3-AR-E7 cells . Two independently isolated control (PC3-AR-V1 and PC3-AR-V2) or HPV-E7 expressing clones (PC3-AR-E72 and PC3-AR-E73) were tested. R1881 was added to exponentially growing cells to a final concentration of 4 nM and cells were further incubated for the indicated times. PC3 cells, which lack AR were used as a negative control. Cell samples were removed A) to determine the glycogen content and B) to determine the viable cell count using the trypan blue assay. Androgen-independent prostate cancer frequently acquires the loss of a functional retinoblastoma protein (pRb) [ 32 ]. Loss of pRb leads to a reduction of androgen-dependent gene expression, which has been interpreted as a possible mechanism for the androgen-independent growth of these cells [ 33 ]. Therefore, we tested whether inactivation of pRb reverses the sensitivity of PC3-AR cells to androgen with respect to glycogen accumulation. PC3-AR cells were constructed that expressed the HPV-E7 protein, which binds and inactivates pRb (PC3-AR-E7) [ 34 , 35 ]. PC3-AR cells transfected with the empty vector (PC3-AR-V) were used as a control. As shown in Fig. 1A , PC3-AR-E7 cells show a similar 2 to 5 fold increase in the glycogen content as do the control cells lacking E7 expression. The two cell lines differ in their basal glycogen content, which is about 50% lower in PC3-AR-E7. At 120 hours we observed less glycogen accumulation in the PC3-AR-E7 cells compared to control. We further compared the number of viable cells using manual cell counts coupled with trypan blue staining when the cell lines were treated with R1881 (Fig. 1B ). We tested two E7 and two control transfectants. A gradual reduction in cell number was observed both in PC3-AR-V1/V2 and PC3-AR-E72/E73 cells upon R1881 treatment. However, the PC3-AR-E7 clones showed slightly higher viable cell counts than the control clones following 72 and 120 hours of R1881 treatment. Growth inhibition was not observed for R1881-treated PC3 cells lacking AR and demonstrates that it requires the presence of AR. Cell cycle arrest for both cell lines 24 hours upon R1881 treatment was identified using FACS analysis (Fig. 2 ). The number of S-phase cells decreased in both R1881-treated PC3-AR cell lines while the number of G1-phase cells increased. Figure 2 FACS analysis of R1881-treated PC3-AR-V and PC3-AR-E7 cells . R1881 was added to exponentially growing cells to a final concentration of 4 nM and cells were further incubated for 24 hours. Cells were then harvested and processed for FACS analysis. GS activity in PC3-AR cells treated with R1881 The increase in glycogen content suggests enhanced glycogenesis in androgen-treated PC3-AR cells. The enzyme catalyzing glycogenesis is GS, which exists in two forms, an active unphosphorylated form, and an inactive phosphorylated form, which can be allosterically activated by G-6-P [ 10 ]. To determine GS activity, cell extracts were prepared from PC3-AR-E7 cells, which were either untreated or treated with R1881 for 72 hours. GS activity measured in the absence of G-6-P was hardly detectable in untreated and R1881-treated cells suggesting that GS was predominantly present in the inactive phosphorylated state despite hormonal treatment of cells (Fig. 3A ). When the GS assay was performed in the presence of G-6-P, which allosterically stimulates GS, an 18 and 35 fold higher GS activity was observed in control and R1881-treated cells respectively. To determine whether the increase in G-6-P activated GS occurs early upon exposure of cells to R1881 PC3-AR-E7 cells were harvested for GS assays in short time intervals (Fig. 3B ). G-6-P-activated GS did not show an increase compared to untreated cells (0) until 48 hours after exposure of cells to R1881 indicating that the increase in GS activity is not an immediate response to androgen. However, the observed twofold glycogen increase within 24 hours upon R1881 treatment suggests the possibility that androgen-treatment results in an increase in the intracellular G-6-P content, which allosterically stimulates GS activity. We determined the G-6-P levels in untreated and in 24 hours R1881-treated PC3-AR-E7 cells. We found a 3 fold increase in the G-6-P content normalized to mg protein (Fig. 3C ). These results indicate that enhanced glycogenesis in R1881-treated could be the result of GS stimulation by G-6-P. Figure 3 Determination of glycogen synthase activities in PC3-AR-E7 cells . R1881 was added to cells to a final concentration of 4 nM. Samples were removed at the indicated times. Activities are in nMol/min/mg protein. A) GS activity was determined from cells treated with R1881 for 72 hours and from untreated cells. GS activities were determined in the absence (-G-6-P) and presence (+G-6-P) of G-6-P. B) GS activities in the presence of G-6-P was determined from cells treated with R1881 for the times indicated. C) The G-6-P content was determined in untreated and 24 hours R1881-treated PC3-AR-E7 cells. GP activity in PC3-AR cells treated with R1881 Glycogen accumulation can also result from inhibition of glycogenolysis, which involves GP. GP exists in two forms, an active phosphorylated (GP-a) and an inactive form (GP-b), which can be regulated by allostery [ 9 ]. We examined the GP-a activity in untreated and R1881-treated cells. The GP-a activity normalized to protein in R1881-treated PC3-AR-V and PC3-AR-E7 cells increased slightly within 24 hours and doubled after 72 hours compared to untreated cells demonstrating activation of GP by androgen (Fig. 4 ). Since despite an increase in GP-activity the glycogen content increased, this suggests steady-state increase in glycogenesis. It is likely that GP-a may be partially inhibited allosterically by G-6-P, which increases in R1881-treated cells. Figure 4 Glycogen phosphorylase activity in PC3-AR-V/E7 cells . R1881 was added to cells to a final concentration of 4 nM and GP activities were determined in the absence of AMP, which reflects the activities of the levels of activated GP. The in vitro enzyme activity for each sample was followed over a time period of 5 min. The activities are expressed in nMol/min/mg protein. Glycogenolysis inhibition by CP-91149 in R1881-treated PC3-AR cells We showed that stimulation of glycogenesis, which exceeds glycogenolysis appeared to be responsible for the glycogen accumulation that inversely correlated with cell growth upon androgen-treatment in PC3-AR-V and PC3-AR-E7 cells. To test whether the inhibition of glycogenolysis further enhances the cellular response to androgen, GP activity was blocked with the selective GP inhibitor CP-91149. CP-91149 allosterically stabilizes the inactive conformation of human liver GP and induces dephosphorylation of GP-a [ 21 - 23 ]. We previously showed that CP-91149 also inhibits brain GP, which is the GP isoform expressed in all cell lines we have so far analyzed including PC3 cells [ 19 ]. PC3-AR-V and PC3-AR-E7 cells were treated with 30 μM CP-91149, which is a sufficient concentration to inhibit GP in several different cell lines including PC3 [[ 19 ] and data not shown]. PC3-AR-V and PC3-AR-E7 cells were both growth-arrested in the presence of CP-91149 without androgen-treatment (Fig. 5B ). In this particular experiment, untreated cells were harvested 24 hours after the start of the incubation to avoid the effect of high cell density while treated cells were harvested after 72 hours. The actual number of cells from the untreated culture would, therefore, be much higher after 72 hours compared to the CP-91149-treated culture (about double). This demonstrates inhibition of cell growth by CP-91149 treatment alone. The cell number of R1881-treated cultures was about 50% smaller compared to CP-91149-treated cultures. When cells were treated with both R1881 and CP-91149 simultaneously, the cell number was reduced to about 50% of R1881-treatment alone consistent with an additive effect of CP-91149 on R1881-treatment. These results indicate that glycogenolysis in cells treated with R1881 is still enough to support growth and/or survival as the intracellular glycogen content correlated inversely to the number of cells (Fig. 5A ). Untreated cells had the lowest glycogen content as normalized to cell count while the glycogen level doubled in CP-91149-treated cells. A 4-7 fold increase in the glycogen content was achieved by R1881-treatment. CP-91149 and R1881-double treatment resulted in 35–40% additional increase in glycogen content. This result further confirmed a tight correlation between glycogen accumulation and cell growth of R1881-treated PC3-AR cells and demonstrated that blockage of glycogenolysis by inhibition of GP-a with CP-91149 enhanced the effect of androgen on both PC3-AR-V and PC3-AR-E7 cells. Figure 5 Determination of viable cell number and glycogen content in PC3-AR-V and PC3-AR-E7 treated with R1881 and/or CP-91149 . Cells were either treated with 4 nM R1881 or 30 μM CP-91149 or both simultaneously. Control cells are those grown in the absence of R1881 and CP-91149 and were harvested after 24 hours. Treated cells were harvested 48 hours later and the total incubation time is 72 hours. A) The glycogen content normalized to the number of cells and B) the number of viable cells was determined. R1881 withdrawal results in glycogen accumulation in LNCaP cells The effects of androgen on the glycogen content was then evaluated in the androgen-dependent prostate cancer cell line LNCaP. The LNCaP cell line expresses a functional AR endogenously and growth arrests in the absence of androgen. LNCaP cells were cultured for seven days in the presence and absence of R1881 with regular medium changes. Cells ceased to grow and the intracellular glycogen content increased compared with cells grown in the presence of R1881 (Fig. 6 ). LNCaP cells were similarly growth-arrested as PC3-AR cells when glycogenolysis was inhibited with CP-91149 (Fig. 6B ). We observed no difference in the glycogen level from control cells grown in the presence of R1881 and absence of CP-91149. The most likely explanation is that control cells were grown to high density, which has been reported to result in decreased GP-a activity in colon cancer cells [ 36 ]. When R1881 was removed from the medium and simultaneously CP-91149 was added, the number of cells further reduced and the cell count normalized to the glycogen level increased to a higher extent than either treatment alone. Thus the inverse relationship between intracellular glycogen accumulation and cell growth and the additive effect of GP inhibition in androgen-deprived cells were reproduced in the androgen-dependent prostate cancer LNCaP cell line. Figure 6 Determination of viable cell number and glycogen content of LNCaP cells deprived of R1881 in the absence or presence of CP-91149 . Control cells were grown in the presence of R1881. Treated cells were either deprived of R1881 and/or treated with 30 μM CP-91149. The total length of the experiment was seven days. A) The glycogen content was determined and normalized to the number of cells. B) The number of viable cells was determined. Discussion In this paper we describe the effect of androgen on glycogen metabolism in different prostate cancers. We used PC3-AR-V and PC3-AR-E7 cells, which ectopically expresses AR and in case of PC3-AR-E7 the HPV-E7 protein. Additionally, LNCaP, which maintains a functional AR, was tested for glycogen content and its correlation to the number of cells. The PC3-AR-V and PC3-AR-E7 cell lines demonstrated a response to androgen leading to G1 arrest with a corresponding increase in the glycogen content (2 to 5 fold). PC3 cells lacking AR did not increase glycogen content in response to androgen. PC3 (AR-) cells demonstrate that intracellular glycogen content corresponds with the growth and/or survival of cells harbouring a functional AR. Intracellular stores of glycogen correlated inversely with the cell number: when cell numbers are low the glycogen content is high. This inverse relationship suggests that glycogenesis participates in growth arrest. However, glycogenesis is most likely not sufficient to induce ATP-dependent apoptosis, as the inhibition of glycogenolysis with the GP inhibitor CP-91149 does not induce cell death in these prostate cell lines. Glycogen content normalized to the number of cells is about 50% higher in PC3-AR cells treated with R1881 than treated with CP-91149. The additional increase in glycogen content of R1881-treated PC3-AR-V/E7 cells upon CP-91149 treatment further results in a reduction of cell number by growth inhibition and reduction in cell viability, which suggests that a certain intracellular glycogen content has to be reached to affect cell viability. Alternatively, certain effects of hormone treatment on cell survival may be enhanced by inhibition of glycogenolysis using CP-91149. Similarly, LNCaP cells responded with glycogen accumulation and growth arrest upon androgen removal, which was further enhanced by the GP inhibitor CP-91149. One explanation of reduced cell viability in conjunction with glycogen content could be the increase in G-6-P in response to androgen. G-6-P is a key metabolite allosterically activating GS [ 12 ]. The G-6-P content was about 3 fold higher 24 hours after androgen addition in PC3-AR cells compared to untreated cells. The increase in the G-6-P level was most probably a consequence of the decrease in activity of some key glycolytic enzymes as well as G-6-P dehydrogenase, which have been reported to be under androgen control [ 3 ]. We can, however, not exclude the presence of another allosteric GS activator nor partial activation of GS, which would sensitize GS to allosteric activation. The increase in G-6-P could also explain why an increase in activated GP (GP-a) in PC3-AR cells upon androgen treatment did not prevent the increase in glycogen content, since G-6-P is a GP-a inhibitor [ 37 , 38 ]. However, the fact that CP-91149 increased the glycogen content twofold more in androgen-treated PC3-AR cells shows that there was still GP-a activity. CP-91149 is known to cause GP-a dephosphorylation and partial GS-activation [ 39 ]. Conclusion Glycogenesis is part of the androgen response in prostate cancer and further inhibition of glycogen phosphorylase by a specific inhibitor reduces the cell number suggesting that glycogenolysis contributes to cell survival. Thus inhibition of glycogenolysis in combination with hormone therapy may be a more effective treatment for advanced prostate cancer than hormone therapy alone. List of abbreviations used GP, Glycogen Phosphorylase; GS, Glycogen Synthase; G-6-P, Glucose-6-Phosphate; AR, Androgen Receptor. HPV-E7, Human Papilloma Virus E7 Competing interests The author(s) declare that they have no competing interests. Authors' contributions J.S. is the corresponding author. His main contributions are the idea to carry out this research, experimental design and carrying out most of the experiments. K.N. has carried out some experiments and contributed conceptually. P.G. has funded some experiments, contributed materials used for the experiments and contributed with his expertise in prostate cancer research. F.G. has contributed with some funding and was a collaborator regarding some experiments. E.M.B. has provided funding, lab space and was a consultant. Pre-publication history The pre-publication history for this paper can be accessed here:

D:\keerthana\PMC001xxxxxx\PMC1079895.xml

1079896

Do Indonesian medical practitioners approve the availability of emergency contraception over-the-counter? A survey of general practitioners and obstetricians in Jakarta

Background Few studies have examined the attitude of medical practitioners towards the availability of emergency contraception (EC) without prescription. In Indonesia, EC (either Yuzpe regimen or Postinor-2) is available by prescription only. We aimed to examine the level of knowledge, attitudes and practices of medical practitioners in Indonesia about EC, in particular their attitudes to the availability of EC over-the-counter (OTC), using a questionnaire. Methods Data were collected by an anonymous structured questionnaire. Questionnaires were distributed to general practitioners in 36 Community Health Centres and 25 private clinics using stratified random sampling according to area in Jakarta, and to obstetricians practicising in 24 government and private hospitals and eight private clinics in Jakarta. Two hundred and five general practitioners and 142 obstetricians and gynaecologists participated; overall response rate was 75%. Results Although most participants were familiar with EC, only 22% received a very good knowledge score (4 or 5/5 answers correct), while 52% received a poor score (0–2/5 correct). Most participants did not support the OTC availability of EC (70%). Logistic regression identified that participants who prescribed EC had an Odds of 3.8 (95% CI 1.90, 7.73) of approving OTC EC, after adjustment for age and speciality. Conclusion Although many organisations are working towards OTC availability of EC, it needs to be recognized and addressed that doctors who do not prescribe EC are unlikely to support the increased availability of EC.

Background Family planning practitioners and women's health advocates have been campaigning for increased availability of emergency contraception (EC) [ 1 - 4 ]. Grimes and colleagues have stated that EC meets all the criteria for over-the-counter (OTC) use such as low toxicity, no potential for overdose or addiction, no teratogenicity, no need for medical screening, self-identification of need, uniform dosage, and no important drug interactions [ 2 ]. In many countries EC is now available OTC from pharmacies, after a consultation with a trained pharmacist, but without a doctor's prescription [ 5 ]. The progestogen-only regimen, using two 0.75 mg levonorgestrel tablets either together or 12 hours apart, is more effective than the Yuzpe regimen in preventing pregnancy (RR 0.51; 95% CI 0.31, 0.83) [ 6 ] and is available pre-packaged as Postinor-2 or Plan B [ 3 , 4 ]. Only a small number of studies have examined the attitude of medical practitioners towards the availability of EC without prescription. Although Postinor-2 is available OTC in Vietnam, the majority of family planning workers participating in focus groups favoured availability by doctor's prescription [ 7 ]. Most health care providers interviewed in Mexico City (n = 40) favoured distribution of EC through hospitals and clinics [ 8 ]. Interviews with family planning providers in Kenya found a mixed response to distribution of EC; some providers expressing concern that pharmacists may not be able to monitor the distribution as well as family planning clinics [ 9 ]. Other studies also found that a low proportion of health care providers believed EC should be available through pharmacies: nine percent of health care providers surveyed in Nigeria (n = 735) [ 10 ] and 22% of GPs and O&Gs in India (n = 130) [ 11 ]. Although only 14% of practitioners surveyed in Minnesota, USA (n = 495), supported OTC availability of EC, 63% of physicians approved a trained pharmacist providing EC after conducting a focused history, making appropriate referrals to a physician for STD screening, sexual assault counselling, physical exam, etc (if indicated) [ 12 ]. In Indonesia, EC is available by prescription only, either the Yuzpe regimen (eg 2 Neogynon pills, repeated in 12 hours) or progestogen-only (Postinor-2) [ 13 ]. This study aimed to examine the level of knowledge, attitudes and practices of medical practitioners in Indonesia about EC, in particular their attitudes to the OTC availability of EC. The only study of providers' knowledge and attitudes to EC in Indonesia which we identified is an unpublished study by Lubis and colleagues cited by the Consortium for Emergency Contraception [ 14 ]. Only 25% of health care providers (GPs, O&Gs, nurse-midwives) and policy makers (family planning program managers, members of professional associations, Ministry of Health officials, and religious and community leaders) were familiar with EC [ 14 ]. Methods Study subjects The study participants were medical practitioners in Jakarta, Indonesia. The researchers aimed to distribute questionnaires to 210 GPs and 250 obstetricians and gynaecologists (O&Gs). In Indonesia patients may self-refer to obstetricians and gynaecologists. The Provincial Health Service-Jakarta (Suku Dinas Kesehatan DKI Jakarta) provided a list of Community Health Centres (CHCs) (Pusat Kesehatan Masyarakat (Puskesmas)) and private clinics in five municipalities in Jakarta. Thirty six CHCs and 25 private clinics were chosen using stratified random sampling according to area, from the total of 328 CHCs at district and village levels and 63 private clinics in Jakarta. A research assistant distributed the questionnaires to the CHCs and clinics along with a culturally-appropriate token of appreciation (souvenir pen), and later collected the completed questionnaires. The Department of Obstetrics and Gynaecology, Faculty of Medicine, the University of Indonesia, Ciptomangunkusumo's Hospital (Bagian Obstetri dan Ginekologi, Fakultas Kedokteran-Universitas Indonesia, Rumah Sakit Ciptomangunkusumo) approved the study and distributed the questionnaires to O&Gs practicing in 24 government and private hospitals and eight private clinics in Jakarta. Survey instrument Data were collected by an anonymous structured questionnaire consisting of 30 questions. The first section collected data about the socio-demographic and work characteristics of the participants. The second section assessed the knowledge, attitudes and practices of the participants about EC. Participants were asked if they had ever heard of the terms "emergency contraception", "morning-after pill" or "post coital contraception" and if they were familiar with the specially packaged emergency contraceptive pill, Postinor-2. Knowledge about Postinor-2 was assessed using five statements, with the responses "true", "false" or "not sure". Five statements regarding participants' attitudes towards the availability of EC as an OTC product were provided with a 5-point Likert scale. In addition, two case scenarios were provided to indicate circumstances in which participants would prescribe EC to their patients, with 5-point Likert scale responses. The final item on the questionnaire was an invitation for participants to make free- text comments: "We are interested in your thoughts about emergency contraception, please write any comments here". After pilotting with a small number of Australian and Indonesian doctors and medical students, some minor alterations were made to the survey. Translated questionnaires were copied in Melbourne and sent to Indonesia for distribution. Participants were given the questionnaire along with a Plain Language Statement and a letter of a support from either the GP or O&G organisation, as appropriate. Data collection took place in February and March 2004, and completed questionnaires were couriered back to Melbourne in April 2004 for data entry and analysis. Analysis EpiData 3.0 [ 15 ]was used for data entry and Epi Info Version 3.2 [ 16 ] and Stata 8.0 [ 17 ] were used for analysis. Frequency and summary statistics were calculated for all variables. Relevant variables were cross-tabulated and Wilcoxon-Mann-Whitney and Kruskall-Wallis tests were conducted to explore the association between the variables. A logistic regression model was developed to look at the factors predictive of acceptance of the OTC availability of EC (dependent variable). Independent variables were speciality, gender, age, marital status, religion, knowledge score, oral contraceptive pill prescriber and EC prescriber. Stata 8 was used for this analysis. Participants' comments were translated and typed in English. Selected comments were used to support and illustrate the main themes emerging from participants' responses. This study was approved by the Human Research Ethics Committee, The University of Melbourne. Results Two hundred and five of the 210 (97.6%) general practitioners (GPs) and 142 of the 250 obstetricians and gynaecologists (O&Gs) (56.8%) completed the questionnaire, yielding an overall response rate of 75% (347/460). Some participants completed the demographic responses fully, but did not complete all the responses about emergency contraception, therefore denominators for responses vary. Demographic and work characteristics of participants are presented in Table 1 . Fifty four percent of participants were male, with a mean age of 42 years (range 24–68). Most participants were married (82%) and the dominant religious affiliation was Islam (68%). Almost all participants worked in Jakarta, the capital city of Indonesia (93%). Table 1 Demographic and clinical work characteristics of participants Characteristics GP (n = 205) O&G (n = 142) Total (n = 347) n % n % n % Gender Male 80 39 108 76 188 54 Female 125 61 34 24 159 46 Age Mean age (years old) 39 45 42 Younger 121 59 51 36 172 50 Older 83 41 91 64 174 50 Missing 1 1 Current martial status Single 55 27 1 1 56 16 Married 146 71 137 96 283 82 Divorced 3 2 3 1 Widow/ widower 4 2 1 1 5 1 Religion Islam 108 53 127 89 235 68 Catholic 39 19 5 4 44 13 Protestant 48 24 9 6 57 16 Hindu 1 0 1 1 2 1 Buddhist 7 3 7 2 Other 1 0 1 0 Missing 1 1 Location of work Jakarta 187 92 131 94 318 93 Other 3 1 6 4 9 3 Both 13 6 2 1 15 4 Missing 2 3 5 Main place of work Government hospital 73 58 73 23 Private hospital 5 3 38 30 43 14 Government clinic 2 1 2 1 Private clinic 80 43 15 12 95 30 Community health centre 99 53 99 32 ( PUSKESMAS ) 1 1 1 0 Other 18 16 34 Provide advice on contraception in clinical work Yes 191 94 139 99 330 96 No 12 6 2 1 14 4 Missing 2 1 3 Prescribed OC in the last six months Yes 133 66 126 89 259 75 No 70 34 15 11 85 25 Missing 2 1 3 All percentages are calculated on valid response only GP: general practitioner, O&G: obstetrician and gynaecologist, OC: oral contraception 1 Younger: 21 – 40 years, older: 41 – 70 years The majority of participants provided advice on contraception in their practices (GP 94%; O&G 99%). A higher proportion of O&Gs had prescribed oral contraception (OC) than GPs in the six months prior to the survey (O&G 89%; GP 66%). More O&Gs had heard of the term "emergency contraception (EC)"or "morning-after pill" or "post coital contraception" or "special pill preventing pregnancy" (O&G 130/141, 92%; GP 148/203, 73%). More than half the GPs who answered this question were familiar with the progestogen-only pill (POP) as an EC method (89/139, 64%). The two most common EC methods mentioned by O&Gs were POP and the Yuzpe regimen (POP 88/127, 69%; the Yuzpe regimen 82/127, 65%). Fifty two percent of GPs (78/150) and 66% of O&Gs (85/128) had heard of Postinor-2 specifically. Participants reported hearing about Postinor-2 from lectures, meetings or seminars (83/273, 30%), journal articles (83/273, 30%) and their colleagues (69/273, 25%). The results of the five knowledge questions are presented in Table 2 . Knowledge scores were regarded as very good (4 or 5/5 correct), good (3/5 correct) or poor (0, 1 or 2/5 correct). Only 22% of participants received a very good knowledge score, while 52% received a poor score. Table 2 Knowledge about progestogen-only emergency contraception GP (n = 151) O&G (n = 131) Total (n = 282) n (m) ♣ % n (m) ♣ % n (m) ♣ % Correct responses to questions about POP, Postinor-2 Dosage: 0.7 5 mg levonorgestrel × 2 (T) 54 (12) 39 71 (10) 59 125 (22) 48 Timing of administration: within 84 hrs (F) 51 (8) 36 48 (8) 39 99 (16) 37 Causes abortion (F) 49 (8) 34 77 (7) 62 126 (15) 47 Fewer side-effects than Yuzpe regimen (T) 27 (7) 19 59 (8) 48 86 (15) 32 Efficacy 85% (T) 99 (7) 69 98 (7) 79 197 (14) 74 Knowledge score Very good (4 or 5 out of 5 correct) 10 7 46 39 56 22 Good (3 out of 5 correct) 39 28 28 24 67 26 Poor (0 or 1 or 2 correct) 90 65 45 38 135 52 Missing 12 12 24 All percentages are calculated on valid response only GP: general practitioner, O&G: obstetrician and gynaecologist, POP: progestogen-only pill, T: true, F: false ♣ The numbers in brackets are the missing values Seventy percent (193/276) of the participants disapproved of the availability of EC as an over the counter (OTC) product (GP 120/149, 81%; O&G 73/127, 57%). Fourteen responded that they were "not sure". Two case scenarios were provided to indicate circumstances in which participants would prescribe EC to their patients. The majority of participants would prescribe EC in the case of contraceptive failure in a married couple (GP 105/146, 72%; O&G 112/130, 86%). Similarly, most participants would also provide EC to a young woman who had been raped (GP 97/146, 66%; O&G 118/130, 91%). More O&Gs reported prescribing EC than GPs in the six months before this survey (O&G 83/126, 66%; GP 74/144, 51%). An equal proportion of GPs and O&Gs had provided the Yuzpe regimen in their practices (GP 54/144, 38%; O&G 49/129, 38%). In addition, 37% (48/129) of O&Gs had also prescribed POP as EC (GP 28/144, 19%). About half the GPs had never prescribed EC in their clinical practices (GP 70/144, 49%; O&G 43/129, 33%). The IUD was also used more by O&Gs than GPs (O&G 18/119, 15%; GP 4/131, 3%). Participants were dichotomized into two age groups (aged 40 years and below, and over 40 years). Older participants were more knowledgeable about EC than younger participants (z score = 2.071; p = 0.0384). However, stratification by speciality resulted in no significant association between level of knowledge and age group (GP, chi-square = 3.0993, p = 0.212; O&G, chi-square = 0.0620, p = 0.969). There was a significant difference in the knowledge level between GPs and O&Gs (z score = 5.486; p < 0.0001), with O&Gs having higher knowledge scores. Participants who prescribed oral contraception (OC) in the six months before the survey were more likely to have a "very good" level of knowledge, especially about Postinor-2, than participants who had not prescribed OC (z score = 3.381; p = 0.0007). The difference in the level of knowledge about EC was not significantly related to gender (z score = 1.765; p = 0.0775) or location of work (chi-square with ties = 4.035; p = 0.2577). Table 3 presents the association between approval of OTC availability of EC and age, gender, speciality, location of work, knowledge score and prescribing EC. More male participants accepted the OTC status of EC (z score = 2.036; p = 0.0418). O&Gs were more likely to accept EC as an OTC product than GPs (z score = 3.410; p = 0.0006). More older participants approved the OTC status of EC product than younger participants (z score = 3.029; p = 0.0025). Most participants disapproved the availability of EC OTC, however participants with "very good" and "good" knowledge score were more likely to approve the OTC status of EC than participants with "poor" knowledge (z score = 2.626; p = 0.0086). However, stratification of attitude by speciality showed that significant difference was only found among GPs (GP, chi-square = 11.7983, p = 0.003; O&G, chi-square = 0.8995, p = 0.638). Table 3 The association between approval for OTC availability of EC and other variables Variables (n = 276) Approves EC as an OTC product Disapproves EC as an OTC product 1 n % n % Age group 2 Younger 23 18 121 82 0.0025 4 Older 43 34 85 66 Missing = 1 Gender Male 47 30 112 70 0.0418 Female 22 19 95 81 Speciality GP 25 17 124 83 0.0006 O&G 44 35 83 65 Location of work Jakarta 63 25 190 75 Other 1 14 6 86 0.8569 Both 4 31 9 69 Missing = 3 Knowledge score 3 Very good 19 34 37 66 Good 22 33 45 67 0.0086 Poor 25 19 110 82 Missing = 18 Prescribes EC Yes 57 37 96 63 <0.0001 No 12 11 100 89 Missing = 5 EC: emergency contraception; GP: general practitioner, O&G: obstetrician and gynaecologist, OTC: over the counter 1 Includes "not sure" 2 Younger: 21 – 40 years, older: 41 – 70 years 3 Very good (4 or 5 out of 5 correct), Good (3 out of 5 correct), and Poor (none or 1 or 2 out of 5 correct) 4 Wilcoxon-Mann-Whitney test was used to examine the association between variables and attitude (Kruskall-Wallis test was used for location of work) The EC practices were different across age group, gender, speciality, knowledge and attitude scores. Older GPs were more likely to prescribe EC in their practices (older 69/112, 62%; younger 75/157, 48%; z score = 2.239; p = 0.0252). Male participants provided EC more commonly than females (male 105/156, 67%; female 52/114, 46%; z score = 3.562; p = 0.0004). In addition, being an O&G was a positive predictor for prescribing EC (O&G 83/126, 66%; GP 74/144, 51%; z score = 2.402; p = 0.0163). Participants who had better knowledge about EC were more likely to provide EC (z score = 3.378; p = 0.0007). However, stratification of EC practice by speciality showed a significant relationship between level of knowledge and prescribing EC only for GPs (GP, Pearson chi-square = 6.6889, p = 0.035; P&G, Pearson chi-square = 3.4616, p = 0.177). Participants who accepted the OTC status of EC were more likely to use EC in their clinical work (accepted OTC status 57/69, 83%; disapproved OTC status 96/196, 49%; z score = 4.854; p < 0.0001). A logistic regression model was developed to look at the factors predictive of acceptance of the over-the-counter availability of emergency contraception. The independent variables (speciality, gender, age, marital status, religion, knowledge score, OC prescriber and EC prescriber) were tested individually against the dependent variable using logistic regression to determine Odds Ratios. The p-values of the Wald statistic for all variables were <0.05, except religion which was 0.179 and marital which 0.693. Independent variables were entered in the model if the p-value of the Wald statistic was = 0.25 [ 18 ], therefore all variables were entered initially except marital status. From the stratified univariate analysis, it was recognized that age was a confounding factor as it was associated with speciality; therefore both were retained in the model. Records with missing data on any of these variables were removed from the sample, leaving 247 records for analysis. Variables were eliminated one at a time using logistic regression, only those with a p-value of the Wald statistic of ≤ 0.05 were retained in the model [ 18 ]. The process was repeated until only significant variables remained. Then the marital variable was added back into the model to check that if it was now significant given the reduced model. Interactions were examined between age and speciality and age and sex. These were not retained in the model as the Wald statistic p-values were > 0.01 (p = 0.623 and 0.525 respectively). To check how closely aligned the predicted and observed data values were, a 'goodness of fit' test was performed (Hosmer-Lemeshow chi2(6) = 1.54, p = 0.9565); the non-significant p value indicates a good fit. The sensitivity of the model, that is, how often it correctly predicted the outcome (y) given the value of a value of a covariate (x) was tested using the area under the ROC curve. To be said to have good discrimination, a model should have a statistical value of = 0.7 [ 18 ]. The lroc test identified that the area under ROC curve was 0.7195. The lstat test showed 72.03% correctly classified. The final model, with Odds Ratios, can be seen in Table 4 . Table 4 Approval for OTC availability of EC Unadjusted Multivariate analysis Odds Ratio (95%CI) Adjusted Odds Ratio (95%CI) EC prescriber No Reference Reference Yes 4.35 (2.18, 8.68) 3.70 (1.83, 7.50) Age (yrs) 21–40 Reference Reference 41–70 2.66 (1.49, 4.76) 1.96 (1.05, 3.66) Speciality GP Reference Reference O&G 2.37 (1.33, 4.23) 1.76 (0.94, 3.28) EC: emergency contraception; GP: general practitioner, O&G: obstetrician and gynaecologist Many participants made comments about the use of EC in the final section of the questionnaire. Some participants stressed the emergency nature of this method: • Agree on providing contraception/emergency contraceptive pill which might be used only in the case of contraceptive failure (forgetting to take regular oral contraceptive pill or condom leakage) (46 year-old male O&G) Some participants argued that EC would be beneficial to reduce the abortion rate and prevent unwanted pregnancy. • Emergency contraception is very useful for people wanting to prevent pregnancy. (It) may reduce the abortion rate as well as the complications of abortion (42 year-old female GP) Participants had concerns about the availability of EC OTC in encouraging intercourse among unmarried couples especially adolescents. • Do not market emergency contraception without prescription because may lead to free sex among teenagers which later causes STD, for example HIV AIDS (50 year-old male GP) Most participants indicated that EC should be available by prescription. • Emergency contraception should be given with prescription , not to be sold as an over the counter drug! (35 year-old female O&G) Another theme was the need for more information about EC for both professionals: • Need symposium for doctors, midwives, health personnel (62 year-old male O&G) and the community: • Need more explanation/ information for public through printed mass media/ TV/ radio (62 year-old male O&G). Discussion This survey of general practitioners (GPs) and obstetricians and gynaecologists (O&Gs) in Indonesia has identified a low level of knowledge about emergency contraception (EC). While the majority of medical practitioners supported the availability and the use of EC, they disapproved the availability of EC as an over the counter (OTC) product. This survey also revealed that EC was prescribed infrequently by Indonesian medical practitioners. The higher response rate among GPs, 98%, compared to O&Gs, 57%, was likely to be due to the different method of recruitment. The research assistant visited GPs' workplaces and gave them a small token (pen), while O&Gs received the questionnaire through their medical organization, without any contact with the researchers. However, although demographic questions were well answered, many participants did not complete all the questions; questions relating to prescribing practices were completed by 70% of GPs (144/205). In our study, O&Gs were more likely to be involved in contraceptive care than GPs, and to be more familiar with the concept of EC. Although O&Gs were more likely to approve EC as an OTC product on univariate analysis, this was not significant on multivariate analysis. Logistic regression identified that participants who prescribed EC had an Odds of 3.7 of approving over-the-counter EC after adjusting for age and speciality. There is a common misconception that EC is an abortifacient. Twenty percent of family planning coordinators in Michigan, USA, believed that EC worked by causing an abortion [ 19 ]. Anna Glasier states "It cannot be stressed too strongly that if hormonal emergency contraception works largely by interfering with ovulation, then it cannot be regarded as an abortifacient" [1 p1063]. A survey involving 100 O&Gs in Massachusetts, United States revealed that 94% had prescribed EC, with 57% reported prescribing it no more than five times per year [ 20 ]. In contrast, only 76% of 96 family physicians reported prescribing EC, and 82% of them prescribed it five times or less [ 20 ]. Lower levels of prescribing have been found in studies in developing countries. In Nairobi, Kenya, only 15% of family planning providers reported prescribing EC [ 9 ] and only 20% of primary health care workers recommended EC in Turkey [ 21 ]. In our survey, 66% of O&Gs and 51% of GPs reported prescribing EC in the previous six months, but as many participants did not complete this question, this should be regarded as an overestimate of prescribing practices. A survey of family physicians and nurses (n = 78) in the USA found that practitioners who prescribed EC were more likely to agree that the benefits outweigh the risks (94%) than nonprescribers (50%) [ 22 ]. All of the nonprescribers (n = 7) agreed that they felt uncomfortable prescribing EC for religious / ethical reasons, compared to 8% of the prescribers [ 22 ]. The low level of support for OTC availability of EC found in our study is similar to the results of other studies [ 7 , 10 , 11 ]. A study in Oxford, UK, in which 76 GPs were interviewed in 1996, found that only eight of the GPs reported unqualified enthusiasm for deregulation of EC [ 23 ]. The four main themes expressed by the GPs were that women would be missing out on the benefits of the consultation; that EC is not the most effective, or safe, form of contraception; that some women may use it frequently; and that the pharmacy has characteristics which make it unsuitable for dispensing EC [ 23 ]. Ziebland has suggested that health professionals may feel uncomfortable with EC, because in contrast to other methods of contraception, the provision of EC is closely associated with a sexual episode [ 24 ]. Conclusion Medical practitioners in Indonesia would benefit from additional education about EC. Medical practitioners should be encouraged to include discussion of EC in their routine consultations with women about contraception. Although many organisations are working towards OTC availability of EC, it needs to be recognized and addressed that doctors who do not prescribe EC are unlikely to support the increased availability of EC. Competing interests The author(s) declare that they have no competing interests. Authors' contributions Both authors participated in the design, conduct and analysis of the study. Both authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:

D:\keerthana\PMC001xxxxxx\PMC1079896.xml

1079897

Public access defibrillation: Suppression of 16.7 Hz interference generated by the power supply of the railway systems

Background A specific problem using the public access defibrillators (PADs) arises at the railway stations. Some countries as Germany, Austria, Switzerland, Norway and Sweden are using AC railroad net power-supply system with rated 16.7 Hz frequency modulated from 15.69 Hz to 17.36 Hz. The power supply frequency contaminates the electrocardiogram (ECG). It is difficult to be suppressed or eliminated due to the fact that it considerably overlaps the frequency spectra of the ECG. The interference impedes the automated decision of the PADs whether a patient should be (or should not be) shocked. The aim of this study is the suppression of the 16.7 Hz interference generated by the power supply of the railway systems. Methods Software solution using adaptive filtering method was proposed for 16.7 Hz interference suppression. The optimal performance of the filter is achieved, embedding a reference channel in the PADs to record the interference. The method was tested with ECGs from AHA database. Results The method was tested with patients of normal sinus rhythms, symptoms of tachycardia and ventricular fibrillation. Simulated interference with frequency modulation from 15.69 Hz to 17.36 Hz changing at a rate of 2% per second was added to the ECGs, and then processed by the suggested adaptive filtering. The method totally suppresses the noise with no visible distortions of the original signals. Conclusion The proposed adaptive filter for noise suppression generated by the power supply of the railway systems has a simple structure requiring a low level of computational resources, but a good reference signal as well.

Background More than 35 years ago external cardiopulmonary resuscitation (CPR) and defibrillation were first described as effective treatments for sudden cardiac arrest. However, survival after out-of-hospital cardiac arrest is still poor. The American Heart Association (AHA) previously addressed this problem by emphasizing the importance of the 'chain of survival' [ 1 ]: early access, early CPR, early defibrillation, and early advanced life support. Because early defibrillation is the single most important intervention, the AHA challenged manufacturers to develop simple, low-cost automatic PADs for use at locations in which large numbers of people congregate: stadiums, airports, railway stations, etc [ 2 ]. It has been reported that the chance of survival of a patient at a state of ventricular fibrillation decreases by approximately 10% with each minute that passes after the time of attack [ 3 ]. There also exists a probability of irreversible brain (cortical) damage due to systemic hypoxia after 5 minutes. However, response time for paramedics or emergency medical technicians to arrive on site with a defibrillator is often more than ten minutes, resulting in average survival rates of less than 5%. Widespread deployment of automated PADs is the only feasible method of achieving early defibrillation. The strategy for time reduction is that defibrillators can be used by non-healthcare personnel (for example, untrained bystanders and trained members of the staff at the public place) before the emergency medical services arrive. A specific problem using the PADs arises at the railway stations. Some countries as Germany, Austria, Switzerland, Norway and Sweden are using AC railroad net power-supply system with rated frequency of 16.7 Hz [ 4 ]. According to the Official journal of the European communities[ 5 ] the interference is frequency modulated from 15.69 Hz to 17.36 Hz. The power supply frequency is magnetically induced and can contaminate the electrocardiogram (ECG). It is difficult to be suppressed or eliminated due to the fact that it considerably overlaps the typical rhythms analysis spectra between 1 to 30 Hz of the ECG. The interference impedes the automated decision of the public access defibrillators whether a patient should be (or should not be) shocked, no matter of the high accuracy reported recently by some authors [[ 6 , 7 ] and [ 8 ]]. Schlimp et al [ 9 ] have tested two automated external defibrillators near high-voltage power lines run by the Austrian railway company with an AC of 16.7 Hz and 15 kV (overhead of railway tracks) or 16.7 Hz and 110 kV (overland). The reported errors are: i) failed operation due to artifacts; ii) misinterpretation of the artifact, resulting in failure to treat an underlying ventricular fibrillation and ventricular tachycardia or failure to recommend proper bystander action in the cases of underlying asystolia; iii) failure to identify clearly detectable rhythm and misinterpretation of the noise as motion artifact or ventricular fibrillation, resulting in a shock advice. Kanz et al [ 10 ] have tested eleven automated external defibrillators with 16.7 Hz, 15 kV AC (S-Bahn) and a subway system powered with 750 V DC (U-Bahn). The authors have recorded 5280 tests concluding that high voltage AC interferes more (shock decision sensitivity reduction of 9.7% and no-shock decision specificity reduction of 10.6%) than low voltage DC (shock decision sensitivity reduction of 0.6% and no-shock decision specificity reduction of 1.4%). The interference is minimized, if patient position is parallel and electrode cables are perpendicular to overhead power lines. Four of the devices have demonstrated an unacceptable performance in respect of accuracy. We could not find any material dealing with 16.7 Hz noise suppression generated by the power supply of the railway systems. That is why we referred to some standard real-time methods used for main interference (50 or 60 Hz) suppression, from the point of view whether it is possible to adopt them to the specific problem: - The QRS suppression introduced by notch filters tuned to reject a band from 15.69 Hz to 17.36 Hz is so high that it makes them unacceptable. - 'Comb' filters averaging in a time interval of the interference from 57 ms to 64 ms are over-smoothing the QRS complexes, making it sometimes undistinguished from the T wave. - The good performance of the mains interference subtraction method [[ 11 - 13 ] and [ 14 ]] is mostly due to the correlation between the interference and the ECG sampling frequency. In the conditions of huge noise modulation the method seems inapplicable. - Adaptive filtering described by some authors [[ 15 , 16 ] and [ 17 ]] does not disturb the electrocardiogram (ECG) frequency spectrum, but requires re-adaptation at any change of the normal ECG course, such as arrhythmias or appearance of an extra systolic ectopic beat. The re-adaptation period is characterized by the appearance of a tail of gradually attenuating unsuppressed interference. The same effect can be observed at any change of the interference frequency [ 16 ]. The aim of this study is to investigate the suppression of the 16.7 Hz interference generated by the power supply of the railway systems. Method The problem of electric train noise suppression could be considered as a particular case of a more common problem of adaptive noise cancellation [ 18 ]. The adaptive filtering method is software based and could be added into the PAD computer algorithm, but does need a special hardware configuration (antenna) within the PAD to sample the electromagnetic interference signal without the ECG data for reference. We assume that a reference noise signal can be easily picked up and used. A block-diagram of adaptive filtering is shown in Fig. 1 . Figure 1 Block-diagram of adaptive filtering. The primary input of the adaptive filter is the active channel d(i) , which is a mixture of a clear ECG signal s(i) and the interference generated by electrical train n 1 (i) , where i is the consecutive number of the sample: d(i) = s(i) + n 1 (i) (1) The noise has a sinusoidal waveform and it can be expressed as: n 1 (i) = b*cos(2π*F/Fd*i + φ) (2) where b , F , Fd and φ are amplitude, frequency, sampling frequency and phase of the noise signal, respectively. The reference input of the adaptive filter is a noise signal collected by an antenna: n 2 (i) = a*cos(2π*F/Fd*i) , (3) which differs from n 1 (i) only in amplitude and phase. The task of the adaptive algorithm depicted in Fig. 1 is to adjust the coefficients w 1 (i) and w 2 (i) to compensate the amplitude and phase difference between n 1 (i) and n 2 (i) . In other words, y(i) should resemble n 1 (i) as close as possible. We rearrange the reference noise signal in the following manner: x 1 (i) = a*cos(2π*F/Fd*i) (4) x 2 (i) = a*sin(2π*F/Fd*i) (5) where x 2 (i) is the noise reference signal phase shifted on 90 degrees by Hilbert transform. Conventionally the Hilbert transformer is realized as a FIR system [ 19 ]. The transition band reduction needs a very high order FIR system, which requires complicated computational structure. Considerable reduction in computational resources is achieved by all-pass structure which highest order is 5. In our structure the Hilbert transformer is realized as a parallel 3 all-pass sections. Two of them are intended to make the 90 degree shift and one - to prevent the distortion in the x 1 (n) signal. The order of the three sections is 4, 5 and 4 respectively. The advantage of this structure compared to the conventional one is that it allows very fine tuning of the signal phase and amplitude which is proved later with the experiments. Then y(i) = w 1 (i)*x 1 (i) + w 2 (i)*x 2 (i) , (6) and the error signal is formed as: e(i) = d(i) - y(i) . (7) We apply a Least Mean Square adaptive algorithm [ 20 ] with a cost-function e 2 (i) and finally get a system of two equations for adapting the coefficients w 1 (i) and w 2 (i) : w 1 (i+1) = w 1 (i) + 2*μ*e(i)*x 1 (i) (8) w 2 (i+1) = w 2 (i) + 2*μ*e(i)*x 2 (i) , (9) where μ controls the adaptation rate. Results The adaptive filtering method was tested with ECG recordings taken from the AHA database. Each recording has duration of 30 min and includes two leads. The sampling frequency is 250 Hz and the resolution is 5 μ V*bit -1 . The experiments were performed by generating a sinusoidal-like noise n 1 having a main frequency of 16.7 Hz with frequency modulation of 20% for 10 s, and adding it to the ECG signals. The same noise but with different amplitude and phase from n 1 was considered as a reference input n 2 for the adaptive filtering method. The method was tested with normal ECG signals (Fig. 2 ), tachycardia (Fig. 3 ) and ventricular fibrillation (Fig. 4 ). The first two plots of any of the figures represent 20 s of the original ECG signal. Plots 3 and 4 present the ECG contaminated by a frequency-modulated noise. Plots 5 and 6 depict the processed signal as well as the frequency of the added interference, shown by red trace and red scale on the right. Figure 2 Adaptive filtering applied on normal sinus rhythm. Plots 1 and 2 – original ECG signal. Plots 3 and 4 – the ECG contaminated with n 1 = 2 mV noise. Plots 5 and 6 – processed signal. The frequency modulation of the added interference is shown by red trace and red scale on the right. Interference amplitude at the reference channel n 2 = 3 mV. Phase shift of 52° between n 1 and n 2 . Figure 3 Adaptive filtering applied on a patient with symptom of tachycardia. Plots 1 and 2 – original ECG signal. Plots 3 and 4 – the ECG contaminated with n 1 = 3 mV noise. Plots 5 and 6 – processed signal. The frequency modulation of the added interference is shown by red trace and red scale on the right. Interference amplitude at the reference channel n 2 = 2 mV. Phase shift of 52° between n 1 and n 2 . Figure 4 Adaptive filtering applied on a patient with ventricular fibrillation. Plots 1 and 2 – original ECG signal. Plots 3 and 4 – the ECG contaminated with n 1 = 3 mV noise. Plots 5 and 6 – processed signal. The frequency modulation of the added interference is shown by red trace and red scale on the right. Interference amplitude at the reference channel n 2 = 2 mV. Phase shift of 52° between n 1 and n 2 . The experiments were performed with phase shift of 52° between n 1 and n 2 . The amplitudes of the noise were n 1 = 2 mV, n 2 = 3 mV in Fig 2 , and n 1 = 3 mV, n 2 = 2 mV in Fig. 3 and Fig. 4 . With no change of the coefficients of the Hilbert transformer, the same adaptive filter structure can be used for 50 Hz (or 60 Hz) power-line suppression. The first two plots in Fig 5 depict 20 s epoch of original ECG signal. Sinusoidal noise with carrier frequency of 50 Hz (frequency modulated at a rate of 0.1 Hz* sec -1 ), and 3 mV amplitude (amplitude modulated at a rate of 0.2 mV*sec -1 ), is presented in plots 3 and 4. The noise added to the original ECG is shown in plots 5 and 6. The same noise, but phase shifted with 90° is applied on the reference channel. The filtered signal is presented in plots 7 and 8. Figure 5 Plots 1 and 2 – original ECG with ventricular fibrillation. Plots 3 and 4 – sinusoidal noise with carrier frequency of 50 Hz (frequency modulated at a rate of 0.1 Hz*sec -1 ), and 3 mV amplitude (amplitude modulated at a rate of 0.2 mV*sec -1 ). Plots 5 and 6 -–the original ECG mixed with the 50 Hz noise. Plots 7 and 8 – processed ECG. Discussion Most often the interfering signal is presented not only with its carrier frequency but also with its third harmonic. The proposed adaptive filtering using a reference input can easily control signals with multiple harmonics by cascade or parallel structures based on the configuration shown in Fig. 1 . No visual distortions of the processed ECG signal due to the superimposed modulated interference are observed. The amplitude and phase difference between n 1 and n 2 affect mostly the initial adaptation time until the coefficients w 1 and w 2 reach some stable levels. With an adaptation rate of μ = 0.0001 the adaptation time is from 0.1 s to 20 s. The increase of μ cuts down the adaptation time but the filter results are getting worse. After the initial adaptation time any amplitude and phase changes between n 1 and n 2 will be small and the system will succeed to readapt at a rate of μ = 0.0001. If a huge difference between n 1 and n 2 appears, the re-adaptation period will depend on the gradient of the change, but in any case will be less than the initial adaptation time. The reaction of the system will be not to suppress enough the 16.7 Hz. In an attempt to decrease the adaptation time we used a stepwise decrease of the adaptation rate after the start of the algorithm. First we make 30 iterations with μ = 0.1, then gradually reduce the step to 0.01 and 0.001 making 10 iteration at each, until a finally μ = 0.0001 is reached. The starting number of 50 iterations was chosen empirically, considering that the amplitude of noise in one of the adaptive filter input should not be 3 times higher than the amplitude of the noise in the other input and the phase shift has to be lower than 60°. The embedding of a reference channel in the PADs presents some practical difficulties. The antenna has to be designed in a way so that the reference noise does not significantly depend on the PADs position or possible people/object moving around the PAD during resuscitation. This needs to be subjected to further investigations following the publication. The final decision should be done after comparison to other methods, usually very complex and inflexible. Conclusion The proposed adaptive filter for suppression of 16.7 Hz noise generated by railroad net power-supply system has an excellent performance and its simple structure requires a low level of computational resources. With no change of Hilbert transformer coefficients, the same filter structure can be used for suppression of the 50 Hz (or 60 Hz) power-line interference.

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1079898

Review of "Statistical Analysis and Data Display" by Richard M. Heiberger and Burt Holland

Statistical analysis methods and applications have traditionally placed relatively little attention to the graphical visualisation of data and information. Such techniques are fundamental not only for summarising the outcomes originating from statistical analyses, but also for facilitating the detection and assessment of relevant findings. Data-intensive application domains such as medical informatics, biomedical engineering and bioinformatics rely on the application of several statistical analysis and information visualisation techniques to support the generation, validation and integration of hypotheses. This requires an understanding of fundamental principles of data sampling, comparison and classification. Heiberger and Holland have produced a comprehensive, well-presented review of essential statistical topics. Furthermore, they emphasise the application of graphical display techniques to summarise data and assess analysis results. This book explains why, how and when to apply particular techniques and tools. Another important feature is the incorporation of examples and exercises directly relevant to three leading statistical languages: S-Plus , R and SAS . Executable functions and macros are also provided for several graphical display methods. After discussing basic statistical concepts, such as probability distributions and hypothesis testing, graphical display techniques are overviewed with an emphasis on scatterplot-based methods. An introductory chapter on inference illustrates how to compare two populations, determine sample sizes and measure goodness of data fit. Chapters dedicated to one-way analysis and multiple comparisons are followed by several chapters on linear, multiple and logistic regression. It also discusses two-way analysis of variance, design of experiments, bivariate and nonparametric statistics. The last section focuses on time series analysis. The authors, collaborators and publishers made a serious effort to achieve a high quality presentation of text, tables and figures (only black and white). Although the book concentrates on the application of S-Plus, R and SAS, it is a relevant resource to users of other software platforms. Overall, this is an excellent book for supporting advanced undergraduate courses on statistics or data analysis. Postgraduate students of machine learning and data mining topics may also be greatly benefited from reading most of its chapters. This is a valuable reference source to researchers from these and related areas including medical decision support, clinical engineering and bioinformatics.

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1079899

Non-rigid registration of a 3D ultrasound and a MR image data set of the female pelvic floor using a biomechanical model

Background The visual combination of different modalities is essential for many medical imaging applications in the field of Computer-Assisted medical Diagnosis (CAD) to enhance the clinical information content. Clinically, incontinence is a diagnosis with high clinical prevalence and morbidity rate. The search for a method to identify risk patients and to control the success of operations is still a challenging task. The conjunction of magnetic resonance (MR) and 3D ultrasound (US) image data sets could lead to a new clinical visual representation of the morphology as we show with corresponding data sets of the female anal canal with this paper. Methods We present a feasibility study for a non-rigid registration technique based on a biomechanical model for MR and US image data sets of the female anal canal as a base for a new innovative clinical visual representation. Results It is shown in this case study that the internal and external sphincter region could be registered elastically and the registration partially corrects the compression induced by the ultrasound transducer, so the MR data set showing the native anatomy is used as a frame for the US data set showing the same region with higher resolution but distorted by the transducer Conclusion The morphology is of special interest in the assessment of anal incontinence and the non-rigid registration of normal clinical MR and US image data sets is a new field of the adaptation of this method incorporating the advantages of both technologies.

Background In a recent study the advances of 3D sonographical imaging techniques to allow a sophisticated study of anal sphincter and levator ani muscle anatomy were described [ 1 ]. Today's common US examiniation techniques using a 7.5 MHz transducer allow a spatial resolution of up to 0.3 mm in each direction [ 2 , 3 ], whereas it is hard to obtain good quality MR images better than 1 mm in a single direction, when imaging the pelvis. Nevertheless, MR is a well established 3D data acquisition technique, which is used as gold standard to describe human anatomy in vivo. It is a clinical necessity to enhance the information contained in imaging for diagnostic and also therapeutic purposes. In the past this led to new imaging techniques (visual representations) which use the information of at least two modalities in order to maximize the benefit for the clinician in diagnosis and treatment [ 4 ]. The registration of MR and US is of special interest because sonography is a diagnostic technique which is easy to handle, widely available, and furthermore economic [ 5 ]. To combine the best of the two worlds we will show that it is possible to match 3D MR and US for the assessment of female pelvic floor morphology. Both introitus sonography and endoanal sonography focus on rectum and anal sphincter muscle morphology [ 6 , 7 ]. A recent publication by Williams et al. [ 8 ] shows a good correlation of endosonographic anatomy with endocoil MR. In contrast to this paper, our case shows the anatomy in a native physiologic state without stretching the tissue with a transrectal probe. No results have been published so far about the combination of introitus sonography and MRI using a standard bodyflex coil used for clinical purposes showing the anatomy in native status. Because the organs in pelvic floor area are movable in position and size, consequently, new imaging techniques in this region should be based on non-rigid registration techniques (techniques considering tissue deformations) rather than on rigid registration techniques [ 9 - 11 ] in order to find the relationship of corresponding data set points. Actually, few cases of non-rigid MR and US image registration in the pelvic floor area are reported (e.g. for the treatment of prostate cancer by Mizowaki et al. [ 12 ]). Non-rigid registration has become a fundamental method for medical image analysis during the past years [ 5 , 13 - 15 , 35 ] and is tested and approved with data from different anatomical regions [ 15 - 18 ]. An important issue in the registration process is the generation of deformation fields that reflect the transformation of an image in a realistic way with respect to the given anatomy [ 19 , 20 ]. Various physically based elastic models and algorithms have been recently described [ 21 - 24 ]. The aim of this paper is to apply an advanced algorithm previously approved for computer-assisted neurosurgery [ 25 ] and tested in corresponding head-neck data sets [ 26 ] now for registration purposes in the pelvic floor area, especially of the anal canal region to create a new visual representation. Information derived from this type of image registration could lead towards new diagnostic (or even therapeutic) methods in the treatment of female pelvic floor dysfunctions using the MR data as a frame for high-resolution US data. Materials and methods For our case report two corresponding 3D data sets (MR and US) of a women attending the outpatient clinics for diagnosis and treatment of urinary incontinence were taken with no specific clinical preference out of an ongoing study. The 3D volume data set was acquired using Voluson 530 D, Kretztechnik, Zipf, Austria as it has been previously described [ 1 ]. The volume data set of the undistended anal sphincter and levator ani muscle was taken with a 7.5 MHz transvaginal probe (opening angle of 105° in tranversal and of 100° in longitudinal direction, isotropic resolution of 0.3 mm in each spatial direction) was placed at the posterior frenulum of labia minora. The MR examination was carried out in sitting position with an 0.5T open configuration MR system, Signa SP GEMS, using a bodyflex surface coil for data acquisition. After a locator sequence, axial and sagittal T2-weighted fast-spin-echo sequences (TR 4000, TE 100, Matrix 256 × 256, slice thickness 7.2 mm, intersection gap 1.2 mm) were acquired and stored in DICOM format. The resolution in the matrix is 1.09 mm, whereby the MR data sets result to be non-isotropic in the three directions in space in contrast to the sonographical data sets having isotropic voxel size. As a base system for both alignment and visualization we used the 3D-Slicer 2 software available free for non-profit organizations on both standard MS Windows platforms and Sun Solaris 5.8 Workstations [ 27 - 29 ]. The 3D Slicer software is designed for both diagnostical visualization and surgical planning, and it integrates several facets of image-guided medicine into a single environment: It provides capabilities for (I) automatic registration (aligning data sets), (II) semi-automatic segmentation, (III) generation of 3D surface models (for viewing the segmented structures), (IV) 3D visualization, and (V) quantitative analysis (measuring distances, angles, surface areas, and volumes) of various medical scans. The processing followed the strategy as described in Fig. 1 . After carrying out an edge enhancement in the 3D US data set using adaptive filter techniques [ 30 , 31 ] both datasets were initially aligned using the standard 3D-Slicer's fiducial alignment method by placing three landmarks as fiducials in two different axial slices of both axial MR and US data set. We chose the mucosa and points in the internal or the external sphincter muscle as anatomical landmarks for the rigid overlay assuming the MR images to be the gold standard for the muscle components [ 8 ]. As a result an affine transformation matrix was determinded for the overlay. Both data sets were cropped to the region of interest (Fig. 3 ) to minimize the calculation time. After resampling using a standard linear interpolation method both data sets have an isotropic resolution of 0.6 mm in each spatial direction. The worst registration errors of the correlation ratio are due to the MR resolution [ 32 , 33 ]. We carried out a visual assessment of the accuracy of the registration and assumed the error for each modality to be of the order of half a voxel size in our case [ 34 ]. The entire processing time for the process shown in Fig. 1 for this feasibility study was one hour not considering the data acquisition times for MR and US. Figure 1 Strategy to register . Strategy to register MR and 3D ultrasound image data sets. Solid lines display the strategy shown in this paper. Dashed lines symbolize optional ways which were followed but which show no further relevant and new details. The symbols on the right refer to the format of the data sets: The series of squares stands for a series of parallel slices and the cube is for a volume block format. Figure 3 MR data with aligned sonographical data . Shown are the corresponding MR data set of the pelvic floor to the US data set from Fig. 2. (a) shows the axial plane from the axial data set; (b) and (c) show the sagittal respectively coronal plane from the corresponding sagittal data set. (c) is shown only for illustration purposes due to the poor resolution. On the right side the original MR data set is shown and on the left side a cutout with the corresponding initial alignment. The capital letters indicate anatomical structures: A: anal region; B: bladder; C: coccyx; F: ischial tuberosity; L: lumbar vertebrae; S: symphysis; V: vaginal region. In this work we present the application of an algorithm for non-rigid registration described previously by two of the authors [ 19 , 20 ]. This algorithm was originally developed for MR techniques and we applied it for new anatomical region assuming that the edge enhancement algorithms are valid for US data sets, too. In order to obtain realistic deformations, we propose a physics-based elastic model. The method does not require segmentation and does not have the drawback that initial estimates of the deformation are only generated for the boundary of a considered structure. Instead, these estimates are calculated based on a template matching approach with a local similarity measure. Furthermore, we incorporated different models for elasticities into our algorithm. The discretization of the underlying equation is done by a finite element technique, which has become a popular method for medical imaging applications [ 24 , 25 ]. The registration process can be described as an optimization problem. Target of the optmization is the minimization of the deformation energy between two data sets, reference and template. The displacement field which describes the correlation of corresponding anatomical structures in both image data sets can be described using the theorema of minimal potential enegy E. With this in a volume Ω a deformation u needs to be determined which minimizes the following equation. F means the external force causing the deformation u. σ is the stress which causes the (local) distortion ε. The relationship between σ and ε can be described using elastomechanical equation σ = Dε with D being an elasticity tensor. Due to the fact that in the dedicated anatomical region mostly muscle tissue is found, the tissue parameter in D used in the biomechanical model is assumed to be homogeneous (according to [ 20 , 22 ]and[ 25 ]). The minimization of the potential energy E then is the registration process devided in two basic steps. The method is described in detail in [ 20 ]. Results Leading structures in both of our data sets are the rectum and the anal canal with the mucosa, the circular as well as the conjoined longitudinal muscle layer of the anorectal junction and the levator ani muscle, especially the puborectalis muscle. These morphological structures can be identified clearly in US as well as in MR images – in the latter case with much less contrast than in the US data sets. In addition to this MR shows up the pelvic bones and skin surface facilitates spatial orientation. The application of adaptive filtering on US data set increased the signal to noise ratio significantly. As a consequence edges appear enhanced in the filtered images showing therefore the anatomical details much clearer. This is shown in Fig. 2 . Both external and internal anal sphincter muscle as well as the anal mucosa can be identified in filtered images. Especially, the internal anal sphincter muscle appears clearly as a hypoechogenic region. The levator ani muscle has a V-form surrounding the external anal sphincter muscle best visible in the axial plane. It can be easily distinguished from the hyperechogenic tissue of the external anal sphincter. Figure 2 Sonographical data . Sonographical documentation method for examination of the pelvic floor in analogon to the usual used MR nomenclature [36, 37]. Shown is the filtered US data set: (a) axial, (left side: not filtered to show the enhancement induced by filtering) (b) sagittal and (c) coronal plane through the anal canal. A: the internal anal sphincter muscle; B: the external anal sphincter muscle (levator ani muscle); C: anal canal mucosa; D: rectum. Fig. 3 (left side) shows the MR data set analog to the US images in Fig. 2 in its different planes. In the right side of Fig. 3 the position and the size of the overlayed US data set and the spatial orientation in the MR data set is shown. Due to the higher resolution of the US data set about three times more data points are visible in the overlay region in comparision to the MR image significantly magnifying the overlay region. Fig. 3b shows the sagittal plane of the MR data set. Few structures are distinguishable clearly in the vaginal and anal region due to the poor resolution and contrast. Even filter techniques could not significantly increase the contrast in this region in sagittal scan and are not shown therefore. In Fig. 4 the registration is explicitly shown for one axial plane. The top of each image indicates the anterior direction. MR is used as the template and US as the reference image. As a result the US data set is displayed in the coordinates of the MR due to the application of the deformation field (Fig. 4c ). At the position of the transducer – best visible in the top of Fig. 4a – the displacement field shows major differences. The compression induced with the transducer head is partially corrected in the registered image Fig. 4c . The displacement field in z-direction perpendicular to the axial slice is ommited due to the poor resolution of the MR data set in this direction and due to the fact that in sagittal planes too few anatomical landmarks could be identified clearly. Figure 4 Registration and displacement field . Axial plane through non-rigid registration of the anal canal: Original data are shown in (a) and (d) – US and MR image, respectively. For simplification of visualization only the difference components in x-direction (b left) and in y-direction (b right) are shown. The difference in z-direction is ommited. (e) shows the difference image of (a) and (d) together with the difference components in x direction (e left) and in y direction (e right). (c) shows the registered image after the application of the displacement field. The non-rigid registration qualifies the visual assessment in axial plane very well. An estimation of accuracy in both sagittal and coronal plane could be quantified not better than half a voxel size. In Fig. 5 the segmentation of the internal anal sphincter muscle is explicitly shown in one axial plane. Analogous to Fig. 4 the top of each image indicates the anterior direction. It is shown for the MR (Fig. 5a ), the US (Fig. 5b ) and the registered image (Fig. 5c ) respectively. The deformation induced by the ultrasound probe placed in anterior position is corrected (Fig. 5d ). Figure 5 Segmentation of the internal anal sphincter muscle . Segmentation of the internal anal sphincter muscle. (a) shows the original axial MR slice with the contour of the internal anal sphincter muscle in pink color, (b) the corresponding US slice with the contour in green color and (c) the registered image with the contour in blue color respectively. (d) shows the areas in comparision. The arrows indicate where the contour of the internal anal sphincter muscle is modified with the algorithm. The registration partially corrects the compression induced by the transducer (the upper arrow shows the position of the transducer). Discussion MR leads to rigid non-deformed data sets in contrast to most sonographical data acquisition techniques. The better structural contrast of the MR data sets allows a better spacial orientation but the quality and resolution especially for soft tissue is higher with 3D US techniques. Due to several factors such as the lack of image structure, the poor signal to noise ratio of the MR data set, the intensity artifacts, the computational complexity and the restricted time frame it is not feasible to quantify the deformation occurring between each voxel of the corresponding data sets directly. Consequently, we chose a physics-based non-rigid registration algorithm to estimate a deformation field only at sparse locations which have to be interpolated throughout the image. Models of this type have become popular for non-rigid registration because they are fast and have the potential to constrain the underlying deformation in a plausible manner. Included in our method is an edge enhancement for the US data set using adaptive filtering. Our sophisticated 3D filter technique requires an isotropic or at least a nearly isotropic resolution of the volume image data sets which is the case for our US data set. Furthermore, most registration methods require to have similar resolution of both images, similar regions with the same image size and only local deformations on a short range scale. Therefore, nevertheless, preprocessing steps were needed. Corresponding clinical data sets of two different modalities usually fail fulfilling those preconditions completely – so do ours. For initial alignment purposes it helps to localize the anatomy. This is demonstrated in Fig. 2 . The difference of resolution in MR's axial direction in comparison to the resolution of ultrasound is huge. Due to this, a misplacement of ultrasound's axial slices in relationship with MR axial slices was of no initial relevance and our error in misalignment assumed to be in the region of half a voxel size in each spatial direction. Fig. 3 shows the poor resolution and contrast in the vaginal and anal region of the MR data sets. Few structures are distinguishable clearly in this region. Even filter techniques could not significantly increase the contrast in this region in sagittal scan and are not shown therefore. This leads to the wish to enhance the clinical information in this region using registration techniques as presented with this paper. MR and US show minor contour differences for all the anatomical structures. The reason for this misplacement is based on the two completely different data acquisition techniques. Even if they were very carefully carried out the prevention of any distortion is impossible and leads to the following effects: 1. The coupling of the transducer to the tissue induces a tissue deformation in any case. 2. The position of the patient varies without placing any external markers if the data acquisition takes place at different places and different times. 3. Different acquisition times induce e.g. different filling levels of the organs with different contours as a result. The registration results shown in Fig. 4 and Fig. 5 demonstrate clearly the feasibility of the non-rigid registration method for the correspondent 3D data sets of the anal canal. The native MR data set is used as a frame for the US data set – which shows the sphincter structures more clearly than the MR. The levator ani muscle cannot be registered with accuracy because the identification in the US data set due to the poor contrast needs an experienced clinician and results impossible for an automated algorithm. The visual representation is limited to slightly distorted tissue structures as proved with this data and shown before in previous attempts in corresponding head-neck data sets. But regions with high contrast and slight deformations can be registered using our method. For further studies MR data sets with higher resolution MR and isotropic voxel size would be desirable. Conclusion The present case report shows the feasibility of the visual representation presented for normal clinical data of the anal canal. The registration partially corrects the compression induced by the transducer, so the MR data set showing the native anatomy is used as a frame for the US data set showing the same region with higher resolution but distorted by the transducer. As a consequence the clinical information for diagnostic purposes is enhanced for resolution (Fig. 3 ) and for position (Fig. 5 ). Obviously, these findings need to be validated with more cases in a future prospective study. As previously emphazised the MR images were assumed to be the gold standard for the contours. Using the non-rigid registration technique described in this paper and developing a more sophisticated data acquisition and registering technique this might be changed in the future and the application of 3D ultrasound (US) has a high potential in the innovative development of future low-cost applications. Authors' contributions JFV developed the registration strategy and did the technical part implementing the visual representation method described here. JW did the data acquisition and the medical part. SKW and JR developed the used linear elastic model and helped to modify and adapt it to the present data. RK provided the computing infrastructure and consulted the development process of the method. All authors read and approved the final manuscript.

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1079900

Review of "Contemporary IMRT: Developing Physics and Clinical Implementation" by Steve Webb

IMRT is Intensity Modulated Radiation Therapy: Irradiating from several directions repeatedly targets a tumor, but less its surroundings, due to differing projections; yet minimising such collateral damage may be cost-controversial. Beams are modulated by retarding compensators and perimeter-blocking collimators. Radiotherapy retards mitosis, especially G2, but responce is tissue-specific, hampered by the likes of tumor hypoxia. X-rays result when an electron beam hits a target, but in therapy, electrons are previously electromagnetically accelerated so resulting X-rays are quite stronger. Despite improved calculation and control, nonintegration remains: Technology mismanagement frequently occurs when rogue specialties treat others as superfluous; yet the more technical and nimble a field, the greater need for cross-disciplinary clarity and integration (eg p. 379). Tradeoffs between nimbleness and safety are political moving targets. Avowedly the least mathematical, but also least focused (in any order, p.xv), of four, this tome surveys developments, more like a managerial update. Curiously the author spawned separate titles instead of revising, expanding editions: Multibook fatigue has lapsed author into shamanistic jargon and acronyms; yet such superfluous obfuscation bears significant blame for modern medical errors. The final sixth of this tome is a rich collection of references. This, or any precursor, nicely rounds off a two semester radiological engineering course with Cember's health physics[ 1 ], Faiz Khan's radiotherapy[ 2 ], Kak & Slaney's tomography[ 3 ], and Pham & Dimov's rapid manufacturing [ 4 ] texts, preceeded by thorough reexcercising of Schaum's laGrangian mechanics [ 5 ]. In the second chapter on rotation IMRT and tomotherapy, pseudo structures flagged for dose minimisation are especially interesting. The third chapter discusses multileaf collimators (MLC) and sequencers (leaf moving algorithms, aka, curiously , interpreters) in terms of leakage, scattering, rounding, edge penumbra and speed considerations. Radiation might be misdirected while leaves are moving and they don't move quite instantaneously, requiring Leaf Motion Calculators ( shamanistically, not sequencers!). The fourth chapter discusses non-MLC techniques: Accuray.com CyberKnife (accelerators, not Gamma Knife cobalt) with real-time imaging, jaws/mask technique and variable-aperture collimators. The fifth chapter cites specific anatomical tumors and attendant clinical evidence: the author laments "evidence-based medicine" circuitously implies not being accepted until one has been accepted enough to have sufficient clinical data; Perhaps physicists disempower trials specialists. Integrated systems might compensate, real-time, for detected radiobiological characteristics like hypoxia. Furthest clinical results are Sloan Kettering's prostate (rectal toxicity declines) and Marsden's (author) breast. 3-dimensional planning spans book's last half, discussing margin definition, distortion correction, contrast agents, fluence smoothing, patient motion (esp respiration, modelled as cosine to power of assymetry) and surface marker gating, and movement correction. Tracking patient motion is preferrable over immobilisation techniques resembling medieval torture. Image importing planning softwares include Nomos Corvus , Nucletron Plato , Nordian MDS Helax TMS, Philips ADAC Pinnacle , Varian CadPlan Helios & CMS Focus . Inverse planning minimises discrepancy from desired spatial dose, constrained by dose (power-law biological cost) to non-targets and then projects beams backwards from each direction. Various optimisation methods are discussed, quite improved from 1960s confinement to linear programming; However, one must avoid that optimiser scourge, local minima. Monte Carlo techniques (MCDOSE, DOSXYZ, MCNP, ETRAN, EGS4; accumulating random beam behaviors defined by Boolean rules instead of differential equations) allow for simulation of geometries not easily described mathematically, but are slow and have noise convergeance errors. Since Cyberknife robot "chases the moving patient" (p. 164), one hopes for more engineering, integrating "planning" with therapy, instead of "batch" methods, which the author prefers, like older automobiles, in his 2001 text, because they do "not remove the human from decision making". Engineers would prototype in MathWorks.Com MatLab (p. 47), but would consider it too slow for " production ". Planning originated with slower computers (before MLC sequencers, p. 42) like Harvard JCRT (Bjarngard, Kijewski, Siddon) [ 6 ], Wachsmann's 1959 pendulum and rotation "Moving Field Radiation Therapy" [ 7 ] and van de Goijn's 1970 3D planning EXTDOS; yet Shirato & Sawada (pp. 335–341) develop real-time planning integration and Tubingen (p. 295) integrates MLC constraints, sequencers and planning. Given mathematical computation now so competent at scavenging useful information from what was previously considered noise (eg Barbour, Nirx.Net ), perhaps one might exploit treatment radiation for some imaging, reducing total exposure, and seek further integration synergies. Competing interests Reviewer holds restricted securities in a developer of a through-the-sputtered-target, minimal-Bremsstrahlung, relativistically-collimated miniaturised X-Ray tube.

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1079901

In Memorium: Herman P. Schwan [1915–2005]

Herman P. Schwan [1915–2005] was a distinguished scientist and engineer, and a founding father of the field of biomedical engineering. A man of integrity, Schwan influenced the lives of many, including his wife and children, and his many students and colleagues. Active in science until nearly the end of his life, he will be very much missed by his family and many colleagues.

Herman Paul Schwan, renowned scientist and engineer, loving and devoted husband and father, died quietly in his home in Radnor, Pennsylvania on March 17, 2005. Herman P. Schwan was born in Aachen, Germany in 1915. He obtained the German superior school certificate with distinction in Goettingen, 1934. He studied mathematics, physics, and engineering in Goettingen and then biophysics in Frankfurt. He obtained his Ph.D. in biophysics at the University of Frankfurt in 1940 with distinction, his teaching certificate at the University and his professional doctorate (Dr. habil) in the fields of physics and biophysics in 1946. Schwan worked in 1936–37 and again in 1938 with Telefunken on high frequency and microwave measuring techniques. He became a research associate with the Max Planck Institute of Biophysics in Frankfurt in 1937, an assistant professor with the University of Frankfurt and associate director of the Max Planck Institute in 1946. In 1947 he came to the United States, working at the Aeromedical Equipment Laboratory of the U.S. Naval Base in Philadelphia. He joined the University of Pennsylvania in 1950. In 1952 he was appointed Head of the Electromedical Division of the Moore School and in 1961 Chairman of the Graduate School of Arts and Sciences Group on Biomedical Electronic Engineering. In 1972 he became Chairman of the Bioengineering Department. He retired as the Alfred Fitler Moore Professor Emeritus in 1983. Over the course of his long career, Schwan published more than 300 scientific papers and gave countless lectures. He received numerous awards in recognition of his contributions, including the Edison Medal of the IEEE and the first d'Arsonval Award of the Bioelectromagnetics Society, membership in the National Academy of Engineering, and several honorary degrees. An extended biography of Schwan was published recently in the Annual Reviews of Biomedical Engineering [ 1 ]. As a scientist, Schwan is best known for many biophysical studies related to electrical properties of cells and tissues, and on nonthermal mechanisms of interaction of fields with biological systems. He discovered or provided important theoretical insights into phenomena such as the large low-frequency dielectric dispersion that is found in biological material, and electrically induced forces on cells. Schwan was also deeply involved in the issue of possible health effects of nonionizing electromagnetic fields. His letter to the U.S. Navy in 1953, proposing a safe limit for human exposure to microwave energy of 100 W/m 2 (based on thermal analysis) became the basis for exposure standards in the U.S. and elsewhere. Among his many other committee activities in this field, he chaired the committee that established the first (1965) U.S. exposure limit for radiofrequency energy, for the American National Standards Institute. This standard evolved into the present IEEE C95.1 standard and was widely influential in the development of exposure limits around the world. Schwan was married in 1949 to Anne Marie Del Borrello of Philadelphia. In addition to his wife, Herman is survived by five children and six grandchildren. He was a mentor to all of them, first and foremost teaching them always to think for themselves and never to just follow the crowd. A man of integrity, Schwan influenced the lives of many, including his wife and children, and his many students and colleagues.

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1079902

Amplifier spurious input current components in electrode-electrolyte interface impedance measurements

Background In Impedance Microbiology, the time during which the measuring equipment is connected to the bipolar cells is rather long, usually between 6 to 24 hrs for microorganisms with duplication times in the order of less than one hour and concentrations ranging from 10 1 to 10 7 [CFU/ml]. Under these conditions, the electrode-electrolyte interface impedance may show a slow drift of about 2%/hr. By and large, growth curves superimposed on such drift do not stabilize, are less reproducible, and keep on distorting all over the measurement of the temporal reactive or resistive records due to interface changes, in turn originated in bacterial activity. This problem has been found when growth curves were obtained by means of impedance analyzers or with impedance bridges using different types of operational amplifiers. Methods Suspecting that the input circuitry was the culprit of the deleterious effect, we used for that matter (a) ultra-low bias current amplifiers, (b) isolating relays for the selection of cells, and (c) a shorter connection time, so that the relays were maintained opened after the readings, to bring down such spurious drift to a negligible value. Bacterial growth curves were obtained in order to test their quality. Results It was demonstrated that the drift decreases ten fold when the circuit remained connected to the cell for a short time between measurements, so that the distortion became truly negligible. Improvement due to better-input amplifiers was not as good as by reducing the connection time. Moreover, temperature effects were insignificant with a regulation of ± 0.2 [°C]. Frequency did not influence either. Conclusion The drift originated either at the dc input bias offset current (I os ) of the integrated circuits, or in discrete transistors connected directly to the electrodes immersed in the cells, depending on the particular circuit arrangement. Reduction of the connection time was the best countermeasure.

Background Measurements carried out with bipolar electrodes can be modeled by a series circuit composed of two impedances, one representing the electrolytic medium (Z m ) and another taking into account the interface (Z i ) between the former and the metal itself. From a physical point of view, the interface region extends from the larger rugosities of the electrode surface to the deeper double molecular layer [ 1 , 2 ]. Its behavior is complex and relates to the electrode macroscopic and microscopic geometric characteristics [ 1 , 3 ], to the electrolyte proper, and to the operating conditions too, as for example, the applied current intensity or frequency [ 4 , 5 ]. However, in spite of its complexity and of the yet not fully known interface events, this region contains and can supply useful electrochemical information [ 6 - 8 ]. For example, in cellular suspensions, it acts as a highly sensitive transducer to monitor microorganism growth [ 7 - 10 ]. In particular, in Impedance Microbiology [ 10 - 12 ], when interface reactance is being recorded, growth curves typically show a maximum drift at the initial point and thereabouts. Thereafter, the drift slowly decays. Growth curves obtained from the medium bulk, instead, are essentially flat from the very beginning [ 11 ]. In the latter reference, such drift effect is barely mentioned. Capacitive growth curves, for example, are used to assess the Minimum Inhibitory Concentration (MIC) of a disinfectant, where either appearance time or decrease to the 20% level is measured [ 13 , 14 ]. Reactance curves, instead of conductance, are more desirable because they show better sensitivity [ 9 ]. In milk, several authors have used culture conductance during bacterial growth for quantitative and qualitative assessment of microbial content. Interface capacitance curves C i have not been used even though their performance is better by far. The above-mentioned drift has no relationship whatsoever with bacterial growth, it does not stabilize with time, it lacks good reproducibility and introduces a distortion in the temporal curves [ 6 ]. In this paper, we show that the drift is due to spurious dc input bias offset currents (I os ) of the integrated circuits or discrete transistors directly connected to the electrodes in the cells. Such unwanted currents slowly charge up the interface capacitance C i , a known phenomenon in the measurement of interface voltages, as for example in pH-meters [ 15 ]. Nonetheless, this slow charging current (which can be considered as a spurious dc within the measurement interval) increases the C i value [ 16 ] and, thus, alters the previous value. In the case of pH, the problem is solved by means of electrometer amplifiers with sub-picoamper I os . Commercial equipment to measure impedance does not take care of this problem and the reactance curves often show significant distortions. To bring down the drift to a negligible level, we have applied three very simple different techniques, i.e., (a) using ultra-low bias current operational amplifiers, (b) using isolating relays instead of analog multiplexers to select the cells, and (c) shortening the time of connection to a minimum and keeping the relays opened after each sample. Our results show that, connecting the circuit to the measurement cell 4 s every 5 min, produced a drift of only 0.17%/hr, turning into negligible the distortion of the growth curves. No temperature effect is seen if its regulation is kept within ± 0.2°C or better. Methods To quantify the interface reactance (Xi = 1/ ωCi) drift, we used a previously described constant current bridge circuit [ 6 ] implemented in such a way that the input preamplifier could be easily replaced (Figure 1 ). Figure 1 Constant current bridge circuit with replaceable pre-amplifier. Z: measured cell; R: series bridge resistance; R v : variable resistor, C v : variable capacitor, PRE: preamplifier, NF: 50 Hz notch filter, OSC: oscilloscope. The cell for the assays with this bridge was a cylindrical glass tube (8 ml volume, 15 mm diameter, 50 mm length), implemented with stainless steel DENTAURUM ® wire electrodes (1 mm diameter, 10 mm length) immersed in 0.9% NaCl solution at 37°C. All the measurements were carried out at 120 Hz with an electrode current density of 30 μA/cm 2 . Three different integrated circuits were used as preamplifier testing units: uA741(I os = 20 nA, Z input = 6 Mohms); LF356(I os = 3 pA, Z input = 1 Tohms); and AD523(I os = 0.25 pA, Z input = 10 Tohms). Each was tried with three cells other than the one described above. Besides, to analyze the effect of the offset current with different instrument systems, culture sterile media and electrodes, we measured X i at 120 Hz during 200 min using the following setups, 1) HP4192A, NB broth at (37 ± 0.3)°C, with DENTARUM ® wire; 2) HP4284, BHI broth at (37 ± 0.3)°C, with cell C1; 3) FRA-PAR, 0.9% NaCl at 26 ± 1)°C, with cell C2; 4) BACTOMETER, 0.9% NaCl at (37 ± 0.1)°C, with BACTOMETER cell; where HP4192A stands for a Hewlett-Packard impedance analyzer, HP4284A is a Hewlett-Packard precision LCR meter, FRA-PAR is a system including a 1255 SOLARTRON frequency response analyzer and a 273A PRINCETON APPLIED RESEARCH (PAR) electrochemical interface; BACTOMETER is a patented laboratory custom made impedance microbiology analyzer. All these four sets measure impedance in the bipolar form. None is internally implemented with input amplifiers in the sub-picoamp range. Current density was ≤ 64 μA/cm 2 . NB and BHI stand, respectively, for Nutritive Broth and Brain Heart Infusion. The culture cells were stabilized for two hours before they were connected to the system. The cell for the first setup (including DENTAURUM ® steel electrodes) was described in a previous paper [ 10 ]. The cell named C1 is an acrylic cylindrical cell (100 mm in diameter and 10 mm in length) with two stainless steel AISI 304 electrodes (diameter = 10 mm) polished to 0.05 μm. Cell C2 had the same electrodes but polished to 0.3 μm. The reasons for choosing 120 Hz can be summarized as follows, • Low frequency is required to separate R m from Z i [ 8 ]. • At that frequency, and also at 1,000 Hz, X i reflects the double layer [ 17 ], while higher frequencies do not allow discrimination between R m and Z i , as stated above. • From the digital viewpoint, lower frequencies make sampling and further processing on-line easier. Higher frequencies imply conversion frequencies unnecessarily elevated. As well known, the interface impedance can be characterized in its simplest form by a series equivalent circuit Z i = R i - j X i ; herein, we used the series reactance because it carries the same information as the resistive component does, but it is easier to measure [ 7 , 10 ]. To make the I os effect negligible, a series of measurements were made with the HP4284A (second setup) by actually disconnecting it after each sample was taken. During the first hour, measurements were made every 5 min, in the second hour every 10 min and, thereafter, every 2 hours. Finally, to monitor the behavior of X i as a function of frequency, we used the cylindrical glass tube with DENTAURUM electrodes immersed in saline solution at room temperature with an LF356 as preamplifier and the constant current bridge circuit. Results The percentage drift curves obtained with each integrated circuit are shown in Figure 2 , where it is seen that, as the bias current was increased (upward shift), the interface reactive component appeared with a larger change. The drifts at 60 minutes in Figure 2 are, 28%, 9% and 2%, respectively, for the operational amplifiers μA741, LF356 and AD523. In all cases, the amplifiers with lower bias current produced a lower drift. Figure 3 displays the effects produced when four different equipment were used. At 60 minutes, the drifts came out to be 16% (HP4192A), 8% (FRA/PARC), 4% (BACTOMETER) and 2% (HP4284A). Those magnitudes distort growth curves, especially when either the reactive component or directly the capacitance is plotted. Figure 2 Curves of percent interface reactance versus time using integrated circuits with different offset currents as pre-amplifiers: μA741 (black squares); LF356 (open circles); AD523 (open triangles), from top to bottom. Three curves for each amplifier. There is a large difference in one set of data (for μA741) compared to the other two data sets for the same device. Such difference becomes smaller for LF356 and AD523 as the input bias offset currents further decrease in these devices. This is very likely due to the large industrial variability of these op-amps. Figure 3 Percent interface reactance versus time using commercial systems. From top to bottom: ◆ Hewlett Packard Impedance Analyzer HP4192A; ◇ FRA or Frequency Response Analyzer 1255, by SOLARTRON, with an electrochemical interface PRINCETON APPLIED RESEARCH (PAR); o BACTOMETER, a patented custom made prototype; ▲ Hewlett Packard Precision LCR meter HP4284A. In Figure 2 , there is a large difference in one set of data (for μA741) compared to the other two data sets for the same device. Such difference becomes smaller for LF356 and AD523 as the input bias offset currents further decrease in these devices. This is very likely due to the large industrial variability of these op-amps, which may reach 1000% in the first two types and up to 50% in the last one. In their book, Firstenberg and Eden [ 11 ] show during the initial transient phase an approximated drift of 1% at 60 minutes in a capacitance growth curve of Escherichia coli growing in BHI broth. Previous experiments in our laboratory were also marred by a similar effect of 4% at 60 minutes [ 10 ]. One possible way of solving the drift problem is to disconnect the culture cell (its electrodes) from the measurement circuit by using a switch relay, during the period when no sample data are collected (dead period). In such situation, the interface capacitance does not charge up. Besides, the longer the interval between measurements, the lower the observed drift. This proposal was evaluated recording the curve of percent interface reactance (X i ) versus time using the HP4284A and disconnecting the equipment from the cells between measurements. Each sample meant a transient connection to the circuit shorter than 4 s. After 6 hrs, the measured total drift was still in the order of 1%, i.e., essentially negligible. Another possible solution is the use of a pre-amplifier input circuit of very low bias current (in the sub-picoamp range) which, combined with the in-between OFF periods described above, would reduce the drift even further down. In Figure 4 , we studied whether the X i changes due to the bias current drift were dependent on the applied frequency. The diagram was plotted at two different times, i.e., at t = 0 and at t = 3 hrs. Regression analysis modeled after a power function led to, Figure 4 Interface reactance X i versus frequency with time as parameter. measurements made at t = 0; + measurements made 3 hours later. The slope did not show any change. Xi t = 0 = A f B = 8,484 f -0.83 (SD = ± 0.5)    [1] Xi t = 3 = A f B = 11,047 f -0.83 (SD = ± 0.5)    [2] where SD stands for Standard Deviation. The percent change of X i between t = 0 and t = 3 hs was +31% within the analyzed frequency range. This behaviour indicates that the temporal C i changes were frequency independent. Discussion The slow drift due to the dc spurious bias current of the input electronic circuitry causes the distortion observed in bacterial growth curves, either of the reactive or resistive type, when the sampling system is connected to the electrodes. Stainless steel electrodes are considered as polarizable, meaning that they tend to behave as a pure capacitance. Moreover, there are operational amplifiers that behave as constant current sources when connected to a circuit. Such current is the net difference of the input bias currents, which is usually named offset current I os [ 16 ]; from this application point of view, this is to be considered a spurious unwanted current. The highly simplified model of Figure 5 illustrates the interaction between the circuit electronics and the bipolar measuring cell. Only C i is included, because the parallel charge transference resistance is much higher than X i [ 7 ] at the working frequency. The series resistance R and the oscillator represent the circuitry applying the signal to the cell. Figure 5 Simplified circuit model of the measuring cell and input preamplifier. R: series resistance; C i : interface capacitance; RL: relay contact; I dc : unwanted distorting current. Connecting the preamplifier to the cell (Fig. 5 ) is equivalent to connecting a constant current dc generator because the input impedance of the amplifier is very high (see Methods Section). Furthermore, since C i depends on the interface dc current [ 18 ], the larger the absolute value of the current, the higher the value of C i . After the circuit is connected, the current through C i takes at first a maximum value to decrease exponentially thereafter towards its previous null level. The non-constant capacitance C i behaves in a similar way, that is, it is maximal at the beginning and, thereafter, it falls off slowly. Experimentally, this fact is easily seen when stainless steel electrodes are immersed in saline solution or in a culture broth to measure the interface capacitance. Thus, the latter discussion would explain the interface reactance behavior observed in Figure 2 ; since X i is inversely proportional to C i , it increases when the current goes down. Frequency is not included here because its influence on X i was constant with time. An important fact is to be underlined. Usually, the first measurement takes place a few seconds after the cell is switched on to the circuitry [ 7 ]. Hence, the initial measured X i is not the true interface value . The true value is the one that existed before connection. The deviation of the first erroneous measurement depends on the magnitudes of the spurious continuous current applied by the associated electronics and the maximum error occurs at the initial moment, when current is maximal . Hence, precautions should be similar to those taken into account in pH measurements, where electrometer amplifiers are used. We believe the I os effect is so notorious in Impedance Microbiology because the times during which the cells and the electronic circuitry are connected are too long. A 12 hr growth curve, with a 2%/hr drift, is significantly distorted by the unwanted I os ; thus, the latter cannot be ignored. In other electrochemical applications such a drift does not show up because the total measurement periods are considerably shorter [ 19 ]. In other words, it seems justifiable to use electrometer amplifiers and to decrease connection times between cells and circuitry in order to minimize the drift due to I os . The shorter this time, the smaller the continuous current change traversing the interface and the smaller the interface capacitance variation. Results obtained (Fig. 6 ) with equipment designed having these concepts in mind showed essentially no drift and no distortion in the growth curves [ 8 , 9 , 20 , 21 ]. Figure 6 Percent interface reactance X i versus time for sulfate reducing bacteria samples growing in Postgate Broth at 37°C. No curve shows any drift, including the reference (sterile) cell. It must be underlined that all the analysis developed herein is only valid for bipolar impedance measurements. We did not make measurements using tripolar or tetrapolar electrode configurations. For this kind of application, the bipolar technique is more practical. One of the reviewers of this paper perceptively commented that an improved model, which includes a parallel faradic resistance (high in the low frequency range, decreasing with an increase in current density), as well as the half-cell potential, might give a better theoretical background of the study. It brings up an interesting point. The interface is, no doubt and as mentioned above, a complex system produced by the interplay between electrochemical processes and the geometry of the electrode surface. A perfectly smooth surface can be characterized by the double layer capacitance C dl in parallel with the series combination of the charge transference resistance R tc and the diffusion Warburg impedance Z w . The whole set, in turn, in series with the medium resistance R m [ 22 , 23 ]. The model becomes rather complex when the surface geometry and the current density are considered [ 24 ] and we think that entering into his kind of intricacies exceeds the purpose and intended reach of the article. Another aspect brought up during the refereeing process referred to the effects of the recording site area or the metal material, even suggesting platinum black as an alternative other than stainless steel. The fact is that the latter does not show any toxic effect, it leads to measurable impedance values for the used geometry and available equipment, and is economic and easy to get without imposing special care. When massive use is the case (as in industrial applications), price becomes of concern, too, and impedance microbiology has a clear industrial facet. Finally, a few comments should be added regarding the connection time, as another arbiter asked whether measuring for only 4s is really practical for most applications or not. In neural recording, for example, this is certainly not enough time. Our solution has meaning just in impedance microbiology, where bandwidths and sampling frequencies are very low. Let us illustrate with some numbers: Nerve action potentials, with a frequency range of 100 to 2,000 Hz, require a sampling frequency in the order of 8 kHz. Conversely, microbiological growth curves, with signals between 0.00027 Hz and 0.0000016 Hz (a growth curve may take 1 hr up to 7 days to reach its maximum, depending on the kind of bacteria and its culture broth) require sampling frequencies of about 0.0016 Hz (1 sample every 10 minutes). Drift current curves (the main concern of this paper) have similar numerical requirements (signal frequency range, 0.00027 Hz to 0.000027 Hz, which covers drifts that take 1 up to 10 hrs to reach its maximum, and sampling frequency of 0.0016 Hz). Summarizing this point: In impedance microbiology, the signal (that is, the growth curves) and the noise (the drift) coincide in their bandwidth. The method herein described permits a significant noise reduction originated in drift. Conclusion Systems not provided with especially designed input amplifiers introduce drifts leading to unacceptable distortions in bacterial growth curves. Those drifts do depend neither on the measurement conditions nor on the applied frequency. They are due to unwanted input amplifier bias currents connected to the cells. The drifts can be minimized by means of electrometer amplifiers (with sub-picoamps biases) and isolating relays to switch off the cells between measurements. Authors' contributions This paper is the result of over 20 years of experience working in Impedance Microbiology, as a team, always trying to improve the records' quality. Contributions are well balanced and ideas came up slowly after many trials and errors.

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1079903

Review of "Mathematical Techniques in Multisensor Data Fusion" by David L. Hall and Sonya A. H. McMullen

Data fusion has been a trend in the field of imaging and signal/image analysis. Although multisensor data fusion is still not regarded as a formal professional discipline, tremendous progress has been made since the publication of the first edition of this book in 1992. With this second edition, the authors have been successful in updating us with state-of-the-art methods and techniques in multisensor data fusion. The book involves both algorithms and software tools, and also covers contemporary subjects like smart agents, cognitive aides, and so on. This book is written according to the Joint Directors of Laboratories (JDL) data fusion group model. There are five levels in the JDL model. The first level deals with association, correlation, estimation, and identification in the data domain. The second and third levels perform knowledge-based processing and utilize expert systems. The fourth level is focused on process monitoring and optimization. The fifth level is devoted to human computer interaction. Chapter 1 serves as an overview. Then, Chapter 2 introduces the JDL model and associated algorithms. Chapters 3–6 cover processing techniques at level one. Chapter 7 gives methods at levels two and three. Chapter 8 targets the control of sensor and information resources at level four. Chapter 9 is for data fusion at level five. Chapter 10 discusses implementation of fusion systems. Chapters 11 and 12 describe emerging applications and information management. The book contains 100 equations, 75 illustrations and key references. The typesetting quality is generally excellent but it would be better if some labels in the figures have been put in larger size. Note that the additons in this book include materials on fusion system control, DARPA's TRIP model, and applications in data warehousing, medical equipment, and defense systems. Hall (associate dean for research, Pennsylvania State University School of Information Sciences and Technology) and McMullen (captain, US Air Force) are well known experts in the field, and should be congratulated for accomplishing such an excellent job in summarizing the up-to-date essential ideas and results on multisensor fusion. Overall, the book is very informative and not difficult to read for electrical and computer engineers as well as technical managers. These types of practitioners can gain solid advice from the book regarding selection of data fusion methods, balance of trade-offs among commercial off-the-shelf tools, development of multisensor data fusion systems and their applications to solve real-world problems. However, to build a sophisticated data fusion system one may need other references for more comprehensive mathematical theories, more rigorous statistical treatments, and more technical details. The cited literature in the book represents appropriate pointers for that purpose. A good example for further reading is the Handbook [ 1 ]. As an engineer in biomedical imaging, I would like to underline the relevance of this book to research on systems biomedicine. Given the completion of the Human Genome Project [ 2 ] and the momentum of the Physiome Project [ 3 ], in 2003 the NIH defined its Roadmap to guide our efforts towards most important biomedical research, including studies on biological building blocks, pathways, and networks using molecular libraries, imaging, bioinformatics and computational biology. Consequently, multi-modality signal sensing and imaging plays a critical role, which often necessarily generates heterogeous sparse/huge datasets that are multi-dimensional, multi-spectral, dynamic, noisy and/or incomplete. The data fusion techniques discussed in this book are closely related to that for biomedical image registration/fusion. Major challenges and new opportunities are enormous ahead of us. In that context, I hope this book will have a significant impact on the biomedical imaging areas, and recommend it to biomedical imaging researchers in particular and other interested engineers in general.

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1079904

Females with paired occurrence of cancers in the UADT and genital region have a higher frequency of either Glutathione S-transferase M1/T1 null genotype

Upper Aero digestive Tract (UADT) is the commonest site for the development of second cancer in females after primary cervical cancer. Glutathione S-transferase ( GSTM1 and / or T1 ) null genotype modulates the risk of developing UADT cancer (primary as well as second cancer). The aim of this study was to evaluate the difference in GST null genotype frequencies in females with paired cancers in the UADT and genital region as compared to females with paired cancers in the UADT and non-genital region. Forty-nine females with a cancer in the UADT and another cancer (at all sites-genital and non-genital) were identified from a database of patients with multiple primary neoplasms and were analyzed for the GSTM1 and T1 genotype in addition to known factors such as age, tobacco habits, alcohol habits and family history of cancer. Frequencies of GSTM1 null, GSTT1 null, and either GSTM1/T1 null were higher in females with paired occurrence of cancer in the UADT and genital site (54%, 33% and 75% respectively) in comparison to females with paired occurrence of cancer in the UADT and non-genital sites (22%, 6% and 24% respectively). The significantly higher inherited frequency of either GSTM1 / T1 null genotype in females with a paired occurrence of cancers in UADT and genital region (p = 0.01), suggests that these females are more susceptible to damage by carcinogens as compared to females who have UADT cancers in association with cancers at non-genital sites.

Introduction Precise risk estimation for the development of second cancers in the Head & Neck, Lung, and Esophagus, also known as the Upper Aero-Digestive Tract (UADT), is difficult due to the multifactorial origin and gene-environment interaction [ 1 ]. The Glutathione S transferase ( GST ) family of enzymes coded for by at least five distinct loci- alpha , mu , theta , pi and gamma detoxify tobacco carcinogens [ 2 ] and play an important role in the gene-environment interaction associated risk for cancer. The results of a recently published meta- and pooled- analysis of 4635 cases and 5770 controls, support the widely studied association of inherited GST genotype with the risk of developing squamous cell carcinomas (SCC) in the UADT. In addition the results support the notion that the risk increases when genotypes at multiple GST loci are considered [ 3 ]. Patients with second (synchronous and metachronous) cancers in the UADT also have a higher frequency of GST null genotype (particularly GSTM1 and T1 ) as compared to those with single cancers [ 4 - 7 ]. Similar to the association seen in UADT cancers, GST null genotype has been associated with the risk of developing Human Papilloma Virus (HPV)-positive primary cervical cancer in females [ 8 , 9 ] and also with other cancers such as primary breast cancer [ 10 ] and primary thyroid cancer [ 11 ]. Females with a primary HPV related cancer (particularly uterine cervix) develop second cancers in the UADT with an increased frequency [ 12 - 14 ]. HPV transmission has been suggested to account in part for the paired occurrence of cancers at these anatomically distinct sites of UADT and cervix. Exposure to tobacco carcinogens has been suggested as synergistic cofactor [ 15 , 16 ]. The mechanism by which HPV infection, necessary for the development of cervical cancer [ 17 ], causes UADT cancers is unclear [ 18 ]. Recently it has been shown that interaction between viral infection (especially HPV) and xenobiotic enzymes such as GSTM1 could modulate cancer risk, however the evidence comes from in vitro studies alone [ 19 ]. There are no studies found in the literature evaluating the association of GST null genotypes and the risk of developing second cancers in the UADT in females, particularly after primary cancers at genital sites. We identified females with a paired cancer in the UADT in association with another cancer (at all sites-genital and non-genital) and analyzed GSTM1 and T1 genotype in addition to known factors such as age, tobacco habits, alcohol habits and family history of cancer. Patients and methods Patients From a registry of 150 patients with Multiple Primary Neoplasms (MPN) at Tata Memorial Hospital, Mumbai, during the period of 1996 – 2004, all the females, who met the following criteria were selected: 1) synchronous or metachronous occurrence of second primary cancer; 2) Histological or cytological confirmation of both primaries; 3) One of the two cancers was a SCC in the UADT. Modified Hong's criteria [ 20 ] was used to differentiate the second primary cancer from metastatic or direct spread from the first cancer. Details about age, site of first cancer, site of second cancer, histology, grade, history of cancer in the first degree relatives, tobacco habits (smoking or smokeless), alcohol intake (regular, occasional or never) and radiotherapy for the first cancer were noted both from patient interviews and medical records. Detailed and exact quantification of exposure to tobacco and alcohol was not available in all cases. Neither were details about reproductive and sexual history available. DNA extraction Having obtained an informed consent, 5–10 ml of peripheral blood sample was collected in sterile EDTA tubes. The DNA was extracted from the leukocyte pellet obtained from the blood by sucrose lysis, purified by phenol: chloroform and suspended in Tris-EDTA buffer and stored at -20°c. PCR Multiplex Polymerase Chain reaction (PCR) was performed to simultaneously genotype GSTM1 or GSTT1 gene and the Human Fibroblast Interferonβ-1 gene ( IFNβ-1 ). The IFNβ-1 gene was used as an internal control for amplification failure. The primer sequences were as follows 1) GSTM1 : F: 5' CTG GAT TGT AGC AGA TCA TGC 3' and R: 5' CTG CCC TAC TTG ATT GAT GGG 3' which generates a 273 bp fragment; 2) GSTT1 : F: 5' TTC CTT ACT GGT CCT CAC ATC TC 3' and R: 5' TCA CCG GAT CAT GGC CAG CA 3' which generates a 459 bp fragment and 3) IFNβ-1 : F: 5' GGC ACA ACA GGT AGT AGG CG 3' and R: 5' GCC ACA GGA GCT TCT GAC AC 3' which generates a 170 bp fragment. The GSTM1 or GSTT1 gene and Interferon gene were co-amplified in a 15 μl reaction mixture containing a total of 50 ng of genomic DNA as the template, 2.6 μM of each GSTM1 primer, or 2.6 μM of each GSTT1 primer, and 2.6 μM of each Interferon primer, 1.2 mM each dNTP, 1 × PCR buffer (which contained 20 mM Tris, pH 8.4, 50 mM KCl), 2 mM MgCl 2 and 0.6 units of Taq polymerase (Amersham). The PCR profile for amplification of GSTM1 gene consisted of an initial melting step at 94°C for 9 min followed by 30 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, with a final step at 72°C for 10 min for elongation. The PCR profile for amplification of GSTT1 gene consisted of an initial melting step at 95°C for 9 min followed by 30 cycles at 94°C for 1 min, 66°C for 1 min, and 72°C for 1 min, with a final step at 72°C for 10 min for elongation. Gel electrophoresis The PCR products were separated either on 2% agarose gels or on 12% polyacrylamide gels and were seen by ethidium bromide staining or silver staining respectively. In the PCR reaction presence of a 170 bp ( IFNβ-1 ) band alone and absence of a higher 273 bp ( GSTM1 ) or 459 bp ( GSTT1 ) band indicated null genotype of the sample. In the PCR assay when both Interferon and GST fragments were absent then it indicated that either the PCR was not successful or the DNA was degraded. Statistical analysis Fishers exact test was used to assess the statistical significance for the difference between proportions of various factors (number of tobacco chewers, GSTM1 null, GSTT1 null, either GSTM1 / T1 null). Results Overall data of all females Forty-nine females with a paired SCC in the UADT in association with another cancer (at genital and non-genital related sites) were identified. The median time to development of the paired cancer (or second cancer) was 4 years. The median age at diagnosis of first cancer in all females was 51 years and that of second cancer was 60 years. The commonest sites of first cancer were oral cavity (n = 17), genital region (n = 15), and esophagus including post-cricoid region (n = 12). Information on family history of cancer was available in 42 women and 6 (14.3%) had a first-degree relative affected with cancer. Of the 45 women where details of habits were available, 25 (56%) were tobacco chewers whereas only 1 (2%) smoked. Of the 29 patients in whom GSTM1 and T1 genotyping was done, the frequencies of GSTM1 null, GSTT1 null and either GSTM1/T1 null were 35%, 17% and 45% respectively. Groupwise analysis Results were further analyzed by dividing the females into two Groups (Table 1 ): Group 1 (n = 19), females who had a paired SCC in the UADT along with a cancer in the genital region and Group 2 (n = 30), females who had a paired SCC in the UADT along with a cancer at other sites (non-genital region). In the majority of the cases (80% in Group 1 and 86% in Group 2), UADT cancer occurred second in chronological order. The median time for the development of second cancer was 5-years (range 0–20 years) for females in Group 1 in comparison to 3-years (range 0–24 years) for females in Group 2. There were no significant differences in the median age at the diagnosis of the first cancer, family history of cancer or tobacco habits between the two groups. Uterine cervix was the commonest genital site of cancer in females in Group 1 (16/19, 84%), followed by vagina in one female, vaginal vault in one and vulva in one. The sites of second UADT cancer in females in Group 1 were: oral cavity (n = 7), oropharynx (n = 2), hypo pharynx (n = 1), trachea and Lung (n = 4), esophagus including post-cricoid (n = 4), thyroid (n = 1). In the females in Group 2, UADT was the commonest non-genital site of cancer (22/30, 73%), followed by breast (n = 6), duodenum (n = 1), and chronic lymphatic leukemia (n = 1). Histology of the cancer in the genital region in females in Group 1 was confirmed as SCC in the majority (18/19). In the remaining female the histology could not be confirmed as the records were missing, however it was confirmed that her cervical cancer was treated with radiotherapy, a treatment that is never given without histological confirmation at our hospital. Table 1 Comparison of host, environment and treatment related parameters in the two Groups of females with second cancer in the UADT. Group 1 [n = 19] Group 2 [n = 30] p value Median age at the diagnosis of first cancer [years] 50 53 0.58 Median time to development of second cancer in years [range] 5 [range 0–20 years] 3 [range 0–24 years] 0.31 Family history of cancer 3/18 3/24 1.00 GSTM1 null 7/13 [54%] 4/18 [22%] 0.12 GSTT1 null 4/12 [33%] 1/17 [6%] 0.13 Either GSTM1 / T1 null 9/12 [75%] 4/17 [24%] 0.01 *Tobacco habits 9/18 [50%] 17/27 [63%] 0.54 * Except one smoker, all other habitués were tobacco chewers and none of them consumed alcohol. Null genotype frequencies for GSTT1 , GSTM1 and either T1 or M1 were more frequent in Group 1 as compared to Group 2. The proportion of females who were null for either GSTM1 / T1 were found to be significantly higher in the Group 1 than in Group 2. Discussion This is the first study evaluating the association of GST genotype with paired occurrence of an UADT cancer and genital site cancer. Of the 49 women with paired occurrence of an UADT SCC with a second cancer at any site, 19 (39%) were in the HPV related genital site. Majority (84%) of the genital cancers were in the Uterine Cervix and in more than half (68%) the genital cancer occurred before or within 6 months of development of the UADT cancer. Association between malignancies of the UADT and genital region (especially uterine cervix) has been observed by various investigators [ 13 , 15 ]. Hemminki et al [ 14 ] studied the pattern of second cancers in females and found a consistent increase in second HPV related cancer when the first cancer was at an HPV related site (anogenital, skin, head and neck, oesophageal and rectal cancers). An excess of oral cancers (including lip, mouth, tongue, larynx and pharynx), as found in this study, followed cervical cancers. Other investigators have also found similar results in the past [ 12 ]. Using incidence data from Surveillance, Epidemiology & End Results (SEER), Spitz et al [ 15 ] found an elevated Standardized Incidence Ratio (SIR) for cervical cancer after an initial buccal cavity cancer and larynx cancer. HPV transmission has been suggested to account in part for the paired occurrence of cancers at these anatomically distinct sites of UADT and cervix and smoking has been suggested as synergistic cofactor [ 15 ]. However, smokeless tobacco is a predominant form of tobacco consumed by females in India [ 21 ] and a recent study from India has shown a positive correlation of smokeless tobacco habits with cervical cancer risk [ 22 ]. Additionally, results from another study from India found more than 80% of oral cancers in females attributable to smokeless tobacco habit as compared to the negligible influence of smoking and drinking on oral cancer in females [ 23 ]. In concordance with these figures, 44% of females in Group 1 in our study had smokeless tobacco habits and none of the patients, except for one, smoked. The role of GST polymorphisms in the development of second cancers in the UADT has been a focus of few studies in various ethnic groups [ 4 - 7 ]. In all of these studies the GST null genotype ( GSTM1 and or GSTT1 ) has been seen with increased frequency in patients with second cancers in the UADT as compared to those with single cancers in the UADT. The results of the recently published meta and pooled analysis support the notion of a greater risk of head and neck cancer when genotypes at multiple GST loci are considered [ 3 ]. The 35% GSTM1 null genotype frequency (in entire population of females with second cancers in the UADT) in this study is very similar to the GSTM1 null genotype frequency of 36% found in our previous study in male patients with second cancers in the UADT from similar ethnic background [ 7 ]. However, the null genotype frequency for GSTT1 in males was 41% vs. 17% in females from this study. Differences in the site of cancer and the form of tobacco use, which predisposes to cancer development at a particular site, between males and females might possibly explain the gender discordance in GSTT1 null genotype frequencies. Nonetheless the 41% and 17% null genotype frequency for GSTT1 in males and females respectively are higher than 8–12% GSTT1 null genotype frequency found in healthy controls from similar ethnicity suggesting a true association [ 24 , 25 ]. GSTM1 and GSTT1 null genotype frequencies (54% and 33%) in the females who had second cancer in the UADT in association with a cancer in the genital region (Group 1) in the present study are higher than a) the respective frequencies (22% & 6%) in females with second cancer in the UADT in association with a cancer at non-genital region (Group 2), b) the respective frequencies (49% & 18%) found in subjects with single UADT cancers in two studies from a similar ethnic background [ 24 , 25 ], c) the respective frequencies (26% & 19%) found in males with single UADT cancers from our previous study [ 7 ], and d) the respective frequencies (33% & 8% and 24% & 12%) found in Indian healthy controls [ 24 , 25 ]. This suggests that the females who developed second cancers in the UADT in association with cancer at genital sites (Group 1 in this study) are possibly more susceptible to damage induced by carcinogens. The association of GST polymorphism and the risk of single primary cervical SCC have been studied in female patients from various ethnicities. GSTM1 null genotype has been found to incur an increased risk for primary cervical cancer in a mixed Caucasian, Hispanic and African-American patient population as compared to controls [ 9 ]. Like wise, in a study on 181 Korean cervical carcinoma patients, Kim et al [ 8 ], showed a positive correlation between GST genotypes and cervical carcinoma risk as compared to controls. In this study by Kim et al , GSTT1 null genotype was associated with an increased risk for cervical cancer. When the patients were stratified according to age, either GSTM1 / T1 null genotype was significantly over represented in patients with cervical carcinoma who were = 40 years old. However, in a study on 190 Caucasian women, Chen et al [ 26 ], determined no association of GSTM1 null genotype with invasive cervical cancer risk. Other investigators have found results similar to that found in the study by Chen et al [ 26 - 28 ]. Differences in GST genotype frequencies in various control populations could account for the differences seen in various studies. For instance, the prevalence of GSTM1 null genotype in Caucasian, American and Korean population is similar (as high as 50%) whereas the prevalence of GSTT1 null genotypes varies from 15–31% in the Caucasians to 50–58% in the Koreans and Chinese. In view of the known differences of the population frequency of null genotypes and its associated risks for cervical cancer across various ethnic groups, it is important to compare our results with those from studies conducted on subjects from a similar ethnic background. GSTM1 and GSTT1 null genotype frequencies (54% and 33% respectively) in the females who had at least one of the cancers in the genital region (Group 1) in the present study were similar to the respective frequencies (57% and 20% respectively) found in females with single (unpaired) cervical cancer cases from India [ 29 ] suggesting that the association between GST genotype and cervical cancer risk in Group 1 females in our study is more likely to be a true association. HPV is undoubtedly the main causative agent in cervical SCC's and the prevalence of HPV, mainly HPV16/18 of more than 95%, has been seen in tumours from cervical cancer patients from India [ 30 ]. Evidence linking HPV to oral carcinogenesis is also growing and one of the highest prevalence rates of 74% of HPV 16 and 18 in oral tumors from smokeless tobacco habitués have been reported from southern India [ 31 ]. As HPV infections are transient, the absence of HPV DNA does not rule out previous exposure [ 32 ]. Interestingly, though most of the patients with cervical cancer are positive for HPV [ 17 ], not all patients who have HPV infection will go on to develop cancer [ 33 ]. Interactions between HPV and GST genotype may be a reason for this. The evidence for this is two-way. Chen et al [ 19 ] evaluating the reason for the lack of association between GSTM1 null genotypes and cervical cancer risk in their epidemiological study demonstrated that viral infection could have potential effect on the activity of xenobiotic metabolizing enzymes, particularly GSTM1 . The results of their study raised a question of how chronic viral infections could affect cellular defenses against carcinogens in general. Evidence from other studies suggests that inherited susceptibility in the form of GST genotype may modulate the risk of developing HPV related cancer as evidenced by GSTM1 null genotype which, in addition to HPV infection and smoking, has been found to increase the risk of developing cervical cancer [ 9 ]. Moreover, in the patients with cervix cancers having a positive association with GST null genotype, as studied by Kim et al [ 8 ], HPV 16 or 18 infections were confirmed in all. Conclusion The findings of our study suggest an association between the GST null genotype and the occurrence of paired cancers in the UADT and HPV related genital sites in females. Though this is the first study to report such an association, further confirmation from larger studies on patients from different ethnic backgrounds is required.

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1079905

Vascular ossification – calcification in metabolic syndrome, type 2 diabetes mellitus, chronic kidney disease, and calciphylaxis – calcific uremic arteriolopathy: the emerging role of sodium thiosulfate

Background Vascular calcification is associated with metabolic syndrome, diabetes, hypertension, atherosclerosis, chronic kidney disease, and end stage renal disease. Each of the above contributes to an accelerated and premature demise primarily due to cardiovascular disease. The above conditions are associated with multiple metabolic toxicities resulting in an increase in reactive oxygen species to the arterial vessel wall, which results in a response to injury wound healing (remodeling). The endothelium seems to be at the very center of these disease processes, acting as the first line of defense against these multiple metabolic toxicities and the first to encounter their damaging effects to the arterial vessel wall. Results The pathobiomolecular mechanisms of vascular calcification are presented in order to provide the clinician – researcher a database of knowledge to assist in the clinical management of these high-risk patients and examine newer therapies. Calciphylaxis is associated with medial arteriolar vascular calcification and results in ischemic subcutaneous necrosis with vulnerable skin ulcerations and high mortality. Recently, this clinical syndrome (once thought to be rare) is presenting with increasing frequency. Consequently, newer therapeutic modalities need to be explored. Intravenous sodium thiosulfate is currently used as an antidote for the treatment of cyanide poisioning and prevention of toxicities of cisplatin cancer therapies. It is used as a food and medicinal preservative and topically used as an antifungal medication. Conclusion A discussion of sodium thiosulfate's dual role as a potent antioxidant and chelator of calcium is presented in order to better understand its role as an emerging novel therapy for the clinical syndrome of calciphylaxis and its complications.

Background Atherosclerosis and vascular ossification – calcification (VOC) date to the time of the ancient Egyptians [ 1 ]. In the initial descriptive phase before the various hypotheses regarding the pathogenesis of atherosclerosis emerged, prominent pathologists of the time emphasized the bone like changes and used the terms ossification and petrification of atherosclerotic lesions. Benivieni A. in 1507 and Fallopio G. (Fallopius) in 1575 were the first to record for posterity their initial descriptions of atherosclerosis. Fallopius, in 1575, described a degeneration of arteries into bone, which physicians of the time called ossification of the arteries [ 2 ]. In 1863, Virchow labeled the vascular changes as "ossification, not mere calcification, occurring by the same mechanism by which an osteophyte forms calcium on the surface of bone" [ 3 ]. During the 1800s a widely held concept was that VOC might be a protective mechanism to prevent aortic aneurysms from rupturing. In 1906, Bunting was able to demonstrate the presence of bone marrow containing its classical cellular contents including osteoclasts within a sclerotic aorta. He frequently debated the finding of ossification within the arterial vessel wall (AVW) and supported the finding that ossification was not a rare occurrence; rather it was just not specifically searched for at the time of autopsy [ 4 ]. It is currently widely accepted that vascular cells play an active role in osteoid formation and the ossification process to result in VOC. [ 5 - 10 ] Newer imaging techniques such as the ultrafast computed tomography, intravascular ultrasound, and emerging data on magnetic resonance imaging are rapidly changing the way we view VOC. These newer techniques (as compared to VOC in radiographs) (figure 1 ) demonstrate that VOC is not just a degenerative end-stage passive process associated with aging, but a dynamic and regulated event, which serves as a marker and occurs early in the natural history of atherosclerosis [ 11 - 13 ]. Currently, it is felt that coronary artery calcification is not a late, degenerative, end-stage process, but is widespread even in the early, positive, outward remodeling phase and is predictive of cardiac events prior to the development of stenotic lesions [ 11 ]. VASCULAR OSSIFICATION – CALCIFICATION (VOC) VOC represents a pathologic secondary ossification site or dystrophic calcification primarily in the arterial vasculature in a response to injury mechanism, just as the body responds to the traumatic event of a fractured bone and/or repair of the chondral injury associated with the pathogenesis of osteoarthritis (table 1 ). In the vasculature this injury is a result of chronic injurious stimuli associated with multiple metabolic toxicities and their associated reactive oxygen species (ROS) generated in the accelerated atherosclerosis (atheroscleropathy) of the metabolic syndrome (metS) and type 2 diabetes mellitus (T2DM), and non-diabetic atherosclerosis (figure 2 ). There is a recapitulation of embryonic genetic memory associated with this reparative – protective mechanism. In atheroscleropathy and non-diabetic atherosclerosis, the multiple metabolic toxicities and the resultant – parallel ROS, via stimulation of the essential and omnipotent nuclear factor (NFkappaB), which activates the downstream inflammatory mediators TNFalpha, IL-1, IL-6, and other inflammatory markers such as C-reactive protein (CRP). VOC in its early stages develops by a process of matrix vesicle formation usually at the base of the lipid core in the intima of atheromatous plaques (figure 3 ). These matrix vesicles are then partially replaced with osteoid synthesized by osteoblast-like cells of mesenchymal origin, such as the vascular smooth muscle cell (VSMC) or pericyte. The subsequent bone mineralization is dependent on neovascularization – angiogenesis from the adventitial vasa vasorum (Vv), which leads to formation of mature bone tissue in type Vb atherosclerotic plaques (figure 4 ) [ 5 , 14 - 16 ]. This orderly transformation of ossification has been demonstrated to occur in vitro by vascular mesenchymal cells, which undergo a transformation of monolayer growth to swirls, to ridges to a labyrinthine structure [ 16 ] The vasa vasorum's guardian angel: the pericyte and its important role in VOC The role of the Vv seems crucial to the development of VOC and its resultant alteration regarding bone formation within the osteoid tissue of the fibrocalcific plaques of atherosclerosis. The medial and intima Vv are derived primarily from the adventitial layer of the artery. The Vv acts as a custom delivery system to supply the media and intimal layers with oxygen and nutrients as the atherosclerotic artery undergoes the positive outward remodeling. These angiogenic vessels also serve as a conduit delivering systemic hormones, cytokines and inflammatory cells to the remodeling media and the atherosclerotic intimal disease of atherosclerosis including ossification. Of particular importance is vital role of the Vv capillary endothelium and its guardian, multipotential, stem cell-like, supportive, mesenchymal cell the pericyte [ 1 , 5 , 17 ] (table 2 ) (figure 1 , 5 ). The location of VOC in these arteries is both in the medial and intimal layers. It is important to note that in health the Vv normally penetrates the media but not the intima. In disease the Vv invade excessively not only the media but also the intima and this may help to explain the detrimental process of intimal ossification. Bone siloprotein synthesized by the osteoblast-like VSMC has recently been shown to be an angiogenic protein capable of inducing neovascularization by interacting with the alpha v beta 3 integrin [ 18 ]. Even more exciting is the finding that the endothelial pericyte is capable of synthesizing this non-collagenous angiogenic factor as well as being related to ossification of atherosclerotic plaques. VOC IN metS AND T2DM Overview Long before the development of overt T2DM there exists a pro-atherosclerotic process within the arterial vessel wall (AVW) [ 15 , 16 ]. The metS is associated with multiple metabolic toxicities referred to as the A-FLIGHT-U toxicities, which result in the production of ROS (table 3 ). Each of these excess toxic substrates is capable of generating ROS but in the metS they may act synergistically to result in perturbations of the AVW and contribute to a premature atherosclerosis, which has been termed atheroscleropathy (ASO) [ 19 , 20 ]. This is why many patients with overt T2DM often present with acute coronary syndromes and why T2DM should be considered a coronary heart disease (CHD) risk equivalent according to the National Cholesterol Educational Program Adult Treatment Panel III (NCEP ATP III) guidelines. Hyperinsulinemia, Insulin-like growth factor-1 (IGF-1), and Amylin (IAPP) Insulin, IGF-1, and amylin are known to be elevated in the insulin resistance (IR) and metS patient [ 21 ]. Insulin has been long known be a growth factor and is a stimulus for hepatic synthesis of IGF-1, which may be implicated in the accelerated ossification in ASO. There is a human gene polymorphism with a phenotype described as the Benardinelle lipoatrophic syndrome more commonly referred to as lipoatrophic diabetes and this syndrome is associated with manifestations of acanthosis nigricans, generalized lipoatrophy, hirsutism, muscle hypertrophy, intellectual impairment, IR diabetes mellitus with major diet-dependent type V hypertriglyceridemia, and an increase in bone density, thus implying osteogenesis [ 22 ]. In addition to insulin and IGF-1, there is another beta cell derived islet hormone that is elevated in IR, metS, prediabetes, and early T2DM termed amylin or islet amyloid polypeptide. Amylin may be considered to be the fraternal twin of insulin and has recently been shown to be a physiologic regulator of bone remodeling through favoring bone formation, while inhibiting bone resorption and thus is osteogenic [ 23 ] (table 3 ). Hypertension (Htn) Htn is the second leading cause of ESRD after diabetes. Hypertension plays an important role in the ASO associated with metS and T2DM, as atherosclerosis is non-existent in the pulmonary arterial tree and the venous system, due to a lower systemic intravascular pressure. Htn may be considered toxic to the AVW and is known to be associated with VOC (table 3 ) [ 19 , 20 , 24 ]. Mehrotra R and colleagues have recently demonstrated that hypertension plays a more putative role in the association of coronary artery calcification than do levels of serum calcium, phosphorus, parathyroid hormone, and 1,25 di-hydroxy vitamin D levels in T2DM patients not requiring dialysis [ 25 ] Hyperlipidemia – Dyslipidemia and the Lipid Triad The lipid triad of the metS and T2DM consists of an elevation in VLDL-C or triglycerides, small dense atherogenic LDL-C, and a decrease in HDL-C. Once these lipids become modified through oxidation in a milieu of increased ROS from the synergistic effect of the associated A-FLIGHT-U metabolic toxicities, they are a predictive determinate of osteogenesis within the media and intima of the AVW. The earliest changes associated of osteogenesis occur at the base of the lipid core in areas of necrotic-apoptotic macrophages and are associated with ROS and inflamation (figure 3 ). [ 1 , 19 , 20 , 26 , 27 ]. Hyperglycemia – Glucotoxicity: Impaired glucose tolerance (IGT) – impaired fasting glucose (IFG) – overt T2DM Elevated glucose levels (glucotoxicity) have been shown to be a direct sensitizer for osteogenesis [ 28 , 29 ]. In vitro studies with VSMCs and high glucose have been shown to induce cell proliferation and expression of osteopontin, while hypoxia seemed to have an additive effect [ 28 , 29 ]. Even though it is clinically known that diabetes is associated with an increase in medial VOC, the pathogenesis is not completely understood. Glucotoxicity seems to play an important role by transforming VSMCs and possibly pericytes into osteoblast-like cells (figure 6 ) [ 19 , 28 , 29 ]. Ishimura E et al have nicely demonstrated the importance of controlling HbA1c levels. They were able to demonstrate that for every 1 percent increase in HbA1c there was a 2.1 fold increase in the risk of VOC [ 30 ]. Poor glycemic control seemed to be of greater importance than calcium and phosphate concentrations. VOC in T2DM involves primarily the media of the AVW, however depending on the degree of underlying ASO; it may frequently involve the intima in those arteries where there is a more extensive involvement of atherosclerosis due to a deeper penetration of the adventitial Vv [ 1 ]. Medial artery calcification has been shown to be a strong independent predictor of cardiovascular mortality in patients with newly diagnosed T2DM [ 31 ]. In a clinical longitudinal study of 4,553 Pima Indians the risk factors for medial artery calcification in T2DM were impaired vibration perception, long duration of diabetes, and high glucose concentrations as compared to non-diabetic matched controls, whose risk factors for VOC were age, male gender, and high serum cholesterol [ 32 ]. Uric Acid Serum uric acid (SUA) has long been known for its association with the metS and also it's role in T2DM has recently been discussed in length [ 33 ]. SUA is an important substrate in the A-FLIGHT-U multiple metabolic toxicities and contributes to the production of ROS with resultant modification of lipoproteins as well as interfering with the normal function of the eNOS enzyme resulting in an uncoupling phenomenon discussed later (table 3 ). Elevated SUA reflects the activity of the xanthine oxidase enzyme and its production of ROS, which subsequently has a positive effect on VOC. Tseng CH has been able to show that SUA is an independent risk factor for the development of peripheral arterial disease in T2DM [ 34 ]. Newer risk factors: Homocysteine (Hcy), inflammation (hs CRP), Microalbuminuria Each of these novel risk factors plays a significant role in metS – T2DM and contributes to the calcification of the AVW through their association with the multiple metabolic toxicities and production of ROS while contributing to the inflammatory process (table 3 ). Hcy is known to be toxic to the AVW resulting in a host of fibrotic remodeling changes as a result of autoxidation, VSMC activation, and proliferation, as well as the production of ROS [ 35 ]. Li J. et al. have been recently able to demonstrate that Hcy potentiates calcification of cultured rat VSMCs [ 36 ]. Mediators of inflammation such as oxidation, carbonyl stress, C-reactive protein (CRP), and cytokines may directly stimulate vascular calcification. Also, inflammation reduces fetuin-A, a naturally occurring inhibitor of vascular calcification, which binds excess mineral in serum [ 37 ]. Increased levels of CRP are significantly associated with the presence of vascular calcification (including atheromatous and medial calcification) in both aorta and hand arteries, which may indicate a relationship between inflammation and vascular calcification in hemodialysis patients [ 38 ]. It is of interest to note that IL-10 plays an important role as an anti-inflammatory cytokine and is known to be decreased in CKD and ESRD. Low monocyte IL-10 synthesis may contribute to a chronic inflammatory state in these patients by defective feedback inhibition of proinflammatory cytokine production [ 39 ]. While inflammation plays a very important role, it is important to note that ROS occurs upstream from inflammation and that ROS are an important mediator of the nuclear factor kappa B [ 33 , 34 ]. Microalbuminuria is recognized as a surrogate marker for systemic endothelial dysfunction [ 40 ]. Recently, microalbuminuria has been shown to be a strong predicting factor of medial arterial calcification independent of nephropathy in patients with diabetes [ 41 ]. VOC IN CHRONIC KIDNEY DISEASE (CKD) Largely due to the prevalence of T2DM and Htn, approximately 11% of the U.S. population has some manifestation of CKD. This spans the proteinuria phase with normal renal function to advanced ESRD requiring renal replacement therapy either by dialysis or transplantation [ 42 ]. To better grasp this effect of CKD, the USRDS 2004 annual report has estimated there are in excess of 400,000 people undergoing some form of renal replacement therapy in the U.S. [ 43 ]. CKD should be considered in the highest-risk category for the development of cardiovascular disease (CVD) and an aggressive team approach of global risk reduction in order to reduce the prevalence and severity of the associated cardiovascular events should be established by following the RAAS acronym (table 4 ). CVD accounts for the largest cause of death in ESRD patients and may range from 10 – 30 fold higher for those patients being treated with dialysis as compared to the general population [ 44 ]. Patients entering dialysis over the past two decades now have a much higher incidence of diabetes and this is up to 59% as a first or second diagnosis with hypertension being the second leading cause of ESRD. Higher Framingham risk scoresand an aging population at the time of entry as a result of current technology and newer medications are preventing earlier coronary events and allowing them to survive and develop other end organ disease, such as ESRD and congestive heart failure, which are partly responsible for the increasing number of these patients [ 45 ]. Is atherosclerosis accelerated in this patient population or is there some other factor(s) related to the cardiovascular toxicity associated with uremia and dialysis? Schwarz U et al. were able to demonstrate an increased size of the medial layer, marked increased calcification, increased number of calcified plaques, and a decreased lumen area in a study of 27 patients with ESRD compared to 27 non-renal disease controls that had died due to coronary atherosclerosis. Thus, there was no evidence to suggest an accelerated atherosclerosis, but instead, a major change in plaque composition with an increase in ossification and medial enlargement [ 46 ]. Just as there are chronic injurious stimuli in atherosclerosis and ASO the pathology of CKD and ESRD include additional injurious stimuli to the vascular cells of the AVW. These include an elevation of the following: Phosphorus (Pi), calcium (Ca ++ ), parathyroid hormone (PTH), hypoxia and resultant ROS formation, and others (table 5 ) (figure 6 ). VOC is the manifestation of a complicated systemic process that begins primarily with medial ossification of larger and medium size arteries and progresses to the smaller arterioles. Concurrently, there is a development of renal osteodystrophy and ossification in the surrounding articulations of multiple joints [osteoatheroitis – osteoarthritis – renal osteodystropthy (table 1 )]. The clinical manifestations of VOC depend on the location of the affected arteries and arterioles [ 47 - 50 ]. Atherosclerotic nephropathy Scoble JE [ 51 ] presented the term atherosclerotic nephropathy in 1999, which includes abdominal atherosclerosis, renal artery atherosclerosis with renal artery stenosis, and atheroembolic disease. In older age populations, it has been estimated that atherosclerotic renal artery disease accounts for 14% (over the age of 50) and 25% of patients (over the age of 60) with end-stage renal failure [ 51 , 52 ]. With the age groups over the age of 50 and 60 (baby boom – senior boom generation) expanding at an exponential rate in western countries we will see an increasing number of patients with ESRD attributable to this abnormality. This atheroembolic phenomenon accounts for untold cases of ischemic nephropathy with functional loss of nephron units. Associated clinical companions of this condition that might alert the clinician would be: rapid increasing proteinuria, rapid deterioration of renal function, and other clinical evidence of peripheral atheroembolic phenomenon in other vascular beds such as the extremities in peripheral vascular disease (blue toe syndrome), mesenteric thrombosis and associated gastrointestinal bleeding, and extra cranial cerebrovascular atherosclerosis with associated transient ischemic episodes or overt cerebral thrombosis. In atherosclerotic coronary artery disease we do not see evidence of intra-myocardial atherosclerosis and likewise, we do not find evidence of intrarenal atherosclerosis. The renal artery may be likened to an epicardial coronary artery when comparing these two diseases. It is currently accepted that atherosclerotic coronary artery disease and hypertension account for the major cause of death and congestive heart failure in developed countries. It is time to consider atherosclerotic renal artery disease with VOC and associated renal artery stenosis, as well as, hypertension as a significant cause of progressive CKD and its progression to ESRD. As present in any atherosclerotic lesion, VOC is known to occur and will increase in the patient with CKD and ESRD (especially the patients with diabetes and ASO). VOC detected by non-invasive spiral CT scanning would alert the clinician to the possibility of renal artery stenosis and additionally alert the clinician to the importance for more aggressive therapeutic measures to control the A-FLIGHT-U toxicities (table 3 ) as well as to control the metabolic toxicities of the increased known sensitizers to VOC (table 5 ) (figure 6 ). VOC IN CALCIPHYLAXIS (CPLX) – CALCIFIC UREMIC ARTERIOLOPATHY (CUA) Any discussion of VOC would be incomplete without discussing the important issue of CPLX – CUA and a better understanding of this condition merits a janus-faced approach. The century old CPLX – CUA clinical syndrome, which once was considered to be a rare clinical phenomenon primarily associated with CKD and ESRD is now being reported and being seen in increasing numbers. Part of this increase may well be due to a better understanding and recognition of this syndrome and previous underreporting; however nephrology and dermatology departments, and dialysis clinics throughout the U.S. are seeing increasing numbers of this morbid – mortal complicated clinical syndrome. As the number of patients with CKD and ESRD continue to grow in numbers the medical community may well see this syndrome in near epidemic proportions (especially considering the current obesity – metS – T2DM epidemic). CPLX is not unique to any one country and is a global phenomenon. As with any new and emerging clinical syndrome the literature is replete with multiple case reports of this entity. In this review, an attempt is made to increase the awareness and understanding of this malady and provide an overview and not reference the multiple rare and unusual case reports, unless they contribute to the understanding of mechanisms or treatment [ 53 - 63 ]. Historical perspective of CPLX Selye's initial experiments in rats set the stage for the description of this clinical entity in humans [ 64 ]. He hypothesized that sensitizers such as PTH or vitamin D might condition the vasculature to VOC. After providing the sensitization he then challenged the rats with known provoking factors including metal salts, albumin, and trauma, which induced calcium deposition and subsequent inflammatory necrosis of the skin and subcutaneous tissue. Over time other studies suggested alternate contributing factors, such as those listed in table 5 . The name originated from the combination of calcium and anaphylaxis, thus calciphylaxis, however this model does not involve IgE mechanisms. Byrant and White (1898) are credited as having described the first clinical presentation in a six-month-old infant, in which there was VOC and what they termed endarteritis obliterans, indicating involvement of the intima [ 65 ]. It was not until 1969 that Rees and Coles used the terminology calciphylaxis to describe this clinical and pathologic syndrome in man [ 66 ]. Coates (1998) coined the term calcific uremic arteriolopathy (CUA) as a replacement term for CPLX, since in many instances elevations of Ca++, Pi or PTH levels are absent [ 67 ]. Over time CUA may become the preferred term, as this newer terminology is only six years old. The term calciphylaxis is almost entrenched in the medical literature at this point in time; however Selye's animal model more closely parallels the dystrophic tumorous soft tissue calcifications of uremic CPLX. His model did not exhibit the histology consistently described with uremic CPLX, i.e. small vessel calcifications and intimal hypertrophy in association with panniculitis (lobular fat necrosis, and inflammatory changes with neurtrophils, lymphocytes and macrophages, and small vessel thrombosis or involve IgE mechanisms [ 54 , 67 - 69 ]. We favor the term: obliterative calcific vasculopathy. VOC in the syndrome of CPLX – CUA is an important topic for discussion, as it is being reported at an exponential rate and especially over the past two years. The devastating complication of medial vascular calcification of small arterioles with resulting skin necrosis is rapidly becoming a morbid complication in both the non-diabetic and especially the large numbers of diabetic patients (T2DM > T1DM) undergoing renal replacement therapy (figure 7 ). The skin in CPLX The skin lesions of CPLX present as painful violacous (livedo reticularis) areas in the extremities, proximal medial thighs, anterior abdomen and lateral flank areas, the breast in females, and frequently overlie or are adjacent to subcutaneous tumorous calcifications in areas of increased adipose tissue. They then progress to a blackened eschar, which later reveals itself on a granulating bed and enlarges at the periphery of the ulcer. They have a tendency to develop in sites of prior skin trauma, such as abdominal scars from previous surgical incisions and may even develop from the trauma of simple scratching of extremities due to ESRD-associated pruritis. It is important to recognize the clinical presentation of the skin color changes and the neuropathic type of pain before ulceration has developed. If we wait until skin ulceration develops we place the CPLX patient at an increased risk of sepsis and premature death. We should carefully assess any palpable calcific tumorous masses in the subcutaneous tissue in patients at high risk for CPLX. Not all skin ulcerations are due to CPLX and therefore we should utilize our differential diagnostic skills in evaluating ulcers in these high-risk patients with CKD, T2DM, and ESRD. A reasonable differential diagnosis might include the following (table 6 ). Even though a skin biopsy may show medial arteriolar calcification and atrophy of medial muscle layers in the arteriole, we should keep in mind that these changes although quite specific are not pathognomonic for CPLX. The recent emergence of a newer skin pathology termed: neprogenic fibrosing dermopathy may appear co-morbidly with CPLX and recently it has been reported that transforming growth factor beta-1 (TGF beta-1) may to be related to both disorders [ 55 ]. By making an accurate and earlier diagnosis of CPLX, a greater effort may be given by the primary care provider and the nephrology team as well as other specialties (dermatology and plastic surgery) to improve the multiple A-FLIGHT-U toxicities in each patient (table 3 ). Earlier treatment of each altered abnormality of the associated multiple metabolic derangements may prevent or delay the progression of this morbid mortal clinical syndrome (table 4 , 5 ). Endotheliopathy, redox stress, and reactive oxygen species. eNOS and eNO The endothelium is an elegant symphony responsible for the synthesis and secretion of several biologically active molecules. It is responsible for regulation of vascular tone, inflammation, lipid metabolism, vessel growth (angiogenesis – arteriogenesis), arterial vessel wall – capillary sub endothelial matrix remodeling, and modulation of coagulation and fibrinolysis. One particular enzyme system seems to act as the maestro: The endothelial nitric oxide synthase (eNOS) enzyme and its omnipotent metabolic product: endothelial nitric oxide (eNO) (figure 2 , 8 ). The eNOS enzyme reaction is of utmost importance to the normal functioning of the endothelial cell and the AVW. When this enzyme system uncouples, the endothelium becomes a net producer of superoxide and ROS instead of the net production of the protective antioxidant properties of endothelial derived nitric oxide (table 7 ) (figure 8 ). There are multiple causes for eNOS enzyme uncoupling: The A-FLIGHT -U multiple metabolic toxicities, oxidative – redox stress, ROS, metS, insulin resistance, prediabetes, T2DM and T1DM, Htn, endothelin, and asymmetrical dimethyl arginine (ADMA) [ 1 , 19 , 20 , 27 , 28 , 34 , 45 ]. The endothelial cell is important in controlling the function of small arterioles and how they may become a net producer of ROS. This endothelial dysfunction may precede the vascular narrowing – closure, prothrombotic state, thrombosis with resulting ischemia of the surrounding subcapillary interstitium, ischemic necrosis of the subcutaneous adipose tissue, and skin changes of livedo reticularis with transformation to painful necrosis, eschar formation, and skin ulceration. In addition to correcting the underlying multiple metabolic A-FLIGHT toxicities and positive regulators of VOC (table 3 , 5 ), a strong consideration to newer therapies should be entertained in order to prevent, slow, and alter the morbid – mortal natural history of the dreaded complications associated with CPLX – CUA. Future Directions Sodium thioSulfate (STS) intravenous therapy for calciphylaxis: its emerging role STS is an effective antibrowning, reducing, and antioxidant agent and therefore readily donates electrons to re-pair unpaired damaging electrons to be an effective antioxidant in addition to it use as a chelator of cations such as the calcium excess in CPLX – CUA (figure 9 ). The following is one of many possible antioxidant reactions as it scavenges ROS and may be responsible for producing glutathione: STS is known to be a chelator of cations and has been used for over a century as an antidote for cyanide toxicity, topical treatments of acne and versicolor, and recently as a chemoprotectant against carboplatin and cisplatin toxicity. Its use in the treatment of calciphylaxis has only recently been described and its future use may become a standard of care in the treatment of the dreaded complications [ 70 ]. Its dual role as an antioxidant and chelator makes STS an excellent choice for the treatment of calciphylaxis and it will be interesting to follow future clinical trials regarding it use. Clinically, STS seems to have a rather intriguing rapid response in the relief of the pain associated with CLPX and it frequently reduces pain within the very first few treatments (personal experience). Additionally, it seems to promote healing of skin ulcerations especially when used in conjunction with hyperbaeric oxygen treatments [ 70 ]. The STS hypothesis STS may restore endothelial cell dysfunction in CPLX through its antioxidant actions and have a positive effect on eNOS uncoupling and eNO generation. We have formed a hypothesis that the rapid reduction in pain may be due to a restoration of endothelial dysfunction associated with this syndrome. The anitoxidant effect of STS, given in the intravenous dosage of 12.5–25 grams intravenous at the end of dialysis may help to restore the dysfunctional endothelial cell and begin restoring the endothelium's natural tendency (in health) to produce eNO (promoting vasodilation) instead of the dysfunctional super oxide and the resultant peroxynitrite (promoting vasoconstriction) (figure 8 ) [ 1 , 19 , 20 , 27 , 28 , 34 , 45 ]. STS could be working in two different vascular beds in the vulnerable subcutaneous tissue, as the peripheral neuronal unit is also involved with dystrophic ossification – calcification (figure 10 ): The vasa nervorum and the endoneurium of the peripheral neuronal unit [ 71 ] and the distal small pre-capillary arterioles. These changes would help to explain the rapid improvement of this morbid neuritic (breathtaking, stinging, burning, and knife-like) type of pain, as the chelation properties of STS would take much longer (months compared to hours or days) to restore the capabilities of the neuro-circulatory vascular beds. Diabetic and peripheral arterial disease patients may be slower to respond as they are plagued with a much more intense endothelial cell dysfunction and endothelial nitric oxide enzyme uncoupling due to the advanced atherosclerotic process and medial calcifications and associated ischemic changes. Undoubtedly the essential cofactor tetrahydrobiopterin (BH 4 ) becomes depleted during this chronic ischemia in the subcutaneous tissue from the multiple metabolic toxicities. STS may have a "folate-like redox shuttle effect" similar to the pleiotropic effects of folic acid supplementation [ 34 ] by allowing the oxidized cofactor BH 3 and BH 2 to undergo a restoration by the antioxidant effects of STS to the requisite completely reduced form BH 4 . This may ameliorate the underlying oxidative stress within the pre-capillary and capillary beds of the interstitium and the endoneurium of the peripheral neuronal units involved. While this is only a hypothesis it seems to be clinically relevant due to the clinical observation of a rapid relief of the pain associated with CPLX. Emerging role of nanobacteria Currently, the multiple roles of nanotechnology and nanobacteria are rapidly emerging and with new findings come new ideas, which may have a significant impact regarding the future of medicine. Nanobacteria may be a causative agent of diseases related to the biomineralization processes. As clinicians and scientists we must be careful as newer hypothesis and treatments emerge. For example even though nanobacteria have been found in multiple disease states associated with calcium deposition, we have yet to fulfill Koch's postulates regarding VOC. Because this information has just evolved it is important to review some of the background in this field of study. We have witnessed the exciting story of H. pylori and its causative role in ulcer disease and followed the C. pneumonia story in atherosclerosis finding it to be one of the burdensome infectious injurious stimuli to the endothelium that results in remodeling and atheromatous changes in the AVW. Likewise, the new information regarding nanobacteria being associated with VOC is exciting and could play a very important role in the causation of the atheromatous process and VOC (figure 2 ). Nanobacteria belong to the family of gram-negative rods (approximately 100 fold smaller in size) and have been recently localized in cardiovascular tissue. Miller VM et al. have found nanometer-scale particles (ranging in size from 30 to 100 nm) similar to those described as nanobacteria isolated from geological specimens and human kidney stones. These ultra small microbes can be visualized in and stained immunohistologically from calcified human cardiovascular tissue [ 72 ]. Jelic et al. have demonstrated the presence of nanobacteria in a mitral valve associated with a localized calciphylaxis within mitral vegetations from a patient with T2DM [ 73 ]. Maniscalco BS et al., have been able to demonstrate a statistically significant improvement in coronary calcium scores (inferring a reversal of VOC) and a reduction in angina in a group of patients with CAD and coronary artery calcifications using thecombination of EDTA by rectal suppository and a combination of multiple known antioxidants with 500 mg tetracycline (nanobacteriocidal) given daily by mouth. The treatment duration was 4 months [ 74 ]. Recently the literature has been replete with the association of periodontal disease and its relation to cardiovascular disease and events. Whether this condition is responsible for the activation of the chronic inflammatory disease response driving the atherosclerotic process or not remains to be seen. The recent isolation of nanobacterium with periodontal disease has fueled the fire of this being a putative microorganism in the development and progression of atherosclerotic disease and VOC [ 75 ]. Osteoporosis and VOC The coexistence of osteoporosis, CVD, and VOC with the aging process in humans and the concept of a shift of calcium from the osseous skeleton towards the AVW have been suggested as one of the explanations for their simultaneous existence [ 76 , 77 ]. Accumulating evidence indicates that there are similar pathophysiological mechanisms underlying both diseases. The A-FLIGHT-U multiple metabolic toxicities and resultant ROS, oxidative stress, endothelial cell dysfunction, endotheliopathy, and inflammation have been central issues in this paper regarding VOC. Each of these is associated with a modulation of vascular and osteogenic cells but in an opposite manner and may act in a synergistic manner. The finding of oxidative stress promoting VOC while promoting osteoporosis may help to explain the paradoxical parallel buildup of VOC and the concurrent loss of bone mineralization in the osseous skeleton [ 78 - 80 ]. The current evidence linking both of these diseases is far from conclusive and additional research is necessary to further characterize the relationship between these two common illnesses as our population of baby (senior) boomers are aging in large numbers. One such exciting and current emerging area in vascular biology involves the RANKL/RANK/OPG system, which belong to the tumor necrosis factor-related family recently discovered to be critical regulators of the immune system and skeletal biology. RANK(L) (receptor activator of nuclear factor kappaB (ligand) may promote and osteoprotegerin (OPG) may protect against vascular calcification (table 5 ). The mouse OPG knockout model displays osteroporosis and increased vascular calcification and human gene polymorphisms of OPG are currently being defined and how they may confer an increased risk of CAD in Caucasian men while interacting with VOC and osteoporosis [ 81 ]. The finding that OPG levels may reflect endothelial cell dysfunction is certainly an area under serious consideration [ 76 , 82 ]. CPLX and T2DM The diabetic patient is already plagued with an underlying accelerated atherosclerosis (atheroscleropathy (ASO)), as well as, medial fibrosis and VOC (even if they do not develop CKD or ESRD requiring dialysis). With the current epidemic of obesity, metS, insulin resistance related T2DM (diabesity) in all age groups and particularly the aged and adolescent youth, a closer examination of this morbid – mortal complication is in order [ 83 - 87 ]. If these T2DM patients do develop CKD and/or ESRD requiring dialysis they could be at a much higher risk of developing an even more severe form of VOC. This dual effect of having not only T2DM related medial calcification but also having the increased risk for the development of medial remodeling and VOC from CKD and/or ESRD requiring dialysis would place these individuals at an extremely high risk for the development of excessive medial arterial remodeling and VOC, as well as arteriole remodeling and VOC, which could possibly increase the risk of developing CPLX (figure 2 , 7 ). Many T2DM patients may have medial arteriolar calcification that could be contributing to anunderlying skin necrosis currently being attributed singularly to peripheral arterial disease and ischemic necrosis or gangrene, when if fact they might have coexisting arteriolar calcification contributing to their skin changes of gangrene. This dual mechanism of VOC in the arterioles and conduit arteries could be playing an important role in development of CPLX. The treatments to date have largely consisted of careful wound management, hyperbaric oxygen therapy [ 88 ], and specific treatment of wound infections, which frequently become systemic and result in deadly sepsis, organ failure, and patient demise. These open ulcerations of the skin are extremely difficult to heal and while they remain open they place the patient at an extreme risk of infection, as this patient population is known to have an impaired immune system from the combination of dialysis and T2DM [ 89 ]. If STS does emerge as a treatment option for those with CPLX then a careful evaluation of the T2DM patient with skin ulceration should be explored and consideration given to the use of STS provided there is evidence of an associated arteriolar calcification in addition too medial calcification of the conduit arteries. The dual effect of STS being an antioxidant and a chelator of calcium could have a positive outcome in promoting healing of skin ulcerations, thus reducing the possibility of systemic infection and furthermore result in limb salvage of these high risk patients who seem to be presenting to dialysis clinics more and more as the obesity – T2DM epidemic continues to progress. Additionally, the use of STS may be considered in the treatment of VOC associated with CHD and peripheral arterial disease independent of CKD and dialysis. Conclusion This review has been an attempt to increase our basic understanding of VOC and its mechanisms in the patient with metS, T2DM, CKD, CHD, and the syndrome of CPLX. By improving our understanding of the mechanisms in play we may be better equipped to prevent many of the complications associated with VOC. VOC is an exciting and complicated field of study and while our understanding of this process has undergone considerable improvement regarding its mechanisms and understanding during the past few years, there remain many questions to be solved and newer therapies proposed to help decrease the morbid – mortal complications of this actively regulated process. The recent publications of case reports regarding the use of STS to treat the complications of VOC and the syndrome of CPLX may result in the necessary clinical trials and the important scientific animal experiments to fully evaluate and understand its effect. Additionally these early published clinical results should instigate the necessary studies to better understand the mechanisms of this emerging treatment for calciphylaxis and its possible role in treating VOC in patients with CHD, atherosclerosis and atheroscleropathy in metS, and T2DM. Additionally, this emerging treatment may have significant applications for diabetic patients with CKD, ESRD. and VOC. Earlier diagnosis of the patient at risk for the development of VOC is important so that a global risk reduction program can be initiated in order to control for the multiple metabolic toxicities by applying the therapeutic RAAS acronym (table 4 ) for the treatment and prevention of complications [ 90 ]. While this remains a daunting task for clinicians it can be undertaken by a team effort approach involving our multiple resources and sub-specialties in medicine. Competing Interests The author(s) declare that they have no competing interests. Authors' Contributions MRH conceived the idea to write this manuscript. MRH, SCT, LGK, JRS, and RK wrote, and edited this manuscript jointly. List Of Abbreviations ASO – atheroscleropathy AVW – arterial vessel wall CHD – coronary heart disease CKD – chronic kidney disease CPLX – calciphylaxis CRP – C-reactive protein CUA – calcific uremic arteriolopathy CVD – cardiovascular disease eNO – endothelial nitric oxide eNOS – endothelial nitric oxide synthase ESRD – end stage renal disease GLA – gammacarboxyglutamate Hcy – homocysteine HDL-C – high density lipoprotein cholesterol hsCRP – highly sensitive C-reactive protein Htn – hypertension IGF-1 – insulin like growth factor-1 IL-6(10) – interleukin 6 and 10 LDL-C – low density lipoprotein cholesterol metS – metabolic syndrome MGP – Matrix gammacarboxyglutamate (Gla)-protein NFkappaB – nuclear factor kappa B OPG – osteoprotegerin PTH – parathyroid hormone RANK – receptor activator of nuclear factor kappaB RANKL – receptor activator of nuclear factor kappaB ligand ROS – reactive oxygen species SUA – serum uric acid TC – total cholesterol T1DM T2DM – type (1) (2) diabetes mellitus VLDL-C – very low density lipoprotein cholesterol VOC – vascular ossification – calcification VSMC – vascular smooth muscle cell Vv – vasa vasorum

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1079906

Differential expression of CCN-family members in primary human bone marrow-derived mesenchymal stem cells during osteogenic, chondrogenic and adipogenic differentiation

Background The human cysteine rich protein 61 (CYR61, CCN1) as well as the other members of the CCN family of genes play important roles in cellular processes such as proliferation, adhesion, migration and survival. These cellular events are of special importance within the complex cellular interactions ongoing in bone remodeling. Previously, we analyzed the role of CYR61/CCN1 as an extracellular signaling molecule in human osteoblasts. Since mesenchymal stem cells of bone marrow are important progenitors for various differentiation pathways in bone and possess increasing potential for regenerative medicine, here we aimed to analyze the expression of CCN family members in bone marrow-derived human mesenchymal stem cells and along the osteogenic, the adipogenic and the chondrogenic differentiation. Results Primary cultures of human mesenchymal stem cells were obtained from the femoral head of patients undergoing total hip arthroplasty. Differentiation into adipocytes and osteoblasts was done in monolayer culture, differentiation into chondrocytes was induced in high density cell pellet cultures. For either pathway, established differentiation markers and CCN-members were analyzed at the mRNA level by RT-PCR and the CYR61/CCN1 protein was analyzed by immunocytochemistry. RT-PCR and histochemical analysis revealed the appropriate phenotype of differentiated cells (Alizarin-red S, Oil Red O, Alcian blue, alkaline phosphatase; osteocalcin, collagen types I, II, IX, X, cbfa1, PPARγ, aggrecan). Mesenchymal stem cells expressed CYR61/CCN1, CTGF/CCN2, CTGF-L/WISP2/CCN5 and WISP3/CCN6. The CYR61/CCN1 expression decreased markedly during osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation. These results were confirmed by immuncytochemical analyses. WISP2/CCN5 RNA expression declined during adipogenic differentiation and WISP3/CCN6 RNA expression was markedly reduced in chondrogenic differentiation. Conclusion The decrease in CYR61/CCN1 expression during the differentiation pathways of mesenchymal stem cells into osteoblasts, adipocytes and chondrocytes suggests a specific role of CYR61/CCN1 for maintenance of the stem cell phenotype. The differential expression of CTGF/CCN2, WISP2/CCN5, WISP3/CCN6 and mainly CYR61/CCN1 indicates, that these members of the CCN-family might be important regulators for bone marrow-derived mesenchymal stem cells in the regulation of proliferation and initiation of specific differentiation pathways.

Background Members of the cysteine-rich61/connective tissue growth factor/nephroblastoma overexpressed (CCN 1–3) family of genes (CCN-family) function in processes such as proliferation, differentiation as well as cell adhesion, migration and survival [ 1 - 3 ]. Additional members of this family are Elm1/WISP1/CCN4, rCop1/WISP2/CTGF-L/CCN5 and WISP3/CCN6 [ 1 , 2 , 4 ]. The proteins mainly represent matrix associated signal molecules and share a common modular structure [ 5 , 6 ]. N-terminal amino acids have been shown to be important for secretion of CYR61/CCN1 [ 7 ]. Although CCN proteins share an insulin-like growth factor binding protein (IGFBP)-like motif, no clear experimental significance exists to suggest a function in the IGF signaling pathway [ 8 ]. The von Willebrand type C domain (VWC), the trombospondin type I domain and the C-terminal module (which is absent in CTGF-L/WISP2) are considered to be important for protein-protein interactions, either oligomerisation (VWC) or interactions with extracellular matrix molecules and receptors. Interaction partners of CCN proteins include integrin receptors [ 9 - 15 ], surface heparan sulfate proteoglycans (CYR61) [ 11 ], decorin and biglycan (WISP1) [ 16 ], and fibulin 1C (NOVH) [ 17 ]. Additional binding partners are likely to exist since interactions of CCN proteins in additional signal transduction pathways such as BMP and TGF-β signaling for CTGF/CCN2 [ 18 ] and CYR61/CCN1, intracellular calcium signaling (NOV/CCN3) [ 19 ], the notch pathway (NOV) [ 20 ] and the Wnt pathway (CYR61/CCN1 have been described [ 21 ]. An important target for CCN proteins could be bone and cartilage since the expression of CCN members in chondrocytes and osteoblasts is known from animal models and human tissues, and the majority of the above mentioned receptors and pathways are also relevant to skeletal homeostasis. CYR61/CCN1 is involved in chondrogenesis in mice [ 22 ], is expressed in human bone at sites of bone remodeling, in hypertrophic chondrocytes at the growth plate [ 23 ], and in the fracture callus in rats [ 24 ]. In human osteoblasts CYR61/CCN1 expression is regulated by a variety of bone-relevant growth factors [ 25 ]. CTGF/CCN2 is also expressed in bone and cartilage [ 8 , 26 ]. The role in skeleletal homeostasis and cartilage development is strengthened by a mouse model with functional inactivation [ 27 ]. CCN5 has been implicated in osteoblast and chondrocyte function [ 28 ]. CCN3 also is expressed in chondrocytes and could play a role in chondrocyte differentiation [ 3 , 6 ]. Mutations in the CCN6 gene are known to be associated with pseudorheumatoid dysplasia [ 29 ]. Therefore, CCN proteins are relevant for skeletal growth and development, e. g. bone and cartilage formation and function and some of the CCN proteins could as well be important in fracture repair and bone remodeling. Bone marrow-derived mesenchymal stem cells (MSC) are versatile cells which can differentiate into various cell types including osteoblasts, chondrocytes and adipocytes. MSC express markers of additional cell types, depending on the environment or cell culture conditions [ 30 - 36 ]. Therefore, this in vitro differentiation system of osteogenic, adipogenic and chondrogenic differentiation allows to investigate for genes which are temporally expressed during differentiation pathways. Here we investigated the expression patterns of CCN family members during these differentiation pathways using human bone marrow-derived MSC. The results indicate that the expression of CCN-family members is dependent on the differentiation status of human MSC in vitro . Results Lineage-specific differentiation of bone marrow-derived MSC MSC obtained from 4 different patients each were used throughout the individual differentiation pathways for this study. As was described previously [ 36 ] monolayer cultures treated with osteogenic supplements showed a high percentage of alkaline-positive cells, stained positive for mineralized matrix deposition and expressed the osteogenic specific marker genes alkaline phosphatase and osteocalcin (Fig. 1 ). Monolayer cell cultures treated with adipogenic supplements showed cytoplasmic lipid droplets and expressed LPL and PPARγ2, characteristic for adipocytes (Fig. 2 ). High density pellet cell cultures were composed of morpholocically distinct, chondrocyte-like cells, surrounded by a sulfated proteoglycan-rich extracellular matrix and expressed collagen type II (Fig. 3 ). Figure 1 Marker analysis of osteoblasts derived from MSC . Human bone marrow-derived MSC were differentiated into osteoblasts according to the methods section. were compared A) RT-PCR results for alkaline phosphatase and osteocalcin, B) Alizarin Red staining of calcium phosphate deposits in differentiated osteoblasts, C and D) Alkaline phosphatase staining of MSC and osteoblasts after 4 weeks of differentiation, respectively, (scale bar = 100 μm). Figure 2 Marker analysis of adipocytes derived from MSC . Human bone marrow-derived MSC were differentiated into adipocytes according to the methods section. A) RT-PCR results for lipoprotein lipase (LPL) and peroxisome proliferators-activated receptor γ2 (PPARγ2), B) staining for lipid droplets (Oil-Red O), (scale bar = 100 μm). Figure 3 Marker analysis of chondrocytes derived from MSC . Human bone marrow-derived MSC were differentiated into chondrocytes according to the methods section. A) RT-PCR analyses for chondrogenic markes as is indicated, B) H & E-staining, C) Alcian blue staining (H & E-counterstaining, D) example of negative control for immunohistochemical analyses (DAPI-counterstained), E) immunohiostochemical staining for collagen type II, F) immunohiostochemical staining for chondroitin sulphate 4, G) immunohiostochemical staining for chondroitin sulphate 6, (scale bar = 100 μm). Expression of CCN family members in undifferentiated MSC Bone marrow-derived MSC prior to the initiation of the various differentiation pathways expressed CYR61/CCN1, CTGF/CCN2, CCN5 and CCN6 genes by RT-PCR analysis at high levels (Fig 4 , 6 and 7 , left lane each). No signals were obtained for CCN3 and for CCN4 expression in undifferentiated MSC as well as during differentiation pathways derived thereof, even after variations of the RT-PCR procedure. Further analyses, therefore, focused on CYR61/CCN1, CTGF/CCN2, WISP2/CCN5 and WISP3/CCN6 (see below). Figure 4 RT-PCR analysis of CCN family members during osteogenic differentiation . Human bone marrow-derived MSC were differentiated into osteoblasts according to the methods section. Total RNA was isolated and reverse transcribed from undifferentiated cells and differentiated cells after 4 weeks. PCR products for CCN family members as indicated were subjected to agarose gel electrophoresis. Figure 6 RT-PCR analysis of CCN family members during adipogenic differentiation . Human bone marrow-derived MSC were differentiated into adipocytes according to the methods section. Total RNA was isolated and reverse transcribed from undifferentiated cells and differentiated cells after 1 and 2 weeks. PCRproducts for CCN family members as indicated were subjected to agarose gel electrophoresis. Figure 7 RT-PCR analysis of CCN family members during chondrogenic differentiation . Human bone marrow-derived MSC were differentiated into chondrocytes for 3 weeks in high-density cell pellets according to the methods section. Total RNA was isolated and reverse transcribed from undifferentiated cells and differentiated cells after 1, 2 and 3 weeks. PCR products for CCN family members as indicated were subjected to agarose gel electrophoresis. Differential expression of CYR61/CCN1 during osteogenic differentiation A marked decrease in the expression of CYR61/CCN1 was observed during osteogenic differentiation. At the mRNA level RT-PCR analysis revealed a 6.9 ± 2.2-fold decrease (n = 4) in CYR61/CCN1 levels after 4 weeks of osteogenic differentiation, compared to undifferentiated MSC (Fig. 4 ). RT-PCR analysis for other CCN-members in this study did not display changes in PCR product intensity during osteogenic differentiation. At the protein level, immunocytochemistry showed a decrease in CYR61/CCN1 signal from differentiated cells compared to undifferentiated MSC (Fig. 5 ). Figure 5 Expression of the CYR61 protein during osteogenic differentiation . Human bone marrow-derived MSC were differentiated into cells of the osteogenic lineage. Undifferentiated cells and differentiated cells after 4 weeks were fixed and subjected to immunohistochemistry as outlined in the methods section (scale bar = 100 μm). Differential expression of CYR61/CCN1, CTGF/CCN2 and WISP2/CCN5 during adipogenic differentiation RT-PCR analysis of the CCN family members during adipogenic differentiation is shown in Fig 6 . CYR61/CCN1 and WISP2/CCN5 were expressed prior to the initiation of adipogenesis and in initial stages of the differentiation pathway. However, in differentiated adipocytes after 2 weeks of differentiation almost undetectable levels were observed (n = 4). Due to the low expression levels densitometry was not possible. RT-PCR for CTGF/CCN2 revealed a slight reduction of the PCR product levels towards differentiated adipocytes (2.1 ± 0.8-fold). WISP3/CCN6 PCR product intensity did not change during adipogenic differentiation. Differential expression of CYR61/CCN1 and WISP3/CCN6 during chondrogenic differentiation In high-density cell pellets prior to the initiation of chondrogenic differentiation RT-PCR analysis for CYR61/CCN1 and WISP3/CCN6 revealed high levels of RNA expression. During differentiation after one week signals for CYR61/CCN1dropped to undetectable levels. (Fig. 7 ). Therefore, densitometry for CYR61/CCN1 RT-PCR products after differentiation was not possible. PCR product intensity for WISP3/CCN6 was reduced 5.5 ± 0.6-fold (n = 4). RT-PCR for CTGF/CCN2 revealed a slight reduction during chondrogenic differentiation of 1.9 ± 0.6-fold. WISP2/CCN5 PCR products did not reveal differences in intensity along the chondrogenic differentiation of MSC. Immunohistochemistry of slides cut through the center of the pellets indicated high levels of CYR61/CCN1 protein prior to the initiation of differentiation. After three weeks of chondrogenic differentiation a reduced signal intensity at the periphery of the pellet was observed (Fig. 8 ). Figure 8 Expression of CYR61 protein in high-density pellets during chondrogenic differentiation . Human bone marrow-derived MSC were differentiated into chondrocytes in high-density cell pellets. Slides from pellets of the undifferentiated cells and differentiated cells after 3 weeks were cut through the center of the pellet, fixed and subjected to immunohistochemistry as outlined in the methods section. (scale bar = 100 μm). Discussion CCN proteins play important roles in growth and differentiation and are involved in cellular processes such as migration, adhesion and survival [ 1 , 2 , 6 ]. A major role for CYR61/CCN1 and CTGF/CCN2 in bone and cartilage development is indicated by animal studies [ 6 , 22 , 27 , 37 , 38 ]. In the adult both CCN-family members have been associated with remodeling and repair in tissues such as bone and cartilage, muscle, the vascular system and the nervous system as reviewed in [ 2 , 3 , 39 , 40 ]. The culture of human bone marrow-derived MSC applied in this study allows the analysis of CCN protein expression and function during differentiation pathways. Particularly, we investigated the expression of CCN family members during the differentiation of human bone marrow-derived MSC into osteoblasts, adipocytes and chondrocytes. Results showed that CYR61/CCN1 in all pathways, and CTGF/CCN2, WISP2/CTGF-L/CCN5 and WISP3/CCN6 in specific pathways were differentially expressed and therefore could participate in MSC function and lineage progression. Quantitative measurements of mRNA levels have not been performed in this study. This limits the possibility to detect subtle differences in CCN mRNA expression. However, the reproducibly observed differences for CYR61/CCN1, WISP2/CCN5 and WISP3/CCN6 expression along differentiation pathways were sufficiently intense to be detected by the conventional RT-PCR procedure applied. Except for NOV/CCN3 and WISP1/CCN4, all CCN family members investigated were expressed in undifferentiated MSC and/or along differentiation pathways derived thereof. Expression of lineage-specific cell markers at the RT-PCR level as well as histochemical analysis for mineralized matrix deposition, lipid droplets and cartilage-specific extracellular matrix revealed that the appropriate conditions for differentiation of MSC were applied. Whereas NOV/CCN3 expression has been described in the murine growth plate and murine chondrosarcomas [ 41 ], our failure to detect NOC/CCN3 along chondrogenic differentiation could rely on species and/or cell specific differences, however, we cannot exclude low expression levels below the detection limit of the conventional RT-PCR procedure. We never observed an increase of PCR product intensity in RT-PCR of any CCN family member during lineage-specific differentiation. Therefore, corresponding steady state mRNA levels of CCN members were highest in undifferentiated MSC compared to differentiated cell types. This might indicate specific functions of CCN proteins in the precursor cells prior to the onset of differentiation pathways. These functions could be associated with the known positive growth regulation capabilities of CYR61/CCN1, CTGF/CCN2 and WISP2/CCN5. Particularly, CYR61/CCN1 could play a significant function in MSC in vitro since its expression was largely decreased during all differentiation pathways under study. The decreased PCR product intensity in RT-PCR analysis during differentiation, however, does not prove the absence of particular functions in differentiated cells. The protein can be stably bound within the extracellular matrix. The results of immunohistochemistry for CYR61/CCN1 expression after osteogenic differentiation indicated lower but still detectable signals and in high-density cell pellet cultures used for chondrogenic differentiation a reduced but significant protein expression was observed. Despite lower expression of CYR61/CCN1 in differentiated cells, the mRNA and protein levels can be upregulated by various bone relevant growth factors [ 25 ]. Our finding of undetectable CYR61/CCN1 expression in MSC-derived chondroctes is in contrast to reports describing the CYR61-dependent regulation of chondrogenesis in mouse limb bud mesenchymal stem cells [ 22 ] and its expression in chondrocytes during fracture repair in rats [ 24 ]. This difference could result from species and/or cell specific differences or depend on the detection limit as is discussed above. CTGF/CCN2 was constitutively expressed at the RT-PCR level during osteogenic differentiation and only slightly reduced during adipogenic and chondrogenic differentiation. CTGF/CCN2 is able to stimulate the expression of markers of differentiated osteoblasts and chondrocytes in vitro [ 8 , 26 , 42 ]. WISP2/CCN5 expression was only decreased during adipogenic differentiation in a pattern that paralleled CYR61/CCN1 expression, but was constitutively expressed in the other differentiation pathways. WISP3/CCN6 expression was downregulated during chondrogenic differentiation. Since mutations in the WISP3/CCN6 gene result in pseudorheumatoid dysplasia [ 29 ] an important role in cartilage formation and/or initiation of chondrogenesis is possible. With regard to the molecular signalling mechanisms, the CCN family members act in a variety of signalling pathways such as notch, BMP, Wnt and intracellular calcium signalling [ 18 - 21 ] as is reviewed in [ 2 , 3 ]. Several of these identified pathways are relevant for differentiation lineages and cell fate decisions. Particularly the notch, BMP and Wnt signaling pathways are relevant for the developing skeleton. The adhesive signalling capabilities, mainly studied for CYR61/CCN1 are of special relevance for tissue homeostasis in the bone and cartilage microenvironment [ 11 , 13 , 43 ]. Mainly CYR61/CCN1 [ 22 ] and CTGF [ 27 , 44 - 46 ] appear to regulate bone and cartilage formation in embryonic development. Tissue repair in the adult could comprehense cellular processes occurring in the developmental stage. Thus, the growing importance of MSC for targeted tissue repair could, in part, rely on CCN-protein function. The differential expression of CCN family members, particularly CYR61/CCN1 mainly in MSC compared to differentiated cells could indicate a functional importance of these proteins for tissue engineering purposes. Conclusion The high expression of CCN family members in MSC and the sharp decline in RNA expression levels during differentiation of MSC into osteoblasts, adipocytes and chondrocytes, particularly for CYR61/CCN1 but also for CTGF/CCN2, WISP2/CCN5, WISP3/CCN6 in selected pathways, could indicate that these members of the CCN-family might be important regulators for bone marrow-derived MSC. MSC possess a growing importance in targeted tissue repair in the adult. Repair processes in the adult could involve cellular events occuring during development. Since the CCN family members (mainly CYR61/CCN1 and CTGF/CCN2) play important roles in the developing skeleton and in angiogenesis, these factors likely could play a role in targeted tissue repair and could improve tissue engineering strategies (Fig. 9 ). Figure 9 CCN proteins could improve tissue engineering strategies . Human bone marrow-derived MSC express and secrete CCN family proteins. Mainly CCN 1, 2, 5 and 6 are expressed in undifferentiated cells. Particularly CYR61/CCN1 and CTGF/CCN2 are important regulators in the developing skeleton and represent angiogenic modulators. Targeted tissue repair in the adult could, in part, rely on this dual role of CCN proteins allowing for improvement of tissue engineering strategies. Methods Materials FCS was supplied by Gibco (Eggenstein, Germany). Taq Polymerase was purchased from Amersham Pharmacia (Freiburg, Germany). An anti mouse CYR61 polyclonal antiserum was obtained from Munin Corp. (Chicago, USA). All other chemicals were of the highest purity available. Isolation of MSC MSC were isolated from human bone marrow obtained from the femoral head of patients undergoing total hip arthroplasty using a modified protocol [ 36 ] originally described by Haynesworth et al. [ 31 ]. Usage of patient material was approved by the local ethics commitee of the University of Würzburg and written consent was obtained from each patient. MSC from 4 patients were used for this study (3 male, 1 female), age between 51 and 62 years. Trabecular bone plugs were harvested from the cutting plane of the femoral head and transferred to 50 ml conical tubes containing DMEM/F12 medium (PAA, Cölbe, Germany). After vortexing and centrifugation (1000 rpm, 5 min) the pellet containing bone plugs and released cells was reconstituted in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS), antibiotics (50 I.U. penicillin/ml and 50 μg streptomycin/ml) and 50 μg/ml ascorbate (complete medium). After repeated washings, the released cells were pelleted (1000 rpm, 5 min), suspended in complete medium, plated at a density of 60 × 10 6 cells per 150 cm 2 tissue culture flask and maintained at 37°C in 5% CO 2 . Non adherent cells were removed after 2 days and subsequently the medium was changed every 2 days until the cell cultures reached confluency. Osteogenic differentiation of MSC Cells were plated in 6-well plates or chamber slides in complete medium. At confluency osteogenic differentiation was initiated using the complete medium supplemented with 10 mM β-glycerophosphate and 50 μg/ml ascorbate. Medium was changed every 2 days up to 4 weeks. To monitor osteogenic differentiation RT-PCR analysis for alkaline phosphatase, osteopontin and osteocalcin was performed as well as stainings for alkaline phosphatase (Sigma, Taufkirchen, Germany, Kit No. 86) and calcium phosphate deposition (Alizarin Red S) were performed as described by Nöth et al. [ 36 ]. Adipogenic differentiation of MSC Cells were plated in 6-well plates or chamber slides in DMEM with 10 % FBS. At confluency, adipogenic differentiation was initiated using the same medium supplemented with 1 μM dexamethasone, 0.5 mM isobutylmethylxanthine, 1 μg/ml insulin and 100 μM indomethacin. Medium was changed every 2 days up to 2 weeks. To monitor adipogenic differentiation RT-PCR analysis for lipoprotein lipase (LPL) and peroxisome proliferator-activated receptor γ2 (PPARγ2) was performed, and a staining for lipid droplets (Oil Red O) as described [ 33 , 36 ]. Chondrogenic differentiation of MSC Cells were cultured as high-density pellet cultures in a serum-free medium as described previously [ 32 , 33 ]. 200, 000 cells were centrifuged for 5 min and 1000 rpm in 15 ml conical tubes and the pellets cultured for 3 weeks at 37°C in 5 % CO 2 . The chondrogenic medium consisted of DMEM supplemented with 10 ng/ml transforming growth factor β1 (Stathmann, Hamburg, Germany), 100 nM dexamethasone, 50 μg/ml ascorbic 2-phosphate, 100 μg/ml sodium pyruvate, 40 μg/ml proline and ITS-plus (consisting of 6.25 μg/ml bovine insulin, 6.25 μg/ml transferrin, 6.25 μg/ml selenous acid, 5.33 μg/ml linoleic acid and 1.25 mg/ml bovine serum albumin; Sigma, Taufkirchen, Germany). Medium was changed twice per week. To monitor chondrogenic differentiation RT-PCR analysis for aggrecan and collagen types I, II, IX and X was performed. Additionally, sections were cut through the center of the pellets and stained with alcian blue and were subjected to immunohistochemical analyses for collagen type II, chondroitin sulphate 4 and 6 as described by Nöth et al. [ 36 ]. RNA Isolation and RT-PCR RNA isolates were obtained from cells within the differentiation pathways as well as control isolates. Controls for monolayer differentiation (i.e. into adipocytes and osteoblasts) represent confluent resting MSC at day 0 prior to the initiation of cell differentiation. Control RNA isolates for the chondrogenic differentiation pathway were done from pellets prior to the initiation of chondrogenic differentiation at day 0. RNA isolation was performed using purification columns (Qiagen, Hilden, Germany) according to the manufacturer. Cells were rinsed with PBS, lysis buffer was added, RNA was column purified and the yield was determined by spectrophotometry. cDNA synthesis was done in a 20 μl reaction using Super Script II Reverse Transcriptase (Invitrogen, Karlsruhe, Germany) with identical amounts of RNA isolated from individual differentiation pathways of MSC (1 μg). PCR was performed using a PTC-200 thermal cycler (MJ Research) in a volume of 30 μl containing 1 μl cDNA with primers (Table 1 ) for CCN family members as well as the housekeeping gene actin. Primers used for cell markers are presented in Table 2 . The PCR buffer consisted of 10 mM Tris (pH 8.4), 50 mM KCl, 1.5 mM MgCl 2 , 0.25 mM dNTP's, 5 pmol forward and reverse primer, and 0.5 units of Taq polymerase. Cycling was 4 min at 94°C, 25 – 40 cycles (25 cycles: actin and E1α, 30 – 37 cycles for CCN members) of 94°C for 30 s, 58 – 64°C for 1 min (depending on the primers) and 72°C for 1 min, with a final 72°C step of 5 min. Sequence analysis was performed by dye terminator sequencing of the PCR products to verify specificity using an ABI 310 Sequencer. 10 μl of PCR products were separated on a 2% agarose gel containing ethidium bromide. In cases of differential PCR product intensities densitometry was performed using an LTF densitometer and the bioprofile software (LTF, Wasserburg, Germany) Table 1 Primers used for RT-PCR gene product primer sequence # in database entry CYR61/CCN1 sense 5'ACCAGTCAGGTTTACTTACG 3' 962 – 981 NM_001554 antisense 5'TGCCTCTCACAGACACTCAT 3' 1679 – 1698 NM_001554 CTGF/CCN2 sense 5'AACACCATAGGTAGAATGTAAAGC 3' 1921 – 1944 NM_001901 antisense 5'CTGATCAGCTATATAGAGTCACTC 3' 2130 – 2153 NM_001901 WISP2/CCN5 sense 5'CACGCATAGGCTTGTATTCAGGAAC 3' 1052 – 1075 NM_003881 antisense 5'CACGCTGCCTGGTCTGTCTGGATC 3' 1379 – 1403 NM_003881 WIS32/CCN6 sense 5'CTGTGTTACATTCAGCCTTGCGAC 3' 846 – 869 NM_003880 antisense 5'CTTGGTTTTACAGAATCTTGAGCTC 3' 1158 – 1182 NM_003880 actin sense 5'CGGGAAATCGTGCGTGACAT 3' 689 – 708 NM_001101 antisense 5'GAACTTTGGGGGATGCTCGC 3' 1381 – 1400 NM_001101 Table 2 Primers used for RT-PCR of markers for MSC-derived differentiation pathways Alkaline phosphatase sense 5'TGGAGCTTCAGAAGCTCAACACCA 3' 368 – 391 NM_000478 antisense 5'ATCTCGTTGTCTGAGTACCAGTCC 3' 798 – 821 NM_000478 Osteocalcin sense 5'ATGAGAGCCCTCACACTCCTC 3' 19 – 39 NM_199173 antisense 5' GCCGTAGAAGCGCCGATAGGC 3' 292 – 312 NM_199173 Lipoprotein lipase sense 5'GAGATTTCTCTGTATGGCACC 3' 1261 – 1281 NM_000237 antisense 5'CTGCAAATGAGACACTTTCTC 3' 1516 – 1536 NM_000237 PPARγ2 sense 5'GCTGTTATGGGTGAAACTCTG 3' 128 – 148 NM_015869 antisense 5'ATAAGGTGGAGATGCAGGCTC 3' 458 – 478 NM_015869 Collagen type II sense 5'GAACATCACCTACCACTGCAAG 3' 4318 – 4339 NM_001844 antisense 5'GCAGAGTCCTAGAGTGACTGAG 3' 4684 – 4705 NM_001844 Collagen type X sense 5'CCCTTTTTGCTGCTAGTATCC 3' 112 – 132 NM_000493 antisense 5'CTGTTGTCCAGGTTTTCCTGGCAC 3' 556 – 579 NM_000493 Collagen type XI sense 5'ACTTCTGACTGCCTCTGCTC 3' 5418 – 5437 NM_001854 antisense 5'GCTTTTGCCATGTGATTCTGCC 3' 5891 – 5912 NM_001854 Aggrecan sense 5'GCCTTGAGCAGTTCACCTTC 3' 1814 – 1833 NM_001135 antisense 5'CTCTTCTACGGGGACAGCAG 3' 2186 – 2205 NM_001135 Chondroadherin sense 5'ACCTGGACCACAACAAGGTC 3' 535 – 554 NM_001267 antisense 5'CACCTTCTCCAGGTTGGTGT 3' 907 – 923 NM_001267 Fibromodulin sense 5'CTTACCCCTATGGGGTGGAT 3' 175 – 194 NM_002023 antisense 5'AAGTAGCTATCGGGGACGGT 3' 792 – 811 NM_002023 Immunohistochemistry MSC undergoing osteogenic differentiation were subjected to immunohistochemical analysis for CYR61 expression. Cells were rinsed with PBS, covered with a glass cover slip and analyzed immediately. As the primary antibody an anti mouse CYR61 polyclonal antiserum (Munin, Chicago, USA) at a 1:100 dilution in PBS was used and the slides were incubated at 4°C overnight. The further steps were carried out at room temperature. Following three 5 min washes with TBS a monoclonal mouse anti rabbit IgG (DAKO, Hamburg, Germany) antibody at a 1:50 dilution in a solution consisting of 100 μl human AB plasma in 700 μl PBS was added and incubated for 30 min. After rinsing with PBS a rabbit anti mouse IgG antiserum (DAKO, Hamburg, Germany) diluted 1:25 was added and incubated for 10 min. After rinsing in PBS cells were incubated with a complex of intestinal alkaline phosphatase and mouse monoclonal anti alkaline phosphatase antiserum (APAAP) (DAKO, Hamburg, Germany) at a 1:50 dilution for 10 min. The final signal intensity was amplified using 2 additional cycles of incubations with the rabbit anti mouse IgG antiserum and the APAAP complex. Signals were developed using a conventional freshly prepared fast-red staining solution (40 mg fast red, 18 mg levamisole and 20 mg naphtol-AS-MX-phosphate, 1 ml DMF and 40 ml propanediol-buffer consisting of 50 mM 2-amino-2 methyl 1,3 propanediol pH = 8.7). Following a 10 min incubation, sections were washed in ddH 2 O and prepared for microscopy. During every analysis a negative control was performed according to Wong et al. [ 22 ]. Sections were incubated using a rabbit serum instead of the primary antibody (rabbit anti CYR61 antiserum) at a similar protein concentration. Microscopic images were acquired using a digital camera and IP-Lab-Spectrum analysis software. Immunohistochemical analyses for markers of chondrogenic differentiation were performed using the antisera. Immunoreactivity was detected using a streptavidin-peroxidase staining as described [ 36 ]. Competing interests The author(s) declare that they have no competing interests. Authors' contributions NS designed and interpreted the experiments, as well prepared the manuscript. UN and CH selected the patients for MSC cell culture, JS isolated the MSC and FJ provided overall guidance.

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1079907

Differential expression of copper-associated and oxidative stress related proteins in a new variant of copper toxicosis in Doberman pinschers

Background The role of copper accumulation in the onset of hepatitis is still unclear. Therefore, we investigated a spontaneous disease model of primary copper-toxicosis in Doberman pinschers so to gain insights into the pathophysiology of copper toxicosis, namely on genes involved in copper metabolism and reactive oxygen species (ROS) defences. Results We used quantitative real-time PCR to determine differentially expressed genes within a target panel, investigating different groups ranging from copper-associated subclinical hepatitis (CASH) to a clinical chronic hepatitis with high hepatic copper concentrations (Doberman hepatitis, DH). Furthermore, a non-copper associated subclinical hepatitis group (N-CASH) with normal hepatic copper concentrations was added as a control. Most mRNA levels of proteins involved in copper binding, transport, and excretion were around control values in the N-CASH and CASH group. In contrast, many of these (including ATP7A, ATP7B, ceruloplasmin, and metallothionein) were significantly reduced in the DH group. Measurements on defences against oxidative stress showed a decrease in gene-expression of superoxide dismutase 1 and catalase in both groups with high copper. Moreover, the anti-oxidative glutathione molecule was clearly reduced in the DH group. Conclusion In the DH group the expression of gene products involved in copper efflux was significantly reduced, which might explain the high hepatic copper levels in this disease. ROS defences were most likely impaired in the CASH and DH group. Overall, this study describes a new variant of primary copper toxicosis and could provide a molecular basis for equating future treatments in dog and in man.

Background Copper is an imperative molecule in life; in contradiction, however, it is highly toxic [ 1 ]. Like zinc, iron, and selenium, copper is an essential trace element in diets and is required for the activity of a number of physiologically important enzymes [ 2 ]. Cells have highly specialized and complex systems for maintaining intracellular copper concentrations [ 3 ]. If this balance is disturbed, excess copper can induce oxidative stress that could lead to chronic inflammation [ 4 , 5 ]. Copper induced hepatitis has been described both in humans (Wilson's disease) as well as in dogs. There are several non-human models of copper toxicosis models, such as the Long-Evans Cinnamon rats and Bedlington terriers. Although the gene underlying Wilson's disease (ATP7B) is deficient in Long-Evans Cinnamon rats [ 6 - 9 ], in Bedlington terriers it has been excluded as a candidate for copper toxicosis [ 10 ]. The recent discovery of mutations in gene MURR1, responsible for copper toxicosis in Bedlington terriers, has given rise to the discovery of a new copper pathway [ 11 ]. Here, we describe in Doberman pinschers a copper associated chronic hepatitis (also called Doberman hepatitis), characterized by micro-nodular cirrhosis with elevated hepatic copper concentrations [ 12 - 15 ]. Doberman hepatitis accounts for 4 % of all deaths in a Dutch population of 340 Dobermans [ 16 ]. Until recently, the role of copper in the development and progression of hepatitis in the Doberman pinscher had been unclear. Recent studies using intravenous 64 Cu clearly show an impaired copper excretion in dogs with hepatitis and elevated copper concentrations [ 17 ]. However, genes ATP7B and MURR1 have been excluded by us as possible candidates by genotyping (data not shown). Therefore, Doberman hepatitis can be seen as a separate form of copper toxicosis and a possible model for other types of copper toxicosis in humans, such as Indian childhood cirrhosis, non-Indian childhood cirrhosis, or idiopathic copper toxicosis. Intracellular copper is always transiently associated with small copper-binding proteins (Figure 1 ), denoted copper chaperones, which distribute copper to specific intracellular destinations [ 18 ]. One of these copper chaperones is the anti-oxidant protein 1 (ATOX1) [ 19 ], which transports copper to the copper-transporting ATPases ATP7A and ATP7B [ 20 ], located in the trans-Golgi network. Copper can then be bound to liver specific ceruloplasmin (CP) [ 21 ] or MURR1 and transferred outside the cell to blood and bile, respectively [ 22 ]. The second chaperone – cytochrome c oxidase (COX17) is responsible for delivering copper to the mitochondria for incorporation into cytochrome c oxidase [ 23 ]. The third chaperone – copper chaperone for superoxide oxidase (CCS) is responsible for the incorporation of copper into Cu/Zn superoxide dismutase (SOD1) – one of the most important cytosolic enzymes in the defence against oxidative stress [ 24 , 25 ]. Also known as ferroxidase or oxygen oxidoreductase, CP is a plasma metalloprotein which is involved in peroxidation of Fe(II)transferrin to Fe(III)transferrin and forms 90 to 95 % of plasma copper. CP is synthesized in hepatocytes and is secreted into the serum with copper incorporated during biosynthesis. Metallothionein 1A (MT1A) is a small intracellular protein capable of chelating several metal ions, including copper. It contains many cysteine residues, which allow binding and storage of copper. Furthermore, MT1A is inducible, at the transcriptional level, by metals and a variety of stressors such as reactive oxygen species (ROS), hypoxia, and UV radiation [ 26 ]. MT1A can donate copper to other proteins, either following degradation in lysosomes or by exchange via glutathione (GSH) complexation [ 27 ]. Figure 1 Schematic overview of intra-cellular copper trafficking in hepatocytes. Copper uptake is mediated by the receptor CTR1. In the cell, copper can bind to copper chaperones such as CCS, COX17, and ATOX1 which in turn deploy to SOD1, the mitochondrial COX, and ATP7A/B, respectively. ATP7A can directly excrete copper or bind it to ceruloplasmin (CP). ATP7B can excrete copper through CP to blood or via MURR1 to bile. Furthermore, metallothioneins (MT) are present in the cytoplasm which can bind and sequester metals. [SCO are metallochaperone proteins with essential, but not yet fully understood, roles in copper delivery to mitochondrial COX.] High hepatic levels of copper induce oxidative stress. There are several important proteins and molecules involved in the defence against oxidative stress. Most of the anti-oxidants can be grouped into either enzymatic defences or non-enzymatic defences [ 28 ]. The enzymatic defence against oxidative stress consists of several proteins that have tight regulations such as SOD1 and catalase (CAT). Non-enzymatic defences against oxidative stress consist of molecules such as α-tocopherol, β-carotene, ascorbate, and a ubiquitous low molecular thiol component – the GSH [ 29 ]. The present study was undertaken to investigate the effect of copper toxicosis on expression of gene-products involved in copper metabolism and oxidative stress in several gradations of hepatic copper toxicosis in Doberman pinschers. Results To gain insight into the pathogenesis of copper toxicosis, we first measured mRNA levels on several important copper binding gene-products by means of quantitative real-time PCR (Q-PCR). Because copper toxicity is often associated with oxidative stress, we also measured several oxidative stress related gene-products. To determine a possible damaging effect of the oxidative stress, we investigated proteins involved in apoptosis and cell-proliferation. Gene-expression measurements on copper metabolism related gene products Several proteins in the Doberman hepatitis (DH) group are reduced compared to healthy controls (Figure 2C ). In all groups the copper chaperone ATOX1 is not affected, whereas COX17 is decreased three-fold in the DH group and remains unchanged in the non-copper associated subclinical hepatitis group (N-CASH, Figure 2A ) and copper associated subclinical hepatitis group (CASH, Figure 2B ). In the DH group, the mRNA levels of both trans-Golgi copper transporting proteins ATP7A and ATP7B are decreased, three- and two-fold respectively. Interestingly, mRNA levels of ATP7A are decreased in the CASH group as well (Figure 2B ). In contrast, ATP7B is not affected in the CASH group but is induced two-fold in the N-CASH group. CP mRNA levels are normal except for the DH group where it is decreased two-fold. The same observation was made with measurements on MT1A mRNA, although this protein is decreased four-fold in the DH group. The protein MURR1 (that transports copper from hepatocytes into bile) is unaffected in the N-CASH group but halved in the CASH and DH groups. Figure 2 Quantitative Real-Time PCR of copper metabolism related genes. mRNA levels of non-copper associated subclinical hepatitis (n = 6 dogs) is shown in (A). mRNA levels of copper associated subclinical hepatitis (n = 6 dogs) is shown in (B). mRNA levels of Doberman hepatitis (n = 6 dogs) is shown in (C). Data represent mean ± 2 SD. Gene expression measurements on oxidative stress markers SOD1 and CAT are reduced 7- and 4-fold (respectively) in the DH group when compared to healthy controls (Figure 3C ). This reduction in mRNA levels can be seen in the CASH group (Figure 3B ), where SOD1 and CAT are halved, but are not lowered significantly in the N-CASH group (Figure 3A ). One of the GSH synthesis enzymes – the glutathione synthetase (GSS) is unaffected in the N-CASH group but reduced 2 to 4-fold in the CASH and DH group, respectively. The glutathione peroxidase (GPX1) responsible for converting oxidized glutathione (GSSG) into its reduced form (GSH) is induced slightly in mRNA expression in the N-CASH group, and is doubled in the CASH and DH groups. The third copper chaperone CCS, responsible for the transport of copper to SOD1, is inhibited 8-fold in the DH group, 2-fold in the CASH group, and remained unchanged in the N-CASH group. Figure 3 Quantitative Real-Time PCR of oxidative stress markers. mRNA levels of non-copper associated subclinical hepatitis (n = 6 dogs) is shown in (A). mRNA levels of copper associated subclinical hepatitis (n = 6 dogs) is shown in (B). mRNA levels of Doberman hepatitis (n = 6 dogs) is shown in (C). Data represent mean ± 2 SD. Gene expression measurements on apoptosis and cell proliferation We measured two anti-apoptotic gene products, viz. Bcl-2, the frequently described anti-apoptotic protein, and a x-linked inhibitor of apoptosis (XIAP) recently associated with MURR1 [ 30 ]. Our apoptosis measurements on Bcl-2 showed no reduction in gene expression in the N-CASH group (Figure 4A ), but is inhibited 4-fold in the CASH and DH groups (Figures 4B and 4C , respectively). XIAP is halved in all groups. The most dramatic changes were found in the mRNA levels of the cell-cycle inhibitor p27KIP which is inhibited 24-fold in the DH group, 12-fold in the CASH group, and 3-fold in the N-CASH group. Figure 4 Quantitative Real-Time PCR of apoptosis and cell proliferation related genes. mRNA levels of non-copper associated subclinical hepatitis (n = 6 dogs) is shown in (A). mRNA levels of copper associated subclinical hepatitis (n = 6 dogs) is shown in (B). mRNA levels of Doberman hepatitis (n = 6 dogs) is shown in (C). Data represent mean ± 2 SD. Western blots analysis on metallothionein proteins during copper toxicosis Measurements on the mRNA levels of MT1A showed a marked decrease in gene expression in the DH group. In order to see whether this decrease was also occurring at the protein level, Western blots were performed in order to confirm decreased mRNA levels. Therefore, the total amount of metallothionein was determined from Doberman pinschers with chronic hepatitis and high copper (DH-group) levels compared to healthy Dobermans. Metallothionein was detected in both samples, where it was present as a single band of 6 kDa (Figure 5 ). Interestingly, the immunoreactive band shows no difference in concentration between the two samples. Figure 5 Western blot analysis of the metallothionein proteins. Immunoreactive bands of total metallothionein of pooled fractions of the Doberman hepatitis (DH) group (n = 6 dogs) versus healthy controls (n = 8 dogs). Total Glutathione measurements during copper toxicosis In order to determine whether the decrease in mRNA levels of GSS decreases the GSH levels, we measured the total amount of GSH. Interestingly, in Figure 6 , the total amount of GSH in the high copper group is halved when compared to healthy controls. Figure 6 Total glutathione (GSH) measurements during copper toxicosis in Doberman. Total GSH levels of pooled protein fractions of the Doberman hepatitis (DH) group (n = 6 dogs) versus healthy controls (n = 8 dogs). Data represent mean ± 2 SD. Discussion In the present study, the expression of a total of 15 gene products involved in copper metabolism of Doberman pinschers was measured. This provided insight into the molecular pathways of a canine copper-associated hepatic disease model ranging from subclinical hepatitis with elevated copper levels (CASH) to severe chronic hepatitis with high hepatic copper levels (DH). Furthermore, these diseases were compared to non-copper associated subclinical hepatitis (N-CASH). Because of the centrolobular accumulation of copper in the hepatocytes during copper toxicosis in the Doberman, a probable defect may be sought in the copper metabolism instead of a secondary effect due to, for instance, cholestasis. Recent findings by Mandigers et al. [ 17 ] indicated that Doberman pinschers with hepatitis and elevated copper concentrations suffer from impaired 64 Cu bile excretion which is, together with other studies, conclusive that copper toxicosis exists in the Doberman pinscher. Furthermore, a double blind placebo-controlled study with the copper chelating agent, D-penicillamine, on Doberman pinschers with CASH showed a marked improvement of liver pathology [ 31 ]; currently, that agent is the only treatment option. If copper is sequestered, in time metallothioneins will store the copper in lysosomes, as described by Klein et al . [ 32 ]. They found that chronic copper toxicity in Long-Evans Cinnamon rats involved the uptake of copper-loaded metallothioneins into lysosomes, where it was incompletely degraded and polymerized into an insoluble material, which contained reactive copper. This copper initiated a lysosomal lipid peroxidation, which led to hepatocyte necrosis. Phagocytosis of this reactive copper by Kupffer cells amplified the liver damage. Histological examination of the DH (Figure 7 ) and CASH group samples revealed copper accumulation in hepatocytes and copper-laden Kupffer cells similar to that described by Klein et al. [ 32 ]; therefore, that can be denoted as benchmarks of chronic exposure to copper. Figure 7 Histological evaluation of Doberman hepatitis. (A) Hepatitis characterised by accumulation of pigmented granules (probably copper) in hepatocytes, and inflammation with lymphocytes and pigmented (probably copper) macrophages. HE staining. (B) Centrolobular accumulation of copper in hepatocytes and band of fibrous tissue with inflammatory cells and copper-laden macrophages. Rubeanic acid staining. P = Portal area, CV = Central vein area. In our study, the gene expression levels of several gene products involved in copper metabolism seem to be reduced in the DH and CASH groups when compared to healthy controls. Short term studies on in vitro models all show an induction of MT1A or CP indicative of a higher efflux of copper from hepatocytes [ 33 , 34 ]. The reductions that are seen in our results could therefore be ascribed to the prolonged or chronic nature of copper accumulation as dogs in the high copper or DH group present clinical signs after 2 years. Therefore, our observations are not directly comparable with the short-term induced copper effects in vitro , but are clinically more relevant, showing the effects of long-term copper accumulation in Doberman hepatitis. However, Western blot experiments on metallothionein, which stores the copper in lysosomes, did not show any reduction at the protein level. This observation could be ascribed to the antibody that binds all metallothioneins, including metallothionein 2 (MT2A), which also is present in the liver. It remains to be proven if this effect is a compensation for the decrease of MT1A. In the earlier stages of copper accumulation, comparable to the CASH group, higher amounts of copper can still be excreted. Interestingly, in the N-CASH group, ATP7B is indeed induced compared to healthy controls, emphasizing a possible higher efflux of copper. Furthermore, from the two subclinical disease groups, the N-CASH group is the only one able to recuperate, whereas the CASH group will eventually turn into clinical hepatitis as seen in the DH group (data not shown). Taken together, our data suggest that in the Doberman pinchers copper accumulates in time and, finally, will have its negative effect on copper metabolism and induce oxidative stress. Oxidative stress has been ascribed to copper toxicosis as one of the most important negative effects [ 35 ]. We can confirm this with four different observations: (i) our measurements showed a decrease in mRNA levels of SOD1 and CAT, indicative of a reduction in the enzymatic defence against oxidative stress in all groups with copper accumulation; (ii) a reduction of GSS mRNA levels (glutathione synthesis), indicative for a reduced glutathione level in these groups which is one of the most important non-enzymatic molecules against oxidative stress; (iii) the mRNA levels of GPX1 were significantly increased, indicating an increase in GSH oxidation; (iv) the decrease in GSH was confirmed by measuring total glutathione levels in the DH group towards healthy Doberman pinschers. A similar decrease in expression of anti-oxidant enzymes was observed in ApoE-deficient mice in response to chronic inflammation [ 36 ], and inflammatory bowel disease (IBD) [ 37 ]. This indicates that chronic inflammation (copper toxicosis, atherosclerosis, IBD) is associated with reduced protection against enhanced exposure to ROS. Other effects of high copper can also be seen in the measurements on apoptosis and cell-cycle. Measurements on Bcl-2 and XIAP indicate a decrease of protection against apoptosis; however, the most affected hepatocytes will go into necrosis due to the formation of hydroxyl radicals by the Haber-Weiss reaction, which is catalyzed by copper [ 38 ]. A striking observation was made measuring p27KIP which was shown to be reduced up to 24-fold in the DH group. This could indicate an induction of cell-cycle compared to healthy controls. This could be ascribed to the renewal of hepatocytes, thus managing the total amount of copper in time. Whether differential gene expression is cause-or-consequence of hepatitis is unknown. However, it is conceivable that the reduction in copper processing gene products might explain copper accumulation and the subsequent oxidative stress. Furthermore, recent Q-PCR measurements on non-copper related hepatitis and extra hepatic cholestasis suggest that ATP7A and CP are not down-regulated by inflammation or cholestasis (data not shown). Therefore, we can conclude that the decreased expression of these gene products is a Doberman hepatitis specific effect. Other important copper associated gene products such as COX17, ATP7B, and MT1A are probably down-regulated due to inflammation. Conclusion This study is the first to show the effect of prolonged exposure to different copper levels on oxidative stress and copper metabolism in canine livers. Our data supports that: (i) Doberman hepatitis is a new variant of primary copper toxicosis; (ii) there is a clear indication of a reduced copper excretion in the Doberman hepatitis group; (iii) there is a clear correlation between high copper levels and reduced protection against ROS; (iv) this Doberman hepatitis could be a good model to study copper toxicosis and its effects for several human copper storage diseases such as Indian childhood cirrhosis, non-Indian childhood cirrhosis, and idiopathic copper toxicosis, and provide the basis for possible future treatments in dog and even in man. Methods Dogs Doberman pinschers were kept privately as companion animals. The dogs were presented to the Department of Clinical Sciences of Companion Animals, Utrecht University, either for a survey investigating the prevalence of Doberman (chronic) hepatitis, as described by Mandigers et al. [ 39 ] or were referred for spontaneously occurring liver disease. All samples were obtained after written consent of the owner. The procedures were approved by the Ethical Committee, as required under Dutch legislation. Groups Animals were divided in groups based on histopathological examination and quantitative copper analysis. Each group contained both sexes from four to seven years of age. [A possible gender effect was later excluded by looking at the individual data.] Liver tissue of all Doberman pinschers was obtained using the Menghini aspiration technique [ 40 ]. Four biopsies, 2–3 cm in length, were taken with a 14-gauge Menghini needle for histopathological examination and quantitative copper analysis and stored for future quantitative PCR and protein investigations. The quantitative copper analysis was performed using instrumental neutron activation analysis via the determination of 64 Cu [ 41 ]. Histopathological biopsies were fixed in 10% neutral buffered formalin, routinely dehydrated and embedded in paraffin. Sections (4 μm thick) were stained with haematoxylin-eosin, van Gieson's stain, reticulin stain (according to Gordon and Sweet), and with rubeanic acid. One experienced board certified veterinary pathologist performed all histological examinations. All diseased groups contained at least six animals that were compared with a group of eight age-matched healthy dogs. Four groups were included in this study (Table 1 ): Table 1 Doberman pinscher group description Group n Hepatic copper Copper concentrations (mg/kg dry matter) Clinical observation Healthy 8 Normal 100 – 200 No abnormalities N-CASH 6 Normal < 300 Subclinical hepatitis CASH 6 Elevated copper levels > 600 Subclinical hepatitis DH 6 Highly elevated copper levels > 1500 Chronic hepatitis 1) Healthy group (n = 8 dogs), clinically healthy dogs with normal liver enzymes and bile acids. Histopathology of the liver did not reveal histomorphological lesions. Liver copper concentrations were below 200 mg/kg dry matter. 2) Non-copper associated subclinical hepatitis group (N-CASH, n = 6 dogs), dogs with liver enzymes and bile acids within reference values. Although histological examination showed evidence of a slight hepatitis, hepatic copper concentrations were within normal levels, i.e. , below 300 mg/kg dry matter. The dogs were classified as suffering from subclinical hepatitis, which most likely was the result of a different etiological factor, such as infections, deficiencies, other toxins, deficient immune status or immune-mediated mechanism [ 42 ]. 3) Copper associated subclinical hepatitis group (CASH, n = 6 dogs), dogs with liver enzymes and bile acids within reference values. At histopathology these dogs showed centrolobular copper-laden hepatocytes, on occasions apoptotic hepatocytes associated with copper-laden Kupffer cells, lymphocytes, plasma cells and scattered neutrophils. These lesions were classified as subclinical copper-associated hepatitis [ 43 , 44 ]. Hepatic copper concentrations were in all dogs above 600 mg/kg dry matter. 4) Doberman hepatitis group (DH, n = 6 dogs), dogs with chronic hepatitis and elevated hepatic copper concentrations. All dogs were referred with a clinical presentation of hepatic failure (apathy, anorexia, vomiting, jaundice, and in chronic cases sometimes ascites) and died within 2 months after diagnosis from this disease. Heparinized plasma liver enzymes (alkaline phosphatase and alanine aminotransferase) and fasting bile acids were, at least, three times elevated above normal reference values. Abdominal ultrasound revealed small irregular shaped echo dense liver, as performed with a high definition Ultrasound system – HDI 3000 ATL (Philips) – with a 4–7 MHz broad band Faced-array transducer. Histopathology showed chronic hepatitis (Figure 7A ) with histological features of fibrosis / micronodular cirrhosis, etc. These lesions are comparable to chronic hepatitis in man [ 42 ]. Rubeanic acid staining revealed copper accumulation in hepatocytes and Kupffer cells / macrophages (Figure 7B ). Hepatic copper concentrations were in all cases above 1500 mg/kg dry matter. RNA isolation and reverse-transcription polymerase chain reaction Total cellular RNA was isolated from each frozen Doberman liver tissue in duplicate, using Qiagen RNeasy Mini Kit (Qiagen, Leusden, The Netherlands) according to the manufacturer's instructions. The RNA samples were treated with Dnase-I (Qiagen Rnase-free DNase kit). In total 3 μg of RNA was incubated with poly(dT) primers at 42°C for 45 min, in a 60 μl reaction volume, using the Reverse Transcription System from Promega (Promega Benelux, Leiden, The Netherlands). Q-PCR of oxidative-stress proteins, copper metabolism and other related signaling molecules Q-PCR was performed on a total of 17 genes involved in oxidative stress and copper metabolism. Real-time PCR was based on the high affinity double-stranded DNA-binding dye SYBR green I (SYBR ® green I, BMA, Rockland, ME) and was performed in triplicate in a spectrofluorometric thermal cycler (iCycler ® , BioRad, Veenendaal, The Netherlands). For each PCR reaction, 1.67 μl (of the 2× diluted stock) of cDNA was used in a reaction volume of 50 μl containing 1× manufacturer's buffer, 2 mM MgCl 2 , 0.5 × SYBR ® green I, 200 μM dNTP's, 20 pmol of both primers, 1.25 units of AmpliTaq Gold (Applied Biosystems, Nieuwerkerk a/d IJssel, the Netherlands), on 96-well iCycler iQ plates (BioRad). Primer pairs, depicted in Table 2 , were designed using PrimerSelect software (DNASTAR Inc., Madison, WI). All PCR protocols included a 5-minute polymerase activation step and continued with for 40 cycles (denaturation) at 95°C for 20 sec, annealing for 30 sec, and elongation at 72°C for 30 sec with a final extension for 5 min at 72°C. Annealing temperatures were optimized at various levels ranging from 50°C till 67°C (Table 2 ). Melt curves (iCycler, BioRad), agarose gel electrophoresis, and standard sequencing procedures were used to examine each sample for purity and specificity (ABI PRISM 3100 Genetic Analyser, Applied Biosystems). Standard curves constructed by plotting the relative starting amount versus threshold cycles were generated using serial 4-fold dilutions of pooled cDNA fractions from both healthy and diseased liver tissues. The amplification efficiency, E (%) = (10 (1/- s ) -1)·100 (s = slope), of each standard curve was determined and appeared to be > 95 %, and < 105 %, over a wide dynamic range. For each experimental sample the amount of the gene of interest, and of the endogenous references glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyl transferase (HPRT) were determined from the appropriate standard curve in autonomous experiments. If relative amounts of GAPDH and HPRT were constant for a sample, data were considered valid and the average amount was included in the study (data not shown). Results were normalized according to the average amount of the endogenous references. The normalized values were divided by the normalized values of the calibrator (healthy group) to generate relative expression levels. Table 2 Nucleotide Sequences of Dog-Specific Primers for Quantitative Real-Time PCR Gene Primer Sequence (5'-3') Tm (°C) Product size (bp) Accession number GAPDH Forward TGT CCC CAC CCC CAA TGT ATC 58 100 AB038240 Reversed CTC CGA TGC CTG CTT CAC TAC CTT HPRT Forward AGC TTG CTG GTG AAA AGG AC 56 100 L77488 / Reversed TTA TAG TCA AGG GCA TAT CC L77489 SOD1 Forward TGG TGG TCC ACG AGA AAC GAG ATG 64 99 AF346417 Reversed CAA TGA CAC CAC AAG CCA AAC GAC T CAT Forward TGA GCC CAG CCC TGA CAA AAT G 62 119 AB012918 Reversed CTC GAG CCC GGA AAG GAC AGT T GSS Forward CTG GAG CGG CTG AAG GAC A 62 131 AY572226 Reversed AGC TCT GAG ATG CAC TGG ACA GPX1 Forward GCA ACC AGT TCG GGC ATC AG 62 123 AY572225 Reversed CGT TCA CCT CGC ACT TCT CAA AA CCS Forward TGT GGC ATC ATC GCA CGC TCT G 64 96 AY572228 Reversed GGG CCG GCC TCG CTC CTC p27KIP Forward CGG AGG GAC GCC AAA CAG G 60 90 AY455798 Reversed GTC CCG GGT CAA CTC TTC GTG Bcl-2 Forward TGG AGA GCG TCA ACC GGG AGA TGT 61 87 AB116145 Reversed AGG TGT GCA GAT GCC GGT TCA GGT ATOX1 Forward ACG CGG TCA GTC GGG TGC TC 67 137 AF179715 Reversed AAC GGC CTT TCC TGT TTT CTC CAG COX17 Forward ATC ATT GAG AAA GGA GAG GAG CAC 60 127 AY603041 Reversed TTC ATT CTT CAA GGA TTA TTC ATT TAC A ATP7A Forward CTA CTG TCT GAT AAA CGG TCC CTA AA 50 99 AY603040 Reversed TGT GGT GTC ATC ATC TTC CCT GTA ATP7B Forward GGT GGC CAT CGA CGG TGT GC 56 136 AY603039 Reversed CGT CTT GCG GTT GTC TCC TGT GAT CP Forward AAT TCT CCC TTC TGT TTT TGG TT 62 97 AY572227 Reversed TTG TTT ACT TTC TCA GGG TGG TTA MT1A Forward AGC TGC TGT GCC TGA TGT G 64 130 D84397 Reversed TAT ACA AAC GGG AAT GTA GAA AAC MURR1 Forward GAC CAA GCT GCT GTC ATT TCC AA 58 122 AY047597 Reversed TTG CCG TCA ACT CTC CAA CTC A XIAP Forward ACT ATG TAT CAC TTG AGG CTC TGG TTT C 54 80 AY603038 Reversed AGT CTG GCT TGA TTC ATC TTG TGT ATG Western blot analysis Pooled liver tissues (n = 6 dogs) were homogenized in RIPA buffer containing 1 % Igepal, 0.6 mM Phenylmethylsulfonyl fluoride, 17 μg/ml aprotinine and 1 mM sodium orthovanadate (Sigma chemical Co., Zwijndrecht, The Netherlands). Protein concentrations were obtained using a Lowry-based assay (DC Protein Assay, BioRad). Thirty five μg of protein of the supernatant was denatured in Leammli-buffer supplemented with Dithiothreitol (Sigma Chemical Co.) for 3 min at 95°C and electrophoresed on 10 % Tris-HCl SDS PAGE polyacrylamide gels (BioRad). Proteins were transferred onto Hybond-C Extra Nitrocellulose membranes (Amersham Biosciences Europe, Roosendaal, The Netherlands) using a Mini Trans-Blot ® Cell blot-apparatus (BioRad). The procedure for immunodetection was based on an ECL western blot analysis system, performed according to the manufacturer's instructions (Amersham Biosciences Europe). The membranes were incubated with 4 % ECL blocking solution and 0.1 % Tween 20 (Boom B.V., Meppel, The Netherlands) in TBS for 1 hour under gentle shaking. The incubation of the primary antibody was performed at room temperature for one hour, with a 1:2000 dilution of mouse anti-horse metallothionein (DakoCytomation B.V., Heverlee, Belgium). After washing, the membranes were incubated with horseradish peroxidase-conjugated chicken anti-mouse (Westburg B.V., Leusden, The Netherlands) at room temperature for one hour. Exposures were made with Kodak BioMax Light-1 films (Sigma chemical Co.). Total GSH assay The total amount of GSH was determined by a modified version of a total GSH Determination Colorimetric Microplate Assay according to Allen et al. [ 45 ], based on the original Tietze macro assay [ 46 ]. Protein samples from Doberman hepatitis (n = 6 dogs) and healthy controls (n = 8 dogs) were isolated as described in Western blot analysis and subsequently pooled. Total protein concentration was measured using a Lowry-based assay (DC Protein Assay, BioRad). In short, 50 μl of the cell-lysate (1 mg/ml) was used in triplicate in a 96-wells plate. The lysates were incubated for 5 minutes with 50 μl of 1.3 mM 5,5'dithiobis-2-nitrobenzoic acid (DTNB), and 50 μl GSH reductase (1.5 U/ml). To start the reaction 50 μl of NADPH (0.7 mM) was added to the wells. Absorbance at 450 nm was measured at start and after 5 minutes. The rate of 2-nitro-5-thiobenzoic acid production (yellow product) was measured in delta absorbance per minute and is directly proportionate with the amount of GSH in the samples. A standard curve was added with known concentrations GSH (0 to 20 μM) in order to determine the GSH concentrations in the samples. Statistical analysis A Kolmogorov-Smirnov test was performed to confirm normal distribution of every group, and a Levene's test checked the homogeneity of variances across groups. After both verifications, the statistical significance of the difference between the control group and each particular non-healthy group was determined by using the Student's t -Test. The significance level (α) was set at 0.05. Authors' contributions BS performed all Q-PCR measurements and wrote the manuscript. PM participated in its design and coordination and helped to draft the manuscript. BA performed the GSH assays and participated with Western blotting. PB performed the Copper measurements on which our groups are based. TI histochemically examined all samples described in this manuscript. GH performed genotyping on Dobermans and provided theoretical background. JR and LP, conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

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1079908

Policymaking based on CERs: changes in costs are not the same as changes in benefits

Background Earlier cost-effectiveness studies showed the cost-effectiveness ratios for pneumococcal vaccination in preventing cases of Bacteremic Pneumococcal Disease (BPD) alone to vary between € 11,000 and € 33,000 per quality-adjusted life year. If vaccination is also assumed to be effective in preventing cases of Non Bacteremic Pneumococcal Disease (NBPD) (at the same level of effectiveness), vaccinating all elderly persons becomes highly cost-effective or even cost saving. Methods The present article examines the effect of partial preventive power of the vaccine against cases of NBPD additional to its preventive power against cases of BPD, and the consequences this has in terms of cost-effectiveness. Results The analysis shows that even a fairly small additional preventive power against cases of NBPD leads to a dramatic and unexpected decrease in the cost-effectiveness ratio. Conclusion Because a Cost-Effectiveness Ratio (CER) is a ratio, changes in costs and changes in effects have rather different influences on its value. There is a linear relation between a change in costs and a change in CER if the effects are kept constant. This linear relation is not found on the effect side. Assuming that costs are constant, a change in effect will be different for low levels of effect than for high levels.

Introduction Data on efficacy or effectiveness are often among the cornerstones of the calculations of Cost-Effectiveness Ratios (CERs) and information on such effect variables is crucial for the decision-making process [ 1 , 2 ]. As an example, this article considers the lack of information regarding the effectiveness of the vaccine for pneumococcal pneumonia. There is rather strong evidence that this vaccine prevents cases of Bacteremic Pneumococcal Disease (BPD), which however represents only a minority of cases of pneumococcal disease (about 15%) [ 3 - 7 ]. There is much less evidence (some experts say no evidence) that the vaccine also prevents cases of Non Bacteremic Pneumococcal Disease (NBPD) [ 8 ]. Several published studies have calculated cost-effectiveness ratios (CERs) of pneumococcal vaccination programmes for the elderly [ 9 - 14 ]. In the earlier studies, the assumption was made that the vaccine was able to prevent cases of NBPD to the same degree as it prevents cases of BPD. The overall conclusion of the studies based on this equal effectiveness assumption was that vaccination of the elderly is highly cost-effective, or indeed in many instances even cost saving. However, the results of these studies did not have a great impact on the use of the vaccine in many countries. The hesitation to use the vaccine can be explained by various factors, including the rigorous assumption of equal effectiveness of the vaccine for BPD and NBPD. Three more recent studies have focused on the BPD subgroup in calculating the CERs of the vaccination programme [ 11 , 13 , 14 ]. If it can be shown that the vaccine is cost-effective by preventing cases of BPD alone, this would mean that the vaccine would definitely be cost-effective if it additionally prevents some cases of NBPD, which form the majority of cases. However, the three studies yielded inconsistent results. Sisk et al. [ 11 ] presented results showing that vaccination would save costs in the US, whereas the other two studies [ 13 , 14 ] presented CERs for European countries ranging from € 10,000 and € 40,000 per quality-adjusted life year (QALY) for different age groups. All of the above-mentioned studies regarded the preventive power of the vaccine against cases of NBPD as a binary variable: either the vaccine prevents no cases of NBPD or it prevents cases of NBPD to the same degree as it prevents cases of BPD. At this point, there is not much empirical evidence regarding this crucial aspect. Moreover, it is not to be expected that more specific information on the efficacy of the vaccine against NBPD will become available in the near future. Due to power rules, such a trial would cost a tremendous amount of money. This means that decisions about the introduction of the vaccine and the further expansion of its use will have to be based on present-day knowledge, with its inherent uncertainty regarding the vaccine's effectiveness. In this article we take one further step in investigating the possible partial preventive power of the vaccine against cases of NBPD additional to its preventive power against cases of BPD, and the consequences of such additional prevention in terms of cost-effectiveness. In an earlier short article [ 15 ], we discussed the results of an additional partial effectiveness of the vaccine against cases of NBPD, and attributed the unexpected results we found merely to the fact that there are far more cases of NBPD than of BPD. Due to space limitations, we were unable to address the technical aspects of the additional analysis in that article, or to discuss the consequences in more detail. Nor were we able to explain the specific influence of the partial effectiveness of the vaccine against cases of NBPD on the resulting CERs. The present article conducts a more profound analysis and discusses the unexpected results in more detail. Additionally, it uses a graphical method to explain the results. It also explores why the results are unexpected, an analysis that might be useful for other investigators who are confronted with similar problems. The next section provides some background information, together with the overall results of the European study (15), an analysis that forms the basis of all further analyses in this article. This is followed by a section explaining the methodological aspects of the additional analysis and presenting the detailed results of this analysis, including the influence of a potential additional preventive power against cases of NBPD. The final section further investigates why these results are unexpected and draws more general conclusions from the analysis. Background The present additional analysis is based on the results of an earlier study of the cost-effectiveness of a vaccination programme in five European countries [ 15 ]. The analysis uses clinical and economic variables that are unique to each country and considers cost savings from simultaneous administration of pneumococcal and influenza vaccines. In the base case analyses, the cost-effectiveness ratios for pneumococcal vaccination in preventing cases of BPD alone vary between € 11,000 and € 33,000 per QALY among the five countries. Using more plausible epidemiological assumptions of the incidence (50 cases per 100,000) and mortality (20% to 40%) of invasive disease, the cost-effectiveness ratios are € 12,000 or less per QALY in all five countries. If vaccination is also assumed to be effective in preventing cases of NBPD (at the same level of effectiveness), vaccinating all elderly persons would be highly cost-effective or even cost saving. The above analysis (16) included only CERs corresponding to extreme positions: either no preventive power against NBPD or the same degree of preventive power against NBPD as against BPD. Our implicit assumption was that intermediate positions between these two extremes could be easily calculated by simple interpolation of the results in terms of CERs. If, for example, the CER for BPD alone in a particular country were 30,000 and the CER for BPD and NBPD were 10,000, we would expect, from a prior point of view, that, starting from the situation 'BPD alone' (CER = 30,000) an additional 10% preventive power against NBPD would lead to a situation characterised by a CER of (say) 28,000 (10% of the difference between the two extremes). We did not anticipate that such a small additional preventive power would in fact have a major impact on the resulting CER. Explaining this huge difference between expectations and reality requires a stepwise analysis. Methods Since this additional analysis was not foreseen at the start of the above European study [ 15 ], it was not possible to apply the existing model ex post to produce the CERs for intermediate levels of effectiveness directly. However, it was possible to derive the CERs for intermediate steps of preventive power in an alternative way. Although the model developed for the European study is a rather complex and sophisticated cohort model, the outcomes of the model in terms of costs and benefits are linearly related to the input variable 'efficacy'. To calculate the costs, for example, the model starts with the incidence of the disease, after which the incidence is multiplied by the efficacy of the vaccine. This result is then multiplied by the protection rate of the vaccine, and the resulting figure is multiplied by the number of cases hospitalised. The result is then multiplied by the number of hospital days, and finally by the cost of one hospital day. This multiplicative model leads to the conclusion that the input variable 'efficacy' shows a linear relation with the outcome variable 'costs', a conclusion that is verified by the model. This linearity is also found for the outcomes in terms of QALY's. The consequence of this linear relation is that increasing the efficacy rate (expressed as a percentage) by a certain step results in the same change in effect, expressed as QALY's or as costs. In algebraic terms the cost can be expressed in the following formulas: 1) C = a + b * x (0<x<1) 2) Q = c + d * x (0<x<1) where: C is costs Q is QALY's a, b, c and d are constant x is vaccine efficacy for cases of NBPD The presence of linearity in the model made it possible to interpolate between the extreme outcomes (zero prevention against NBPD and same efficacy against NBPD as against BPD). As an example, let us discuss the results for Scotland in more detail. The data for 'step 0' and 'step 10' include the existing data on the costs and benefits for Scotland, which were used for the calculation of the CERs [ 15 ]. Step 0 corresponds to the situation where the vaccine is assumed to prevent only cases of BPD, whereas step 10 assumes that the vaccine prevents cases of NBPD to the same degree as it prevents BPD. The 10 intermediate steps are interpolated, based on these two extremes. Results The first surprising observation that can be made from Table 1 is that in the first step, the CER drops from an initial level of about € 15,000 per QALY to a level of € 7,000 per QALY. In the next step, where the vaccine is assumed to prevent an additional 10% of potential cases of NBPD, the CER drops to a level of about € 4,000. Alternatively, it can also be concluded in this case that an improvement in the effectiveness from say 70% to 100% would have not much impact on the resulting CER, as it would decrease from € 705 per QALY to € 242 per QALY! This makes predicting the influence of a change in effectiveness on the CER virtually impossible. The linearity that was present in the separate calculations of costs and benefits (see the first columns of the table) is no longer found in the CERs in the final column. In this specific decision environment, this would imply that if a decision maker would refuse to finance a vaccination programme based on the hard evidence for vaccine efficacy for BPD alone, a small additional preventive power of the vaccine against cases of NBPD would suffice to make it a (very) cost-effective intervention. Table 1 Cost-effectiveness ratios for intermediate degrees of vaccine efficacy in preventing cases of NBPD in Scotland Assumed preventive power for NBPD Costs in € Million* QALY's* CER Reduction in CER as percentage 0% effectiveness for NBPD 147.7 9923 14892 Step 1 (10% increase) 135.6 19777 6858 53.9 Step 2 123.4 29630 4167 18.1 Step 3 111.3 39483 2820 9.1 Step 4 99.1 49337 2010 5.4 Step 5 87.0 59190 1470 3.6 Step 6 74.9 69044 1084 2.3 Step 7 62.7 78897 795 1.9 Step 8 50.6 88750 570 1.5 Step 9 38.4 98604 390 1.2 Same effectiveness against NBPD as against BPD 26.3 108457 242 1.0 *Cost and benefits of intermediate steps in preventive power against NBPD interpolated based on the linearity of the model This phenomenon is by no means specific to Scotland. Table 2 shows it to be present in all five European countries, although the effect is larger in some countries than in others. Table 2 Cost-effectiveness ratios for intermediate degrees of vaccine efficacy in preventing cases of NBPD in 5 European countries. Assumed preventive Power against NBPD Belgium France Scotland Spain Sweden 0% effectiveness against NBPD 25907 19182 14892 10511 32675 Step 1 (10% increase) 13678 15801 6858 4383 10158 Step 2 (20% increase) 8313 13085 4167 2126 4217 Step 3 (30% increase) 5300 10857 2820 954 1473 Step 4 (40% increase) 3370 8995 2010 235 Cost saving Step 5 (50% increase) 2028 7415 1470 Cost saving Cost saving Step 6 (60% increase) 1041 6058 1084 Cost saving Cost saving Step 7 (70% increase) 285 4881 795 Cost saving Cost saving Step 8 (80% increase) Cost saving 3849 570 Cost saving Cost saving Step 9 (90% increase) Cost saving 2938 390 Cost saving Cost saving Step 10 (same effectiveness against NBPD as against BPD) Cost saving 2127 242 Cost saving Cost saving From a decision analysis point of view, the changes in the first steps are particularly relevant, because they imply that a fairly small additional preventive power against NBPD makes the vaccine cost-effective. With the exception of France, the CERs in all countries decrease by 50% or more in the first step (10% preventive power against NBPD cases). In the second step (20% preventive power against NBPD) the CERs are reduced further, to even more attractive levels. This additional analysis leads to the general conclusion that a relatively small preventive power of the vaccine against cases of NBPD is enough to make vaccination for pneumococcal disease a very attractive investment compared with other health care interventions. Discussion At the start of our European study of the cost-effectiveness of the pneumococcal vaccine [ 15 ], we did not have a clue about the existence of the phenomenon described above, nor would we have thought that the impact of a small additional preventive power against NBPD would be as great as it turned out to be. This is why only the two extreme situations for the effectiveness of the vaccine were considered in our original analysis. None of the earlier articles on cost-effectiveness investigated or even mentioned this phenomenon. What is the reason for the huge differences between reality and (our) expectations at this point? We will answer this question in two different ways: an algebraic and a graphical way. For the algebraic analysis the formula's 1 and 2 have to be specified a little bit further. The costs as a function of vaccine efficacy (x) can be expressed as follows: 3) C = C 0 - x * (C 0- C 1) where: C 0 is costs if x = 0 C 1 is costs if x = 1 C 0 and C 1 (for Scotland) can be found in table 1 and are € 147.7 million and € 26.3 million respectively. The cost as a function of vaccine efficacy for NBPD is therefore: 4) C = 147.7 * 10 6 - x * 121.4 * 10 6 The number of QALY's as a function of vaccine efficacy (x) can be expressed as follows: 5) Q = Q 0 - x * (Q 0- Q 1) where: Q 0 is number of QALY's if x = 0 Q 1 is number of QALY's if x = 1 Q 0 and Q 1 (for Scotland) can be found in table 1 and are 9923 and 108457 respectively. The QALY's as a function of vaccine efficacy for NBPD is therefore: 6) Q = 9923 - x * 98534 The CER as a function of vaccine efficacy for NBPD has the following form: 7) f(x) = (C 0 - x * (C 0- C 1) )) / (Q 0 - x * (Q 0- Q 1) )), (0<x<1). For x is 0 the value of the function becomes C 0 / Q 0 , which is our original ICER for zero vaccine efficacy for NBPD. For x is 1 the value of the function becomes C 1/ Q 1 , which is our original ICER for 100% vaccine efficacy for NBPD. It is obvious that in general de function f(x) is an exponential declining function, which explains, to a certain degree, the specific behaviour of the CER; huge declines in CER for low values of x and small declines when x reaches the value 1. An impression of this can be based on the data of table 1 , where the CER's (of Scotland) as a function of vaccine efficacy for NBPD are given in column 4. A graphical representation allows us to clarify the causes of this phenomenon in another way. Figure 1 shows a graphic representation of the data for Scotland. One extreme represents the costs and benefits under the assumption that the vaccine prevents only cases of BPD (S 0 ) while the other extreme represents the assumption that cases of NBPD are prevented to the same degree as those of BPD (S 10 ). The other points represent the costs and benefits in the consecutive steps. Due to the linearity of the model, these 10 steps are positioned on a straight line, and indicated as S 1 , S 2 etc. Figure 1 Costs and benefits of intermediate steps of partial effectiveness against NBPD for Scotland But why do the CERs for S 0 to S 10 show non-linear behaviour? We have to show that equal steps in costs and/or benefits do not automatically lead to equal differences in CERs. The first step (10% preventive power against NBPD) takes us to point S 1 . Starting from S 0 , there are numerous alternative ways in which a CER of this level could be reached. One of these ways would be to lower the cost from S 0 to level A 1 (see figure), another to increase the benefits from S 0 to B 1 , but other combinations are also possible. A 1 , S 1 and B 1 are on the same line through the origin and therefore have the same CER. Starting from S 0 , a rather small increase in benefits has the same effect (in terms of CER) as a rather large decrease in costs. Looking at the steps from S 9 to S 10 , we see the opposite: in this area a rather small decrease in costs has the same impact as a rather huge increase in benefits. It will be clear from this observation that the ratio between costs and benefits changes in each of the 10 steps. This phenomenon can be visualised in a different way, using the structure of CERs. As many authors have suggested, CERs can be visualised in graphic representations [[ 1 , 2 , 16 - 18 ], and [ 19 ]]. These graphs may help to identify the most cost-effective interventions within a set of compatible (mutually non-exclusive) alternatives. Graphs can also be helpful in identifying the best candidate in a set of incompatible (mutually exclusive) alternatives [ 17 ]. Often there is no need to pay attention to the precise underlying structure of CERs. For exemple, Laupacis et al. [ 16 ] in their original article drew CER lines in a rather arbitrary way. The above observations do not hamper decision-making, as long as only 'ordinal' conclusions are drawn, for example, that intervention A is more cost-effective than intervention B. However, absolute changes in the position of an intervention in the graph might lead to unexpected results, as will be shown below. In figure 2 the exact position of different CER-lines are drawn, based on figure 1 . The figure clearly shows that different incremental increases in preventive power of the vaccine must lead to different effects on the resulting CER. The first increase in preventive power against cases of NBPD (step 1) cuts from a CER of (about) € 15,000 per QALY through a whole series of nearby CER lines and ends up at a CER of € 7,000 per QALY. The next step would bring this down further, to a CER of € 4,000, a relatively minor decrease compared with the previous step. In absolute terms, however, both steps are identical, in that they consist of the same decrease in costs and benefit. Figure 2 CERs for technologies characterised by costs per QALY of € 1,000, € 2,000, € 3,000 etc. can be easily constructed using the constant vertical distance (a) between the lines The above analysis allows the conclusion that a particular change in costs and benefits will not lead to a fixed change in the CER. The impact of such a change in costs and/or benefits on the resulting CER depends on the starting point in the graph. In our example, it could be said that the position of the line S0-S10 determines the resulting CER. This is the reason why the effects of a partial effectiveness of the pneumococcal vaccine against NBPD are different in different countries. Conclusion If the pneumococcal vaccine has even a small preventive power against NBPD, this would make vaccination against pneumococcal pneumonia a very cost-effective intervention. While it is very unlikely that the vaccine is as effective in preventing cases of NBPD as it is in preventing cases of BPD, it is also unlikely that the vaccine does not prevent any cases of NBPD. The above analysis shows that a very small additional preventive power against NBPD (about 5 to 10%) changes the cost-effectiveness of this vaccination programme, on average, from a level just below the maximum value per QALY (€ 20,000 per QALY) to a level of € 10,000 per QALY, a level that in most industrialised countries would be considered a good health investment. Without the above analysis, policy-making would be very complicated, because of the uncertainty about the preventive power and the uncertainty about the maximum value per QALY. With this analysis, there is less uncertainty and policymakers can be more confident in deciding to introduce the vaccination programme for the population aged 65 and older. Is there a more general principle behind the above phenomenon? To answer this question we have to look back to the concept of CER itself. CER is defined as (incremental) costs divided by (incremental) effects. It is obvious that a CER increases as costs increase, if the effects are kept constant. An increase in cost by an amount x will lead to an increase in CER of y, independent of the volume of the costs. There is a linear relation between a change in costs and a change in CER if we keep the effects constant. This linear relation is not found on the effect side. Assuming that the costs are constant, a change in effect will be different for low effect levels than for high levels. Adding 1 QALY to an intervention that produces 1 QALY leads to a reduction of 50% in CER, whereas adding 1 QALY to 10 QALY's results in a 10% reduction in CER. With regard to effects, a relative increase will always lead to the same change in CER, whereas with regard to the costs, an absolute change will lead to the same change in CER. In the above case of a partial increase in preventive power, the costs and benefits increase by the same amount in each step, but we now see that it is merely the effects that lead to the unexpected results: merely changing the costs by a particular amount would not have this effect. We stated above that our model was a multiplicative model as regards costs and effects. We used this property of the model only to calculate costs and benefits corresponding to intermediate steps in preventive power in an easy manner. We can therefore conclude that the above results are not restricted to this type of linear model, but are to be expected in general. The above phenomenon depends on the ratio approach, not on the model used. The above analysis leads to several observations In the first place, intuition might be not a good instrument to predict the CER consequences of changes in costs and (especially) benefits. In the above example the consequences of partial effectiveness of the pneumococcal vaccine for the CERs were totally unexpected. In the example, the effectiveness determines both costs and benefits of the CER, which makes prediction of effects more complicated. Researchers who are thinking of performing sensitivity analyses should be aware of the fact that in some cases even very small changes in variables could have huge impacts on the resulting CERs. It will not always be easy to anticipate or predict this impact, due to the exponential character of the CER itself. Laupacis et al. (1992) were among the first to use graphs to represent CERs of different interventions, although the principle had already been described as early as 1986 (See for example Anderson et al., 1986). Gold et al. suggested that researchers should make use of such graphs, because they are handsome, concise and easy to understand. In their original graph, Laupacis et al. represented two CERs: one for interventions with a CER of $20,000 and the other with a CER of $100,000. The two lines in their figure are drawn rather arbitrarily, whereas in reality the position of the two lines should be fixed (see above). In analysing the results in a graph, one should understand the underlying structure of the CERs (see figure 2 ), especially in cases where interventions are presented in absolute positions, as was the case in our example of the pneumococcal vaccine. If the graph is used to prioritise compatible alternatives according to cost-effectiveness, using the wrong graph would be no big problem. However, if one is interested in identifying the best candidate within a set of incompatible alternatives, using the right framework becomes crucial. A last observation of our analysis has to do with the term 'cost-effective' itself. It has already been recognised before that the term 'cost-effective' is rather unspecified. What is the real meaning of the expression: intervention A is more cost-effective than intervention B? This becomes even more troublesome if one considers different interventions in a QALY League Table. People are often inclined to think that the difference between interventions with CERs of 10,000 and 20,000 is the same as that between interventions with CERs of 50,000 and 60,000, because the difference in CER is the same in both situations. In reality, it is impossible to decide which of the two interventions is better as long as we have no separate information on costs and benefits. This observation also leads to the conclusion that it is impossible to say whether it is better to improve the cost-effectiveness of one particular technology from (for example) € 10,000 to € 20,000 than to improve the cost-effectiveness of another technology from € 50,000 to € 60,000. This means that the cost-effectiveness scale in itself is not a cardinal scale but only an ordinal scale. Needless to say, the incremental-net-benefit parameter recently proposed by many authors behaves in a much more straightforward way in this respect than the above incremental CER, providing another argument in favour of the net benefit approach. Competing interests The original study was supported by a grant of Aventis Pasteur MSD. The above study was performed totally independent from Aventis Pasteur MSD. Authors' contributions The authors worked together in close cooperation on the concepts and methodological aspects of the study. The first author produced the first drafts, whereas the second author produced many valuable contributions.

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1079909

Muscle oxidative metabolism accelerates with mild acidosis during incremental intermittent isometric plantar flexion exercise

Background It has been thought that intramuscular ADP and phosphocreatine (PCr) concentrations are important regulators of mitochondorial respiration. There is a threshold work rate or metabolic rate for cellular acidosis, and the decrease in muscle PCr is accelerated with drop in pH during incremental exercise. We tested the hypothesis that increase in muscle oxygen consumption ( o 2mus ) is accelerated with rapid decrease in PCr (concomitant increase in ADP) in muscles with drop in pH occurs during incremental plantar flexion exercise. Methods Five male subjects performed a repetitive intermittent isometric plantar flexion exercise (6-s contraction/4-s relaxation). Exercise intensity was raised every 1 min by 10% maximal voluntary contraction (MVC), starting at 10% MVC until exhaustion. The measurement site was at the medial head of the gastrocnemius muscle. Changes in muscle PCr, inorganic phosphate (Pi), ADP, and pH were measured by 31 P-magnetic resonance spectroscopy. o 2mus was determined from the rate of decrease in oxygenated hemoglobin and/or myoglobin using near-infrared continuous wave spectroscopy under transient arterial occlusion. Electromyogram (EMG) was also recorded. Pulmonary oxygen uptake ( o 2pul ) was measured by the breath-by-breath gas analysis. Results EMG amplitude increased as exercise intensity progressed. In contrast, muscle PCr, ADP, o 2mus , and o 2pul did not change appreciably below 40% MVC, whereas above 40% MVC muscle PCr decreased, and ADP, o 2mus , and o 2pul increased as exercise intensity progressed, and above 70% MVC, changes in muscle PCr, ADP, o 2mus , and o 2pul accelerated with the decrease in muscle pH (~6.78). The kinetics of muscle PCr, ADP, o 2mus , and o 2pul were similar, and there was a close correlation between each pair of parameters (r = 0.969~0.983, p < 0.001). Conclusion With decrease in pH muscle oxidative metabolism accelerated and changes in intramuscular PCr and ADP accelerated during incremental intermittent isometric plantar flexion exercise. These results suggest that rapid changes in muscle PCr and/or ADP with mild acidosis stimulate accelerative muscle oxidative metabolism.

Background Skeletal muscle respiratory control is a cardinal issue in the field of muscle energetics. Early work on isolated mitochondria identified ADP as an important stimulator of mitochondrial respiration [ 1 ]. Thereafter, it has been verified that ADP is a control signal of muscle oxidative phosphorylation in many studies [ 2 - 7 ]. During steady state phase of muscle contraction, muscle O 2 consumption ( o 2mus ) linearly correlates with intramuscular phosphocreatine (PCr) concentration at varying intensities under relatively stable muscle pH conditions [ 8 - 10 ]. It has also been demonstrated that muscle PCr and pulmonary oxygen uptake ( o 2pul ) show similar kinetics during the transition from rest to steady state exercise in humans in a non-steady state condition [ 11 - 13 ]. In addition, Rossiter et al. [ 14 ] demonstrated that muscle PCr and slowly developing supplementary component (slow component) of o 2pul show similar response during a high intensity constant load exercise with decreased pH condition. Therefore, it has been thought that intramuscular ADP and PCr concentrations are important regulators of skeletal muscle oxidative metabolism [ 1 - 14 ]. Although o 2pul has been used as an indicator of muscle oxidative metabolism [ 11 - 14 ], it does not specifically indicate oxygen consumption each of the exercising muscle group(s). Near-infrared continuous wave spectroscopy (NIR cws ) has unique capability for non-invasively evaluating of O 2 kinetics in an objective portion of tissue with high-time resolution. NIR cws was first applied to the study of exercising skeletal muscle in humans in 1991 [ 15 ]. Since then, many more groups have applied this technique [ 16 - 20 ]. o 2mus can be determined using NIR cws with transient arterial occlusion [ 8 ], and its validity was confirmed [ 21 ]. The rate of decrease in oxygenated hemoglobin and/or myoglobin (HbO 2 /MbO 2 ) under conditions in which interruption of the O 2 supply to the muscle (arterial occlusion) reflects o 2mus [ 8 , 21 , 22 ]. Therefore, this NIR cws technique enables us to determine o 2mus during exercise where metabolic condition changes diversely. It has been reported that there is a threshold work rate or metabolic rate for cellular acidosis (pH T ) and that, above pH T , the decrease in muscle PCr is accelerated during incremental exercise [ 23 - 25 ]. If muscle oxidative metabolism is closely related with muscle PCr even under acidotic condition, it would be predicted that acceleration in increase in o 2mus coincided with decrease in pH. However, there is no evidence for the effect of decrease in pH on muscle oxidative metabolism during incremental exercise. The aim of this study was to measure o 2mus , ADP, and PCr during incremental exercise where muscle pH changed from stable to decreasing condition. We hypothesized that the increase in o 2mus , increase in ADP and decrease in PCr occurred similar kinetics throughout incremental exercise. When exercise intensity increased above pH T , there is a possibility that the accelerative decrease in PCr stimulates accelerative increase in muscle oxidative metabolism during incremental exercise. To test the second hypothesis that with decrease in pH accelerative decrease in PCr could be responsible for the increase in o 2mus , we identified the inflexion point of pH, PCr, ADP, cytosolic free energy of ATP hydrolysis (ΔG ATP ), o 2mus , and o 2pul during incremental exercise. We predicted that when exercise intensity increased above the level which decrease in pH occurred, PCr, ADP, ΔG ATP , o 2mus , and o 2pul would show greater change than that obtained during stable pH condition during incremental exercise. Methods Subjects Five male volunteers, aged between 22 and 34 years, participated in this study. All subjects were healthy, non-smokers, and free of known diseases. All subjects were fully informed of the risks involved in this study, and we obtained written informed consent from each. This study was approved by the Institutional Committee for the protection of human subjects. Experimental design Each subject sat on a platform in an upright sitting position with his right leg positioned horizontally. The subjects performed the same exercise procedure five times on different occasions: once (day 1) with the 31-phosphorus-magnetic resonance spectroscopy ( 31 P-MRS) measurement, twice (day 2, 3) with the respiratory gas analysis, once (day 4) with the NIR cws measurement for determination of o 2mus , and once (day 5) with the EMG record. With the exception of the 31 P-MRS measurement, the other four measurements were performed outside the MRS magnet. During these four measurements the subjects inserted a leg into a cylindrical plastic pipe of the same diameter and length as the bore of the MRS magnet. For each measurement, whether in the magnet or the plastic pipe, the leg was held in a fixed position by a cradle. Exercise Protocols On occasions of the experiment, maximal voluntary contractions (MVC) was measured prior to the principal experiment, and each subject's exercise load was set based on the MVC of each. The MVC of isometric plantar flexion was measured by pushing against a foot pedal with connected force transducer. MVC was measured three times with sufficient rest (> 3 min) between each performance. The maximum value was used as the MVC. After sufficient rest in an upright sitting position, the subjects performed repetitive intermittent isometric plantar flexion exercise with the right leg in the same position. One duty cycle of contraction and relaxation consisted of a 6-s contraction and a 4-s relaxation. With the use of a visual feedback meter, the subjects were directed to perform using the prescribed force. Additionally, the experimental director continuously verified force. Exercise intensity was increased incrementally every 60 s by 10% MVC, starting from 10% MVC to an intensity at which the subject could no longer maintain the required force. A backrest was placed behind the subject during exercise. To fix position of the subject and to limit involvement of muscles other than calf muscle, the contact area of the backrest and subject's body was limited as small as possible. The height of backrest was 21 cm, and area of contact against subject's body was limited to lower back only. The subjects were instructed not to exert muscles other than the calf muscle to the best of their ability during the exercise, and they were fully familiarized with the exercise prior to the experiment. 31 P-MRS 31 P-MRS signals were obtained by an NMR spectrometer (Otsuka Electronics Co. Ltd.) with a 2.0-T superconducting 26-cm bore magnet. A double tuned ( 1 H and 31 P), 3.0-cm diameter radio frequency surface coil tuned to 34.58 MHz with 60-μs pulse width was used for the phosphorus signal. Pulse repetition time was 2 s. Five pulses were averaged to obtain a free induction decay (FID). Therefore, a spectrum was obtained every 10 s. Twelve spectra were averaged during the pre-exercise resting period, and three spectra were averaged during exercise. The surface coil was placed on the medial head of the gastrocnemius muscle (m.MG), and the coil and leg were held in a fixed position in the magnet by a cradle. All 31 P-MRS spectra were fitted to a Lorentzian line shape using the least-squares method. The relative area and frequency of the individual peaks were determined (Otsuka Electronics software) to calculate the areas of PCr, inorganic phosphate (Pi), and β-ATP peaks. The PCr and Pi intensities were normalized using the sum of PCr and Pi to avoid influence from possible changes in the sensitivity of 31 P-MRS signals. Saturation correction was performed using saturation factors of PCr, Pi, and β-ATP peaks, which were calculated by comparing the data from the 2-s and fully relaxed spectra. The saturation factors of PCr, Pi, and β-ATP peaks in this study were 1.330, 1.081, and 1.184, respectively. The intracellular pH was calculated from the median chemical shift between the P i and PCr peaks [ 26 ]. Changes in muscle PCr are expressed as a percentage of the pre-exercise resting value. To convert peak areas to concentrations, the β-ATP peak was assumed to represent total ATP and was set at 8.2 mM [ 27 - 29 ]. [PCr] and [Pi] could then be estimated as the product of the areas to ATP (as PCr to β-ATP and Pi to β-ATP) and 8.2 mM. Total creatine (TCr) was assumed to be equal to the sum of PCr and Pi ([TCr] = [PCr] + [Pi]), and TCr was assumed to be constant throughout the experiment [ 10 ]. ADP was calculated with the assumption that equilibrium of the Cr kinase (CK) reaction [ 23 , 30 , 31 ]: [ADP] = {0.74 [ATP]([TCr] - [PCr])} / {(1.66 × 10 9 )(10^-pH obs ) [PCr]}     ( 1 ) The constant 0.74 is the estimated monovalent ion activity coefficient [ 31 ] that corrects for the fact that pH obs is an activity, subscript obs indicates observed factors, and 1.66 × 10 9 is the equilibrium constant for CK. Free magnesium was assumed to be 1 mM and unchanging throughout the experiment [ 32 ]. Cytosolic free energy of ATP hydrolysis (ΔG ATP ) was also calculated [ 23 , 30 , 31 ]: ΔG ATP = ΔG O + RT ln ([ADP] [Pi] / [ATP]) + RT ln [10^-(pH obs -7)]     ( 2 ) ΔG O is Gibb's free energy, R is gas constant, and T is absolute temperature. ΔG O is taken to be -32 kJ/mol at pH7.0[ 31 ], RT at 37°C is 2.58. NIR spectroscopy NIR signals were obtained by NIR cws (HEO-200, OMRON Co. Ltd.). The NIR cws probe contained a light source and an optical detector with a distance of 3.0 cm between the light source and detector to provide sensory input for the unit. A pair of two-wavelength light emitting diodes, with wavelengths of 760 and 840 nm, was used as the light source. A silicon photodiode was used as the photodetector. The NIR cws probe was placed on the m.MG, and the probe and leg were held in a fixed position by a cradle in a plastic pipe that mimicked the bore of the MRS magnet. Changes in HbO 2 and/or MbO 2 , deoxygenated Hb and/or Mb, and total hemoglobin and/or myoglobin (THb/TMb) were calculated by the least squares method using data from the changes in the absorbance of these different wavelengths of light. The sampling time of the data was 0.1 s. o 2mus was measured using NIR cws with the transient arterial occlusion technique described previously in detail [ 8 , 21 , 22 ]. o 2mus was determined by the rate of decrease in HbO 2 /MbO 2 during arterial occlusion. Since the changes in HbO 2 /MbO 2 measured by NIR cws show a dynamic balance between O 2 supply and O 2 consumption, the rate of decrease in HbO 2 /MbO 2 during arterial occlusion reflects the o 2mus [ 8 , 21 , 22 ]. Arterial occlusion was performed for 1 min during rest, and for 6 s once every 30 s during isometric contraction. Timing for arterial occlusion during exercise took place at the third and the sixth contraction of each intensity i.e. at 20–26 s and 50–56 s of each minute. The o 2mus was expressed as a value relative to that obtained at rest (fold of resting). Respiratory gas analysis o 2pul was measured during the pre-exercise resting period and throughout the exercise period by the breath-by-breath gas analysis method using an Aeromonitor AE-280 (Minato Medical Science Co. Ltd.) [ 33 ]. This system consists of a microcomputer, a hot-wire flow-sensor, and oxygen and carbon dioxide analyzers (zirconium element-based oxygen analyzer and infra-red carbondioxide analyzer). Prior to the experiments, the flow-sensor and gas analyzers were calibrated with a known volume of room air at several mean flow rates and gas mixtures of known concentration, respectively. To improve the signal-to-noise ratio of o 2pul , each subject performed the exercise session for o 2pul measurement twice on different days, and the dual measurement data were subsequently averaged. Surface electromyograms Surface electromyography (EMGs) were obtained from the m.MG, lateral head of the gastrocnemius muscle (m.LG), and soleus muscle (m.SOL) using a bipolar, silver-silver chloride electrode (10 mm diameter sample area) with a fixed inter-electrode spacing of 30 mm (Nihon Koden Co., Japan) during incremental plantar flexion exercise. The EMG signal was sampled at a rate of 2000 Hz using available software (BIOPAC Systems, Inc., USA) and stored on computer disk for later analysis. The root mean square of the EMG signal (rmsEMG) was calculated. Prior to the principal experiment the subjects performed MVC, and the rmsEMG was normalized as 100% at MVC. Data analysis Analysis of each parameter was performed every 30 s as the procedure is shown in figure 1 . Except for o 2mus , all data were averaged over 30 s. The data for o 2mus were obtained at the third (20–26 s) and sixth (50–56 s) contractions of each intensity. The reason o 2mus was measured only once during three contraction phases was to avoid the limitations to exercise performance caused by interrupting the blood flow. The value of the third contraction was used to represent the first 30 s of each minute, and the value of the sixth contraction was used to represent the last 30 s of each minute. All averaged data were shown from pre-exercise rest to the first 30 s at 80% MVC exercise at which every subject was able to perform. The logarithms of the individual metabolic parameters (pH, PCr, ADP, ΔG ATP , o 2mus o 2pul ) were plotted against exercise intensity in order to determine a break point of metabolic change based on the method of determining lactate threshold [ 34 ]. These plots were best fit by a piecewise linear regression model with a breakpoint. Figure 1 Procedure for data analysis . Each parameter was analyzed every 30 s. Muscle phosphocreatine (PCr), inorganic phosphate (Pi), pH, estimated ADP and free energy of ATP hydrolysis (ΔG ATP ), pulmonary oxygen uptake ( o 2pul ), and electromyogram (EMG) were averaged over 30 s. The data for muscle oxygen consumption ( o 2mus ) were obtained during the third (20–26 s) and sixth (50–56 s) contractions at each intensity. The o 2mus value of the third contraction was used to represent the first 30 s of each minute, whereas the o 2mus value of the sixth contraction was used to represent the last 30 s of each minute. Division of data analysis (30s). o 2mus measurement (6 s; once per three contraction phases). Confirmation of reproducibility Since the subjects performed the same exercise procedure five times, we were able to obtain five sets of performance data. The maximal exercise intensity the subjects were able to perform during the exercise protocol was 80–90% MVC (450–510 s). The maximal intensity at which each subject was able to perform was the same throughout five exercise sessions. The coefficient of variation for exercise duration was 0.91%. Regarding time course change and peak value, o 2pul did not differ significantly during the two measurements. There was a significant correlation between each time measurement for individual pulmonary o 2 (r = 0.981~0.993, p < 0.001). Statistical analyses Data are expressed as means ± SD. The data were compared to determine significant changes in the values of each parameter every 30 s compared with the values obtained during the first 30 s of exercise (the first 30 s at 10% MVC), and the 30 s of exercise immediately before. One-way analysis of variance (ANOVA) for repeated measures was used to determine the significance of time course changes in each parameter, and Fisher's PLSD post hoc comparisons were used to determine the significance of differences of each parameter every 30 s. A linear regression analysis was used to examine the relationship between each parameter. P < 0.05 was defined as statistically significant. Results Fig. 2 shows the time course changes in normalized rmsEMG of m.MG, m.LG, and m.SOL. The rmsEMG in those muscles increased similarly with increasing exercise intensity. The rmsEMG of m.MG for each of the first 30 s at 20%, 30%, 50%, 60%, 70%, and 80% MVC differed significantly from that during the 30 s of exercise immediately before (i.e., prior intensity) (p < 0.05). Throughout the exercise, the change in rmsEMG of m.MG was largest in the three muscle groups. Figure 2 Changes in root mean square of EMG (rmsEMG) during incremental intermittent isometric plantar flexion exercise . Changes in rmsEMG at (A) the medial head of the gastrocnemius muscle (m.MG), (B) the lateral head of gastrocnemius muscle (m.LG), and (C) the soleus muscle (m.SOL) during incremental intermittent isometric plantar flexion exercise. Data are represented as relative values obtained during maximal voluntary contraction (MVC) as 100%. Values shown are means ± SD of 5 subjects. * p < 0.05, ** p < 0.01 vs. the value during the first 30 s at 10% MVC (first 30 s of exercise). #p < 0.05 vs. the value obtained during the 30 s of exercise immediately before. Fig. 3A shows the time course of changes in intramuscular pH. We found that pH was relatively constant, from resting values (7.06 ± 0.01) until 60% MVC (7.04 ± 0.08), but it decreased significantly (p < 0.05) at 70% MVC and with exercise progression, being 6.78 ± 0.22 at the end of exercise. Figure 3 Changes in muscle pH, PCr, ADP, and ΔG ATP during incremental intermittent isometric plantar flexion exercise . Changes in (A) pH, (B) PCr, (C) ADP, and (D) ΔG ATP during incremental intermittent isometric plantar flexion exercise. A dotted line in each panel B, C, and D represents a linear regression line which is drawn to obtain the highest correlation coefficient above 40% MVC, at which significant difference was observed when compared with the value obtained during the first 30 s at 10% MVC. Values shown are means ± SD of 5 subjects. * p < 0.05, **p < 0.01 vs. the value during the first 30 s at 10% MVC (first 30 s of exercise). #p < 0.05 vs. the value obtained during the 30 s of exercise immediately before. Fig. 3B shows the time course changes in intramuscular PCr. We found that there were significant differences after the last 30 s at 40% MVC when compared with the value obtained during the first 30 s at 10% MVC (p < 0.05), and that PCr decreased with progression of exercise. Above 70% MVC, the values were significantly different when compared with those obtained during the 30 s of exercise immediately before. A linear regression line was drawn to obtain the highest correlation coefficient above the last 30 s of 40% MVC, at which significant difference was observed when compared with the value obtained during the first 30 s at 10% MVC. The PCr deviated downward from the regression line above 70% MVC. Fig. 3C shows the time course changes in estimated ADP. We found that ADP slightly increased from rest (8.8 ± 0.9 μM) until the last 30 s of 30% MVC (13.0 ± 3.2 μM), whereas above 40% MVC, these values differed significantly from those obtained during the first 30 s at 10% MVC (p < 0.05). Thereafter, ADP increased with progression of exercise, being significantly different above 70% MVC compared with the value obtained during the 30 s of exercise immediately before. At the end of exercise, ADP was 46.6 ± 12.8 μM. A linear regression line was drawn to obtain the highest correlation coefficient above the first 30 s of 40% MVC, at which significant difference was observed when compared with the value obtained during the first 30 s at 10% MVC. The ADP deviated upward from the regression line above 70% MVC. Fig. 3D shows the time course changes in estimated ΔG ATP . We found that ΔG ATP changed only slightly from rest (-63.8 ± 0.5 kJ/mol) until the last 30 s of 30% MVC (-62.9 ± 1.1 kJ/mol), whereas above 40% MVC, these values differed significantly from those obtained during the first 30 s at 10% MVC (p < 0.05). Thereafter, ΔG ATP increased with progression of exercise, being significantly different above 70% MVC compared with the value obtained during the 30 s of exercise immediately before. At the end of exercise, ΔG ATP was -55.1 ± 1.9 kJ/mol. A linear regression line was drawn to obtain the highest correlation coefficient above the first 30 s of 40% MVC, at which significant difference was observed when compared with the value obtained during the first 30 s at 10% MVC. The ΔG ATP deviated upward from the regression line above 70% MVC. Fig. 4 shows the time course changes in o 2mus . o 2mus also showed slight changes during exercise below 40% MVC. Above 40% MVC, however, there were significant differences when compared with the value obtained during the first 30 s at 10% MVC. o 2mus subsequently increased with progression of exercise, and the values obtained during the last 30 s at 70% MVC and the first 30 s at 80% MVC differed significantly from the value during the 30 s of exercise immediately before. The peak value of o 2mus was 21.3 ± 5.2 fold higher than its resting value. A linear regression line was drawn to obtain the highest correlation coefficient above the first 30 s of 40% MVC, at which significant difference was observed when compared with the value obtained during the first 30 s at 10% MVC. The o 2mus deviated upward from the regression line above 70% MVC. Figure 4 Changes in o 2mus during incremental intermittent isometric plantar flexion exercise . A dotted line represents a linear regression line which is drawn to obtain the highest correlation coefficient above the first 30 s of 40% MVC at which significant difference was observed when compared with the value obtained during the first 30 s at 10% MVC. The o 2mus is expressed as a value relative to that obtained at rest (fold of resting). Values shown are means ± SD of 5 subjects. *p < 0.05, **p < 0.01 vs. the value during the first 30 s at 10% MVC (first 30 s of exercise) # p < 0.05 vs. the value obtained during the 30 s of exercise immediately before. Fig. 5 shows the time course changes in o 2pul . We found that o 2pul changed only slightly, and there was little difference relative to exercise intensity up to 40% MVC. When the exercise intensity was raised above 50% MVC, the value of o 2pul differed significantly from that obtained during the first 30 s at 10% MVC. Thereafter, o 2pul increased with progression of exercise, and the values obtained during the last 30 s at 70% MVC and the first 30 s at 80% MVC were significantly different from the value obtained during the 30 s of exercise immediately before. The peak value of of o 2pul was 684.8 ± 64.8 ml/min, which different from the resting value of o 2 pul (Δ o 2pul ) by 364.8 ± 74.3 ml/min. A linear regression line was drawn to obtain the highest correlation coefficient above the first 30 s of 50% MVC, at which significant difference was observed when compared with the value obtained during the first 30 s at 10% MVC. The o 2pul deviated upward from the regression line above 70% MVC. Figure 5 Changes in o 2mus during incremental intermittent isometric plantar flexion exercise . A dotted line represents a linear regression line which is drawn to obtain the highest correlation coefficient above the first 30 s of 50% MVC at which significant difference was observed when compared with the value obtained during the first 30 s at 10% MVC Values shown are means ± SD of 5 subjects. * p < 0.05, **p < 0.01 vs. the value during the first 30 s at 10%MVC(first 30 s of exercise), # p < 0.05 vs. the value obtained during the 30 s of exercise immediately before. When we examind the relationship between the averaged muscle PCr and the averaged o 2mus , we observed a significant inverse correlation between the two (r = 0.980, p < 0.001) (Fig 6A ). There was also a significant inverse correlation between averaged muscle PCr and averaged o 2pul (r = 0.969, p < 0.001) (Fig 6B ). Figure 6 Relationships between muscle PCr and o 2mus , muscle PCr and o 2pul . Relationships between (A) averaged muscle PCr and o 2mus and (B) averaged muscle PCr and o 2pul . Values shown are means ± SD of 5 subjects. We also determined the relationship between the averaged ADP and the averaged o 2mus (Fig. 7A ) and between the averaged ADP and the averaged o 2pul (Fig. 7B ). There was a significant correlation betwene ADP and o 2mus (r = 0.983, p < 0.001) and between ADP and o 2pul (r = 0.971, p < 0.001). Individual correlation between ADP and o 2mus (r = 0.916~0.963, p < 0.001) and between ADP and o 2pul (r = 0.902~0.974, p < 0.001) were seen in all subjects (Figures not shown). Additionally, there was a significant positive correlation between averaged o 2mus and averaged o 2pul (r = 0.975, p < 0.001) (Figure not shown). Figure 7 Relationships between muscle ADP and o 2mus , muscle ADP and o 2pul . Relationships between (A) averaged muscle ADP and o 2mus and (B) averaged muscle ADP and o 2pul . Values shown are means ± SD of 5 subjects. The logarithms of individual metabolic parameters (pH, PCr, ADP, ΔG ATP , o 2mus , o 2pul ) were best fit by the piecewise regression model with an inflexion point ranging from 60 to 70% MVC, and individual break points for all metabolic parameters were the same intensity in all subject. There were significant intra-individual correlations between each pair of metabolic parameters (r = 0.971~0.988, p < 0.001). 0.001) Discussion The main finding of this study was that increase in o 2mus accelerated coincidentally with drop in muscle pH over 70% MVC during incremental intermittent isometric contraction. Changes in muscle PCr ADP, ΔG ATP , and o 2pul also accelerated simultaneously with drop in pH. In addition, the kinetics of each metabolic parameter was similar, and there were significant correlations between each pair of parameters (r = 0.969~0.983, p < 0.001). It has been thought that intramuscular ADP and PCr concentration are important regulators of mitochondrial respiration [ 1 - 14 , 35 , 36 ]. However, there is no evidence that examined relationship between muscle oxidative metabolism and muscle PCr or ADP during incremental exercise where muscle pH changed from stable to decreasing condition. According to the PCr shuttle hypothesis [ 37 ] and other biochemical hypotheses [ 38 ], control of respiration is exerted linearly at the mitochondria by the declining PCr and concomitant rise in cytosolic Cr. These hypotheses [ 37 , 38 ] are based on observations of linear changes in muscle respiration relative to increasing contraction intensity under relatively stable pH conditions. The greater rate of breakdown of PCr under acidotic conditions [ 23 - 25 ], if still tightly coupled to oxidative phosphorylation, would predict that o 2mus increases nonlinearly with increasing contraction intensity. In this study, the increase in o 2mus is actually accelerated with rapid decrease in PCr during a conspicuous drop in pH, to ~ 6.78. Additionally, the accelerated increase in o 2mus coincided with abrupt increase in ADP. Consequently, our results indicated that muscle oxidative metabolism is closely related with muscle PCr and ADP even under mild acidotic conditions. Therefore, it is suggested that rapid changes in muscle PCr and/or ADP, coincided with drop in pH, are factor(s) that accelerate muscle oxidative metabolism during incremental intermittent isometric contraction. The accelerated changes in PCr, ADP, o 2mus , o 2pul , and the calculated ΔG ATP above 70% MVC coincided with the decrease in pH, indicating that metabolic demand changes nonlinearly with increasing exercise intensity (Fig. 3 , Fig. 4 , Fig. 5 ). In contrast, others have shown that, although above pH T muscle PCr rapidly decreases, ΔG ATP increases linearly with increasing intensity throughout dynamic plantar flexion exercise [ 23 ]. o 2pul rises linearly with increasing work rate during bicycle exercise [ 39 , 40 ], and ΔG ATP shows a linear increase with increasing work rate during dynamic plantar flexion exercise [ 23 ] consistent with pulmonary o 2 [ 39 , 40 ]. One possible explanation for the different results between the earlier studies [ 23 , 39 , 40 ] and ours is the difference of load setting. Previous dynamic exercise studies were incremented by prescribed work rate, until either the frequency of contraction/s or the full range of motion could no longer be sustained [ 23 ] or until maximal o 2pul was attained by increments of 15–30W/min ramp loaded bicycle exercise [ 39 , 40 ]. In contrast, we loaded using % MVC and reached 80–90% MVC at the end of exercise. Although % MVC was not expressed in those previous studies, it is possible that the peak intensity attained in our study was higher in those previously reported [ 23 , 39 , 40 ]. It is therefore conceivable that a larger amount of type II fibers were recruited in our study during exercise above the intensity where drop of muscle pH occurred. Since the energy cost of type II fibers is larger than that of type I fibers [ 41 , 42 ], an increase in type II fiber recruitment may produce greater changes in muscle PCr, ADP, o 2mus , o 2pul and ΔG ATP above the intensity during which a decrease in pH occurs. Another explanation for the different results between the earlier study [ 23 ] and ours (i.e. linear vs. nonlinear increase in ΔG ATP ) may be the difference in types of muscle contraction (i.e. concentric vs. intermittent isometric contraction). A nonlinear relationship between heat production, an indicator of ATP turnover rate, and force production during voluntary isometric contractions has been reported, although EMG activity continued to increase linearly with force production [ 43 ]. In addition, it is impossible to determine mechanical work for this type of static contraction. Therefore, voluntary isometric contraction does not necessarily show linear relationships between energy demand and exercise intensity or muscle electrical activity. One might criticize that despite the increasing exercise intensity in the initial phases, up to 30–40% MVC, there were only small changes in energy metabolism. At the onset of exercise, o 2pul showed a steep increase, which remained stable until 40% MVC. o 2pul and heart rate often exceed their steady state levels at the onset of exercise (phase I) during very low work rates [ 44 ]. This abrupt increase in o 2pul is due to the rapid elevation of cardiac output that drives mixed venous blood through the lungs [ 44 ]. It is possible, therefore, that phase I o 2pul exceeded the oxygen demand from the initial phase of exercise in this study. PCr, ADP, ΔG ATP , o 2mus , and o 2pul also changed only slightly during exercise below 30–40% MVC. We found however, that rmsEMG of m.MG, the same site as 31 P-MRS and NIR cws measurements, increased with increasing exercise intensity, and that rmsEMG of m.LG and m.SOL changed similarly with m.MG (Fig. 2 ). These results indicate that, although energy consumption changed slightly below 30–40% MVC, muscle electrical activity changed significantly with increased exercise intensity. It has been demonstrated that heat production increased only moderately with increasing contraction intensity during isometric contraction at low intensities, though EMG increased relative to contraction intensity [ 43 ]. Therefore, it appears that little metabolic change during exercise at low intensities is a characteristic of isometric contraction. One limitation of our study is that the bore diameter of the 31 P-MRS magnet used in this study was small (26cm), and it only permitted us to perform intermittent isometric plantar flexion. Isometric contraction is sensitive to occlude blood flow [ 45 ]. Therefore, one concern is that limited blood flow affected the results of this study. However, as far as we observe EMGs, the subjects fully relaxed between contractions even at highest intensities. In addition, metabolic parameters (pH, PCr, ΔG ATP ) of this study reached at the end of exercise were approximately same levels as reported data which performed incremental dynamic plantar flexion exercise [ 23 ]. The result obtained in this study could be comparable to the previous study that used dynamic exercise [ 23 ]. Although EMGs of plantar flexor muscles increased with increasing exercise intensity it is impossible to entirely eliminate the possibility that increases in o 2pul could include o 2mus from other muscles besides the plantar flexors. These should include muscles that maintain posture during exercise especially at high intensity. However, we observed a linear relationship between calf o 2mus and o 2pul (r = 0.975, p < 0.001). Moreover, the o 2pul kinetics was similar to muscle ADP and PCr which are thought to be important regulators of muscle oxidative metabolism. Therefore, we believe that the increase in o 2pul primarily derives from the increase in o 2mus in the active calf muscle. Conclusion o 2mus changed similarly with PCr and ADP throughout incremental intermittent isometric plantar flexion exercise. The increase in o 2mus accelerated under mild acidosis during exercise at high intensity. The point of acceleration coincided with rapid changes in muscle PCr and ADP. The results of this study suggest that rapid decrease in PCr (concomitant accelerative increase in ADP) under mild acidotic condition stimulates accelerative muscle oxidative metabolism during incremental intermittent isometric exercise at high intensity. Authors' contributions Toshiyuki Homma conceived the experimental design, carried out the experiment, and drafted the manuscript. Takafumi Hamaoka, M.D., Ph.D, participated in the design and coordination of the study, and directed throughout the study. Takayuki Sako, Ph.D., Motohide Murakami, M.D., Ph.D., Kazuki Esaki and Ryotaro Kime, Ph.D, participated in the study design and carried out the experiment. Toshihito Katsumura, MD., Ph.D, participated in the design and coordination of the study. All authors read and approved the final manuscript.

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1079910

Muscle oxygenation trends after tapering in trained cyclists

Background This study examined muscle deoxygenation trends before and after a 7-day taper using non-invasive near infrared spectroscopy (NIRS). Methods Eleven cyclists performed an incremental cycle ergometer test to determine maximal oxygen consumption (VO 2 max = 4.68 ± 0.57 L·min -1 ) prior to the study, and then completed two or three high intensity (85–90% VO 2 max) taper protocols after being randomly assigned to a taper group: T30 (n = 5), T50 (n = 5), or T80 (n = 5) [30%, 50%, 80% reduction in training volume, respectively]. Physiological measurements were recorded during a simulated 20 km time trials (20TT) performed on a set of wind-loaded rollers. Results and Discussion The results showed that the physiological variables of oxygen consumption (VO 2 ), carbon dioxide (VCO 2 ) and heart rate (HR) were not significantly different after tapering, except for a decreased ventilatory equivalent for oxygen (V E /VO 2 ) in T50 (p ≤ 0.05). However, during the 20TT muscle deoxygenation measured continuously in the vastus medialis was significantly lower (-749 ± 324 vs. -1140 ± 465 mV) in T50 after tapering, which was concomitant with a 4.53% improvement (p = 0.057) in 20TT performance time, and a 0.18 L·min -1 (4.5%) increase in VO 2 . Furthermore, when changes in performance time and tissue deoxygenation (post- minus pre-taper) were plotted (n = 11), a moderately high correlation was found (r = 0.82). Conclusion It was concluded that changes in simulated 20TT performance appeared to be related, in part, to changes in muscle deoxygenation following tapering, and that NIRS can be used effectively to monitor muscle deoxygenation during a taper period.

Background Near infrared spectroscopy (NIRS) is a non-invasive optical technique that is based on the differential absorption properties of hemoglobin (Hb) and myoglobin (Mb) in the near infrared (700–1000 nm) range. During the last decade, NIRS has been used extensively to measure muscle oxygenation both qualitatively [ 1 - 3 ] and quantitatively [ 4 - 7 ] during exercise. NIRS has become an appealing research tool due to its non-invasive nature for the measurement of localised blood volume and oxygenation. In these exercise studies, NIRS has been used to estimate the lactate threshold [ 8 , 9 ], to predict [ 10 ] and objectively [ 11 ] measure the ventilation threshold, to reflect exercise intensity [ 8 , 12 ], and to monitor muscle [ 13 ] and blood [ 14 ] metabolites. Validation [ 6 , 15 , 16 ] and reliability [ 1 , 2 , 17 ] studies have also been undertaken, and a theoretical model for muscle oxygenation (i.e., oxidative metabolism) measured by NIRS has been presented [ 18 ]. More recently, NIRS has been used to examine the effects of short-term endurance training on muscle oxygenation in a group of healthy untrained subjects [ 19 ], and competitive well-trained cyclists [ 20 ]. These studies showed that NIRS was a viable non-invasive technique to monitor muscle oxygenation, and to reflect the physiological adaptations in peripheral skeletal muscle following a short-term endurance-training program. Tapering is a training method performed by athletes during the days (3-21d) immediately prior to competition to create a "rebound" effect to improve performance. Physiological adaptations have also been reported during a period of tapering [ 21 - 26 ]. Both central cardiovascular (i.e., VO 2 max) and peripheral muscular (i.e., oxidative enzyme) changes have been documented and are correlated with improvements in performance following the taper period. It is likely that the enhanced oxidative capacity following tapering would elicit significant changes in muscle oxygenation. However, this has not been tested prospectively. The focus of this pilot study (due to the limited sample size in each group) was to test the hypothesis that muscle oxygenation changes, measured by NIRS, will occur following a period of tapering in a group of trained competitive cyclists. Furthermore, the possibility exists that NIRS can be used to monitoring the training status of an athletic and/or non-athletic population. Having a non-invasive technique that allows one to monitor peripheral changes and training adaptations occurring in the local muscle tissue will have a great impact on the understanding of local muscle tissue metabolism and haemodynamics. Methods Subjects These data are based on eleven competitive male cyclists between the ages of 19–34 yr that volunteered to participate in this study. Some of these subjects completed more than one taper, explaining why each group had an equal number of five subjects per group. Two subjects completed all three tapers, while three subjects completed two of the tapers. Physical characteristics for the eleven subjects were: age (Mean ± SD) = 22.6 ± 4.7 yr, height = 177.0 ± 5.0 cm, body mass = 70.3 ± 5.0 kg, thigh skinfold thickness = 9.0 ± 1.8 mm, VO 2 max = 4.68 ± 0.57 L·min -1 (66.5 mL·kg -1 ·min -1 ). Each subject completed a written informed consent after a thorough explanation of the protocols and procedures involved in accordance with an institutional ethics review committee. Testing Prior to the taper phase, each subject completed an incremental VO 2 max test on a calibrated Monark cycle ergometer (Model 818E, Varberg, Sweden) as described previously [ 27 ]. Following a two minute rest period while sitting stationary on the cycle ergometer, each cyclist began pedalling at an initial work rate of 80 watts (W) for 2 min, followed by 45 W increments every minute up to 260 W. Thereafter, work rate was increased each minute by 20 W increments to volitional fatigue. This test lasted approximately 10–14 min in duration. Expired gases were collected and analysed by open circuit spirometry by using an automated metabolic analysis system (Vmax, Sensormedics, California, USA). The data were averaged in 20 sec intervals. The gas analysers were calibrated with primary standard gas (16.0% O 2 , 4.0% CO 2 , balance N 2 ) before and after each individual test. The pneumotach was calibrated using a 3L syringe. The following criteria were used to verify VO 2 max: a respiratory exchange ratio (RER) greater than 1.12, and an increase in VO 2 uptake less than 100 mL with an increase in work rate [ 28 ]. All subjects achieved these criteria. Heart rate (HR) was monitored continuously by telemetry (Polar monitors, Electro, Finland). Because of the endurance characteristics of these cyclists (both road and mountain bike), a simulated 20TT performance ride was used as the criterion test (i.e., index of performance) before and after tapering to evaluate the physiological and performance effects of each taper protocol. Each cyclist was asked to complete the ride as fast as possible, with no feedback provided on how well he was performing until the end of the test. The pre-taper 20TT was used as the last workout prior to tapering. The description of this test has been reported in detail previously [ 27 , 29 ]. Briefly, each cyclist used his own bicycle mounted on a set of aluminium cast wind-loaded cycling rollers fitted with a stabilizing bar that was attached to the handle bar of the bicycle for safety. The air pressure of the tires was checked before and after each ride to ensure that maximum pressure was maintained. The same set of cycling rollers was used for all simulated 20TT rides with the rollers being interfaced with a computer to record velocity, distance, and cycling time. This device was calibrated by measuring the circumference of the rollers (and thus the distance was a product of the circumference and rpm, which was recorded by the computer [ 29 ]). Respiratory gas exchange responses (averaged over 20 sec) were monitored for 2–3 min every 5 km during the pre-and post-taper simulated 20TT. The same calibration procedures were performed on the gas analysers as previously stated above for the VO 2 max test. HR was monitored continuously throughout the duration of the 20TT using telemetry (Polar, Finland). The Rating of Perceived Exertion (RPE) was also taken at the end of each work rate throughout the VO 2 max test, and during each 5 km interval using the Borg Scale [ 30 ]. Taper Protocols The experimental design for this study is illustrated in Table 1 . A 3-week high-intensity endurance-training period preceded the taper for consistency in training volume between cyclists. This involved indoor stationary cycling (Blackburn Magnetic Turbo Trainer, USA) 4 sessions·wk -1 , for 60 minutes·session -1 at 85–90% VO 2 max. Heart rate was monitored for all cycling rides using telemetry, and each ride was supervised to ensure that the cyclist maintained their required training intensity. Prior to this consistency period, these cyclists were road training approximately 150–250 km·wk -1 . Before the 7-day taper began, each cyclist was randomly assigned to one of three taper protocols: T30 = 30% reduction in weekly training volume (n = 5), T50 = 50% reduction in weekly training volume (n = 5), T80 = 80% reduction in weekly training volume (n = 5). However, for those cyclists that completed more than one taper protocol, a 3-week baseline training period was implemented to avoid any interference between tapers [ 31 ]. Training volume in this study was defined as a combination of duration and frequency (see Table 1 ). Table 1 Experimental design for the 7-day (DO to D7) taper protocols. Time is expressed in minutes, and denoted in the table with an apostrophe ('); 20TT = simulated 20 km time trial performance ride. TAPER DO D1 D2 D3 D4 D5 D6 D7 T30 (n = 5) 20TT Rest 60' 55' 50' 45' Rest 20TT T50 (n = 5) 20TT Rest 45' 40' 35' 30' Rest 20TT T80 (n = 5) 20TT Rest 30' 15' 10' 5' Rest 20TT Near Infrared Spectroscopy (NIRSCWS) A continuous dual wavelength spectrometer (NIR CWS ) (RunMan, NIM Inc., PA, USA) was used to measure tissue absorbency (Hb/Mb-O 2 ; mV) of the right vastus medialis muscle at 760 nm and 850 nm in each subject during the 20TT. The procedures and protocols involving the use of the NIR CWS during this experimentation have been reported previously [ 3 , 27 ]. Briefly, the NIR CWS probe was calibrated at 760 nm and then 850 nm according to the manufacturer's protocol and specifications prior to each test. This was done while the subject was seated on his bicycle with the leg in a relaxed position at the lowest point of the pedal. The electrical output of the NIR CWS unit was adjusted by initially setting the balance at 0 ± 10 mV and then adjusting the gain control to read between 600 to 1000 mV at 760 nm. The absorbency measurements were then checked at 850 nm to ensure that the values were negative in value and within 10% of those observed at 760 nm. A settling time of approximately 20 sec was allowed at each wavelength to allow for stability of the readings. The calibration was repeated twice before each test to ensure accuracy of the data. In this study the NIR CWS unit was interfaced with a computer as previously described [ 2 , 11 ] with the data viewed on-line during each test. The inter-session reliability of the minimum deoxygenation value for this muscle group during cuff ischaemia in this laboratory is 0.95 [ 2 ]. To record the NIR CWS signal, the probe was placed over the right vastus medialis muscle, approximately 14–20 cm from the knee joint along the vertical axis of the thigh [ 1 ]. At this time, a thigh skinfold measurement (Harpenden Skinfold Calipers) was also taken to record subcutaneous adiposity to ensure that skinfold thickness was not a confounding variable for the measurement of muscle oxygenation (Table 2 ). After measuring and locating the desired anatomical position, a permanent ink mark was placed on the leg of each cyclist, and recorded for subsequent post-taper NIR CWS measurements. A piece of clear plastic wrap was placed around the probe to cover the photo-detectors to prevent distortion of the signal caused by sweating during exercise. The signal was recorded at a depth of approximately 1.5 cm (i.e., separation distance between the emitter and detector sensors was 3 cm), and at sampling frequency of 30 Hz using an A/D board and customized software interfaced with the computer [ 11 ]. Measurements were undertaken continuously for two minutes at rest (i.e., baseline value), throughout exercise (25–30 minutes) and for six minutes of recovery. During the first two minutes of the recovery period the subject pedalled their bicycle at a comfortable intensity (active recovery), while during the remaining four minutes the cyclist sat on the bicycle with the right foot in a relaxed position at the bottom of the pedal (passive recovery). The raw NIR CWS signal (mV) was used to reflect the trend in tissue oxygenation, and this method has been reported previously in the literature [ 20 , 27 ]. Furthermore, both the oxy-Hb (850 mV) and the deoxy-Hb (760 mV) signals were examined from this set of data. These signals demonstrated an opposite trend, indicating that changes in the oxygenation (Hb/Mb-O 2 ) and deoxygenation (Hb/Mb) most likely reflect changes in oxygen extraction. Therefore, the Hb/Mb-O 2 signal was used to represent the findings from this study. This signal was averaged every 20 seconds to correspond with the metabolic measurements. For comparison of the NIR CWS data before and after tapering, a delta score was calculated by subtracting the initial resting baseline value (averaged over 2 minutes of rest) from each 20 second averaged value throughout the complete test [ 20 ]. This subtraction eliminates basal muscle metabolism [ 18 ] and allows a direct comparison before and after tapering, and among individuals. Table 2 Physiological responses during the simulated 20 km time trial (20TT) Pre- and Post-Taper. Values are Mean (SD). T30 (30% reduction in weekly training volume; n = 5), T50 (50% reduction in weekly training volume; n = 5), T80 (80% reduction in weekly training volume; n = 5). * indicates significantly different from Pre-test, p ≤ 0.05; #indicates p = 0.057 from Pre-test. Variable Pre-Taper Post-Taper T30 T50 T80 T30 T50 T80 Time (min) 27:03(1:29) 26:34(0:56) 26:10(2:58) 27:40(2:34) 25:25(1:23)# 25:31(2:07) VO 2 (L·min -1 ) 3.28 (0.54) 4.04 (0.24) 4.09 (0.59) 3.17 (0.56) 4.22 (0.11) 4.15 (0.39) %VO 2 max 74.6% 86.9% 84.7% 72.1% 90.7% 85.3% VCO 2 (L·min -1 ) 3.39 (0.95) 3.82 (0.26) 3.84 (0.49) 3.31 (0.93) 4.07 (0.21) 4.07 (0.31) V E (L·min -1 ) 81.0 (17.2) 102.1 (12.2) 103.1(13.2) 81.7 (16.4) 95.0 (5.9) 106.1 (10.2) V E /VO 2 24.7 (1.9) 25.3 (2.2) 25.2 (1.1) 25.8 (1.0) 22.5 (1.7) * 26.1 (1.2) RER 1.03 (0.07) 0.95 (0.02) 0.94 (0.03) 1.04 (0.03) 0.97 (0.04) 0.98 (0.03) HR (b·min -1 ) 169 (5) 175 (9) 173 (13) 173 (4) 174 (13) 179 (5) O 2 pulse (mL·b -1 ) 19.3 (2.8) 23.2 (2.4) 23.6 (1.8) 18.3 (3.3) 24.3 (2.2) 21.9 (1.4) Hb/Mb-O 2 (mV) -455 (270) -731 (155) -770 (183) -403 (170) -1167 (224) * -847 (106) RPE 13.9 (2.3) 16.2 (1.6) 16.3 (1.3) 14.9 (2.0) 16.8 (1.5) 16.3 (1.2) Thigh Skinfold (mm) 8.9 (1.8) 9.2 (1.7) 9.0 (1.9) 9.0 (1.9) 9.1 (1.6) 9.2 (1.8) Statistic analysis An analysis of variance model (ANOVA) was used initially to determine the homogeneity of variance of the data. A Levine's test (on absolute deviations) for Group × NIRS was violated (df = 2,387; F = 17.11; p = 0.000), and therefore a Mann-Whitney U non-parametric test was used to determine significant differences between pre- and post-taper trials. For the purpose of this study, the alpha level for significance was set at p ≤ 0.05. All statistical analyses were performed using Statistica 6.0 software program (Stats Soft, Tulsa OK, USA). All values are expressed as Mean ± SD. A significant Group main effect was apparent, but post hoc analysis revealed non-significant means pre- to post-taper for all dependent (cardiovascular) variables measured except for a significant different V E /VO 2 ratio in the T50 group. Results Cardiovascular responses All subjects attained their individual VO 2 max according to the criteria established (see Methods). The average exercise intensity during the 20TT was between 72% and 90% VO 2 max. Table 2 illustrates the physiological responses of these cyclists during the simulated 20 km time trials before and after tapering, showing that a significantly lower V E /VO 2 ratio (25.3 ± 2.2 to 22.5 ± 1.7; p ≤ 0.05) occurred in the T50 group. Performance time was improved on average by 1:09 min:sec in the T50 group (26:34 ± 0:56 to 25:25 ± 1:23; p = 0.057), but unchanged in the other groups (Table 2 ). Muscle Oxygenation Trends The trend in mean muscle oxygenation (Hb/Mb-O 2 ) during the 20TT before and after the taper for each group is shown in Figure 1 . A consistent pattern in tissue deoxygenation was found for all performance rides. Tissue Hb/Mb-O 2 showed a rapid decrease during the first 2 km of the simulated race, followed by a gradual decline or plateau until the end of the race (range = 25 to 30 minutes). Mean tissue oxygenation values for the complete test (Table 2 ) was not different (U = 2038, z = -0.347, p ≥ 0.05) for the T30 protocol (-455 ± 270 to -403 ± 170 mV), and the T80 (U = 1691, z= 1.96, p ≥ 0.05) protocol (-770 ± 155 to -847 ± 106 mV), but was significantly greater (i.e., increased muscle deoxygenation) following the 50% reduction (T50) training volume taper (U = 642, z = 6.85, p ≤ 0.05) protocol (-731 ± 155 to -1167 ± 224 mV). It was interesting to note that the T50 group had a decreasing (p ≤ 0.05) trend in tissue Hb/Mb-O 2 with a concomitant 0.18 L·min -1 (4.5%) increase in oxygen consumption after tapering during the 20TT. Figure 1 Group mean (± SD) tissue oxygenation (Hb/Mb-O 2 ; Absorbency, mV) during the simulated 20 km time trial performance ride Pre- and Post-taper in the three taper groups. Panel T30 (30% reduction in weekly training volume; n = 5); Panel T50 (50% reduction in weekly training volume; n = 5); Panel T80 (80% reduction in weekly training volume; n = 5). * denotes significance (p ≤ 0.05) between pre- vs. post-taper trials. All three groups of cyclists also showed a delay in tissue Hb/Mb-O 2 during the recovery phase following the 20TT ride. In fact, muscle oxygenation did not return to baseline before or after tapering, and remained well below baseline during this recovery phase (Figure 1 ). In general, the trend in muscle deoxygenation followed the metabolic response of each cyclist during the simulated ride. Also, it was interesting to note that there was a moderately high correlation (r = 0.82) between the change in tissue Hb/Mb-O 2 vs. 20TT taper performance time when all cyclist were combined (Figure 2 ). The delta value was calculated by post-taper minus pre-taper score. Figure 2 Relationship (r = 0.82) between the change (delta) in Tissue Hb/Mb-O 2 (mV) vs. 20TT Taper Performance time (minutes) for all subjects combined (n = 11) after tapering. Note that a negative value for performance time indicates an improvement in performance (i.e., a faster 20TT ride). Delta scores were determined by post-taper minus pre-taper values. Each symbol represents a different taper group (● T30; ■ T50; ▲ T80). Discussion The results of the present study are the first to show that peripheral adaptations in muscle oxygenation can occur with a proper preparation period prior to competition (i.e., taper), and that these adaptations can elicit improvements in performance. For example, the T50 protocol resulted in a significantly greater muscle deoxygenation after the taper with a concomitant increase in O 2 uptake (4.5%) and an improvement (4.53%) in performance (Table 2 ). It was also interesting to note that when the change in tissue Hb/Mb-O 2 was plotted against the change in 20TT taper performance time for all subjects combined (n = 11), a moderately strong correlation was found (Figure 2 ; r = 0.82). This suggests that metabolic changes that occur in the vastus medialis muscle are, in part, responsible for changes in performance. The decrease in oxygen levels presented in this paper is consistent with an increase in oxygen extraction (i.e., more oxygen uptake for a given blood flow). Previous studies have shown both oxygen delivery and oxidative metabolism to improve with training [ 19 , 32 ], and now, presumably with a taper. This is consistent with previous research on tapering which showed that changes in oxidative enzyme activity (cytochrome oxidase) correlated (r = 0.84) with changes in 40 km time trial performance time [ 33 ]. As indicated earlier, the intent of this study was to examine the effects of tapering on muscle oxygenation using NIR CWS , and not to test the differences between groups per se. One limitation of this study was the small sample size in each of the taper groups (n = 5). Notwithstanding this limitation, it was still evident from these data that NIR CWS successfully measured the trends in muscle oxygenation during the three different taper protocols. Using non-parametric statistics (Mann-Whitney U), NIR CWS demonstrated that the T50 protocol (50% reduction in weekly training volume) resulted in a significantly greater muscle deoxygenation (i.e., a difference in the balance between O 2 delivery and utilisation) after the 7-day linear taper. This increase in muscle deoxygenation resulted in a concomitant increase of 0.18 L·min -1 of O 2 uptake during the time trial for the T50 group, which translated into a 4.53% improvement in 20TT performance time (26:34 ± 0:56 to 25:25 ± 1:23 min:sec; p = 0.057). A number of physiological adaptations to tapering have been reported in the literature. For example, the oxidative capacity of the muscle is enhanced as a result of an increase in oxidative enzyme activity [ 21 , 31 , 33 ]. As well, an elevated muscle glycogen concentration [ 21 , 31 ], and, improved haematological and biochemical profiles following a period of tapering in competitive athletes [ 23 , 34 ] have been documented. More recently, Trappe et al. [ 35 ] showed physiologic changes in single muscle fibres after tapering. Furthermore, tapers in which a systematic step-wise reduction in training volume was maintained at high exercise intensity showed greater improvements in performance [ 22 , 24 , 31 , 36 ]. Thus, the results of the present study also support the contention that peripheral adaptations can occur with a proper preparation period prior to competition (i.e., taper), and can elicit improvements in performance. Physiologically, acute changes in tissue Hb/Mb-O 2 are related to changes in muscle blood flow, O 2 extraction and venous return [ 1 ]. An increased O 2 extraction [i.e., arterio-venous O 2 difference; (a-v)O 2diff ] during the taper is most likely the result of an increase in oxidative enzyme activity, and/or an improvement in the haematological parameters, both of which have been shown to improve with tapering [ 21 , 23 , 31 ]. The (a-v)O 2 diff itself can be affected by the rate of diffusion of oxygen from the blood into the myocyte, the [Hb], and by the oxyhemoglobin dissociation curve. It is reasonable to assume that the significant increase in muscle deoxygenation in the T50 group after the 7-day taper period in the present study was related to the above mentioned cellular and biochemical changes. An increased (a-v)O 2diff results because of the structural adaptations in skeletal muscle in response to alterations in metabolic demand. However, a second limitation of this study is that we do not have muscle biopsy or blood data to confirm our hypothesis. Albeit, the physiological consequence of these peripheral adaptations would mean that the working skeletal muscle can extract greater amounts of O 2 , which in turn should allow for an improved endurance performance [ 37 ]. Endurance performance on the 20TT for the T50 group reached a significance level of p = 0.057 when comparing the pre- to post-taper test results, although previous research in our laboratory has demonstrated that 20TT performance was significantly increased in a slightly larger group (n = 6) of well-trained cyclists when using a similar protocol [ 38 ]. Furthermore, research has demonstrated that skeletal muscle adaptations (i.e., increased oxidative capacity and mitochondrial activity) can coincide with positive changes in performance following a taper period [ 21 , 31 , 35 ]. However, it is unknown whether the changes in muscle deoxygenation measured by NIRS CWS correlate with changes in oxidative enzymes during the taper, although Puente-Maestu et al. [ 32 ] showed that changes in the recovery deoxygenation signal (τHbO 2 ) correlated (r = 0.76) with changes in citrate synthase enzyme activity in muscle biopsy tissue. Another possibility for the increased deoxygenation during the taper maybe related to increases in blood flow to the skeletal muscle. Green et al. [ 39 ] speculated that alterations in blood flow might be one of the earliest adaptive responses to training, and possibly capable of eliciting the same physiologic advantage as the increase in the muscles enzymatic machinery. Research is available to demonstrate that increases in plasma volume and red blood cell volume occur after tapering [ 40 ]. This would allow a greater blood flow to be available for the contracting muscle post-taper. Unfortunately, we do not have this data available to confirm this hypothesis. Therefore, whether the increased muscle deoxygenation from pre- to post-taper (T50) is related to the alteration in muscle blood flow, and simultaneous increases in oxidative enzymes and blood (cell) volume, is yet to be determined. It is now possible using spatially resolved spectroscopy (NIRS-SRS) to examine the contribution of blood volume. Research has also shown that changes in blood lactate, both acute [ 8 , 9 ], and chronic [ 19 ], can also influence the NIR CWS signal during exercise. The data from this study is also important as it reflects individual cyclists' responses to each particular taper protocol. For example, in the T50 protocol four of the five cyclists demonstrated significant muscle deoxygenation trends after tapering. As well, an examination of the T80 individual results showed that four of the five cyclists demonstrated this decline in muscle oxygenation following tapering although no significant difference occurred post-taper in the T80 group. Future research is warranted to determine the reason(s) for this. In general, those in the T50 and T80 taper protocols showed a greater muscle deoxygenation, which coincided with their personal changes in performance and physiological (VO 2 , VCO 2 , V E , HR, RER) measurements (also see Figure 2 ). A closer examination of Figure 2 illustrates that the T50 group had faster performance times and a great deoxygenation. However, four of the five cyclists in the T30 taper protocol showed the opposite effect (i.e., greater muscle oxygenation and an unchanged or slower performance time). These results would suggest that the minimal 30% reduction in weekly training volume during the taper was not enough to elicit physiological or performance adaptations, possibly due to residual fatigue accompanying the excessive training during the taper phase. This is consistent with the philosophy that sufficient rest is needed to express the adaptations concomitant with training, and, supports previously published literature that optimal amounts of rest and exercise are needed during the taper [ 21 , 24 - 26 , 31 , 33 ]. The improved performance time during the simulated 20TT in the T50 taper group was consistent with the decreased tissue Hb/Mb-O 2 (NIR CWS ) data (-731 ± 155 to -1167 ± 224 mV; p ≤ 0.05). This supports recent work by Nioka et al. [ 41 ] and Bae et al. [ 8 ] who showed that the degree of muscle deoxygenation varies in accordance with the intensity of exercise and the level of training. Thus, it appears that NIR CWS was sensitive enough to detect the changes in muscle oxygenation following the different taper protocols, and NIR CWS is a viable non-invasive technique to monitor muscle oxygenation in peripheral skeletal muscle following a taper period. Puente-Maestu et al. [ 32 ] concluded that "NIRS may be a useful non-invasive tool for detecting the adaptations of skeletal muscle to training". Another important observation from this data was that muscle re-oxygenation during recovery from the prolonged high-intensity exercise during the 20TT was delayed and did not return to baseline after 6 minutes of (active and passive) recovery. Although some reports have shown that Hb/Mb-O 2 resaturation is not affected by pH when using small muscle mass [ 42 ], a delayed re-oxygenation has been shown in highly oxidative muscle following isometric contractions [ 43 ]. However, these conditions are different from the current study which used whole body exercise and involved a greater muscle mass (i.e., vastus medialis). Im et al. [ 44 ] showed that muscle desaturation was related (r = 0.83) to whole body VO 2 during dynamic exercise involving a large muscle mass. Therefore, taken together, these data suggest that a number of possible factors may be influencing this re-oxygenation including, but not limited to, muscle fibre type, type of muscular contraction and mode of exercise, capillary recruitment immediately post exercise, a high intramuscular temperature, and the presence of muscle metabolites (i.e., [H + ], increased lactic acid). Further research is needed to identify the relative contribution of those factors that are associated with the delayed recovery as measured by NIRS. Conclusion In summary, these results are somewhat unique and suggest that a positive adaptation to taper training is that blood volume and metabolic changes lead to an increase in O 2 extraction. Furthermore, that increased oxygen extraction is associated with increased performance. Therefore, it appears that NIRS CWS can be used to record the peripheral metabolic changes in trained endurance cyclists after a period of taper training, and this research has contributed further to our understanding of the application of NIRS to exercise sports science [ 45 - 47 ]. Authors' contributions JPN and DCM conceived the study and wrote the research grant; YNB participated in its design, coordination and provided the NIRS equipment and expertise; JPN and YNB carried out the testing and data analysis of the study. All authors read and approved the final manuscript.

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1079911

Distributional interaction: Interpretational problems when using incidence odds ratios to assess interaction

It is well known that the incidence odds ratio approximates the risk ratio when the disease of interest is rare, but increasingly overestimates the risk ratio as the disease becomes more common. However when assessing interaction, incidence odds ratios may not approximate risk ratios even when the disease is rare. We use the term "distributional interaction" to refer to interaction that appears when using incidence odds ratios that does not appear, or appears to a lesser degree, when using risk ratios. The interpretational problems that arise from this discrepancy can have important implications in epidemiologic research. Therefore, quantification of the relationship between the interaction odds ratio and the interaction risk ratio is warranted. In this paper, we provide a formula to quantify the differences between incidence odds ratios and risk ratios when they are used to estimate effect modification on a multiplicative scale. Using this formula, we examine the conditions under which these two estimates diverge. Furthermore, we expand this discussion to the implications of using incidence odds ratios to assess effect modification on an additive scale. Finally, we illustrate how distributional interaction arises and the problems that it causes using an example from the literature. Whenever the risk of the outcome variable is non-negligible, distributional interaction is possible. This is true even when the disease is rare (e.g., disease risk is less than 5%). Therefore, when assessing interaction on either an additive or multiplicative scale, caution should be taken in interpreting interaction estimates based on incidence odds ratios.

Introduction It is well known that the incidence odds ratio approximates the risk ratio when the disease of interest is rare, but increasingly overestimates the risk ratio as the disease becomes more common. Therefore, it may not be surprising that odds ratios can give the impression of effect modification when none exists among the corresponding risk ratios. Despite this intuition, there is little formal discussion of this phenomenon in the epidemiologic literature. In what follows, we address this issue in an effort to enhance our understanding of the influence of effect measure selection on the interpretation of study results. The risk ratio, which compares the probability of disease in the exposed and unexposed, is a conceptually appealing measure of effect. A risk ratio (RR) of 2, for example, is easily understood as a doubling of risk. This is in stark contrast to the interpretational obscurity of the incidence odds ratio (OR) [ 1 ]. When we use the term "incidence odds ratio," we are referring to the OR calculated from data on disease status obtained at the end of the observation period. This is the type of odds ratio calculated from fixed cohort studies and from case control studies that use cumulative sampling methods (see endnote 1). Despite this interpretational problem, the OR is frequently used because of its appealing statistical properties. The interpretational hurdle is overcome by treating the OR as if it were a RR, by understanding an OR of 2, for example, as a doubling of risk (see endnote 2). This interpretational license is innocuous as long as the RR is reasonably small and the probability of disease in the unexposed is reasonably low. Several formulas have been developed to quantify the discrepancies between the RR and the OR and thus prevent misinterpretation [ 2 - 5 ]. The general principle that the OR approximates the RR under the rare disease assumption is well known. The implications of the "rare disease assumption" for the interpretation of interaction (i.e., effect modification) on the multiplicative or additive scale when using ORs have received less attention. However, interpreting the OR as if it were an RR can be particularly misleading when examining effect modification [ 6 ], as illustrated in the hypothetical cohort study presented in Table 1 . For simplicity, we constructed this and all our examples with no confounding and no sampling or measurement error. Table 1 Illustrative example: a hypothetical cohort study of the relationship between low aspirin intake and polyps among individuals with and without the high-risk COX-2 genotype Individuals with high-risk COX-2 genotype Individuals without high-risk COX-2 genotype Polyps No Polyps Polyps No Polyps Low aspirin intake 75 25 30 70 High aspirin intake 25 75 10 90 Risk Ratio 3.0 3.0 Odds Ratio 9.0 3.9 In this illustrative example, the RR for the relationship between low aspirin intake and the presence of polyps is the same for individuals with and without the high-risk COX-2 genotype. That is, the RRs do not provide evidence for effect modification on a multiplicative scale. Among those without the high-risk genotype, for whom the incidence of the disease is low, the OR is a close approximation of the RR. Among those with the high-risk genotype, however, where the disease is common, the OR is not a good approximation of the RR. This leads to considerable heterogeneity of the OR that would be interpreted as evidence of effect modification. Morabia et al. referred to this discrepancy as "the interaction fallacy" [ 7 ]. However, as Walter notes, this is not a statistical fallacy, since there is indeed effect modification of the OR [ 8 ]. Nonetheless, there is an interpretational fallacy if the conclusion reached based on these results is that low aspirin intake increases the risk (on a multiplicative scale) of polyps more for individuals with the high-risk COX-2 genotype than for individuals without the high-risk genotype. The effect of the risk factor appears to differ between strata due solely to a difference in the disease risk between strata. That is, this artifact appears because the disease is differentially distributed across strata. We therefore refer to this phenomenon as "distributional interaction" (see endnote 3). To avoid this interpretational problem, we need to know the conditions under which effect modification of the OR is likely to approximate effect modification of the RR and when it is not. This understanding necessitates clarification of the elements that cause this discrepancy. Although Morabia et al. reported the existence of this phenomenon, these elements have not been previously explicated in the literature. Furthermore, this discussion has not been extended to additive interaction. To address this problem, we 1) provide a quantification of the relationship between effect modification of the RR and the OR on both multiplicative and additive scales, 2) examine the sensitivity of these relationships to variations in their determinants, and 3) use an example from the literature to highlight the causes and consequences of distributional interaction on either the multiplicative or additive scale. Analysis Multiplicative interaction is commonly assessed by comparing the relative effect estimates for one risk factor across strata of another risk factor. Heterogeneity of the effect across strata is evidence of interaction. Here, we will use an equivalent method of assessing multiplicative interaction that we find more conceptually meaningful. This notion of interaction refers to the extent to which the joint effect of the two risk factors on disease differs from the independent effects of both factors. The joint effect is the effect of the presence of both factors on disease. Each factor's independent effect is its effect in the absence of the other factor. A comparison between the joint effect and the product of the independent effects provides a measure of multiplicative interaction. This calculation of multiplicative interaction (i.e. the ratio of the joint effect to the product of the independent effects) using RRs is shown below. Throughout this paper, we refer to an interaction between a genetic and an environmental factor, as in the example of COX-2 genotype and aspirin in causing polyps described above. This discussion could be extrapolated to an interaction between any two factors (i.e. two genetic factors, two environmental factors, etc.). Notation Here, G represents the genetic factor, E represents the environmental factor and D represents the disease. All three factors are dichotomous. For these factors, upper-case letters indicate the presence of the factor and lower-case letters indicate the absence of the factor. Assuming a multiplicative scale, the causal effect of the G-E interaction on D measured using RRs will be referred to as GxE RR . Similarly, the causal effect of the G-E interaction on D measured using ORs will be referred to as the GxE OR . The baseline risk of disease is represented by p(D|ge). This refers to the probability of disease [p(D)] among those who have neither the genetic nor the environmental risk factor. Assessment of Multiplicative Interaction Population N is a population that underlies a hypothetical cohort study. The members of this population are categorized according to their exposure to G and E and followed for incident disease. Because these two exposures are dichotomous, individuals are classified according to four exposure categories. This hypothetical cohort study is illustrated in figure 1 . Figure 1 Schematic representation of study cohort In this cohort study, those exposed to both G and E are represented by N 11 , N 10 represents the sub-population that is exposed to G and unexposed to E, N 01 corresponds to the sub-population that is unexposed to G and exposed to E, and N 00 corresponds to the sub-population that is unexposed to both factors. These individuals can be further classified according to their D status. The cells labeled a 1 to d 1 represent those exposed to G and the cells labeled a 2 to d 2 represent those unexposed to G. To demonstrate the calculation of the interaction between G and E using RRs, we have organized the cells a 1 to d 2 into the two-by-two tables presented in figure 2a . The joint and independent effects of G and E, and the calculation of the gene-environment interaction are shown in figure 2b . The baseline risk of disease, or the risk of disease attributable to factors other than G and/or E, is represented by (c 2 / N 00 ). The RR for the joint effect of G and E (RR GE ) is calculated by comparing the risk of disease among those who are exposed to both G and E to the baseline risk. The RR for the independent effect of G (RR Ge ) compares the risk of disease among those who are exposed to G and unexposed to E to the baseline risk. Similarly, the RR for the independent effect of E (RR gE ) compares the risk of disease among those who are unexposed to G and exposed to E to the baseline risk. Using these components, the gene-environment interaction based on RRs (GxE RR ) is measured by the ratio of the joint RR to the product of the independent RRs. Figure 2 Assessment of multiplicative interaction using risk ratios and odds ratios. Using the same notation and the two-by two tables shown in parts a and b of figure 2 , the joint and independent effects of G and E measured by ORs can also be derived (see figure 2c ). Just as the joint and independent effects measured by RRs are used to calculate the GxE RR , the joint and independent effects measured by ORs are used to calculate the GxE OR . Relationship between the GxE RR and the GxE OR Several algebraically equivalent formulations of the relationship between the OR and the RR have been discussed in the literature [ 2 - 5 ]. This relationship can be expressed in terms of two components – the size of the RR and the magnitude of the baseline risk, i.e. the risk of the disease in those who are unexposed (equation1) [ 4 ]. Similarly, the relationship between the GxE OR and the GxE RR depends in part on the magnitude of the GxE RR and the baseline risk of disease. Equation 1 can be used to calculate the OR for each of the three components of the GxE RR , resulting in the OR GE , OR Ge and OR gE . Consequently, the relationship between the GxE RR and the GxE OR can be described using equation 2, shown below. A rearrangement of this formula is shown below in equation 3. Equation 3 illustrates the factors that determine the relationship between the GxE RR and the GxE OR : the size of the GxE RR , the magnitude of the baseline risk of disease, and the strength of the independent effects. For illustration, the data for the relationship between aspirin, COX-2 genotype and polyps shown in table 1 can be rearranged according to the three tables depicted in figure 2 . In this example, the GxE RR is 1 and the baseline risk of disease (i.e. the probability of disease among individuals without the high-risk genotype and with high aspirin intake) is 10%. Additionally, the RR for the independent effect of low aspirin intake (among individuals without the high-risk genotype) is 3 (RR Ge = 3), the RR for the independent effect of the high-risk COX-2 genotype (among those with high aspirin intake) is 2.5 (RR gE = 2.5), and the RR for the joint effect of low aspirin intake and the high-risk COX-2 genotype is 7.5 (RR GE = 7.5). When these four elements are entered into equation 3, the GxE OR is 2.3. Effect of Disease Risk on the Relationship between GxE OR and GxE RR In the absence of independent effects, equation 2 reduces to equation 1. That is, the relationship between the GxE OR and the GxE RR is equivalent to the relationship between the OR GE and the RR GE. This relationship can be fully expressed in terms of the RR GE and the baseline risk. When the RR is 1, the OR is also 1, regardless of the baseline risk of disease. Similarly, when the GxE RR is 1 and there are no independent effects, the GxE OR is also 1, regardless of the baseline risk of disease. In the absence of independent effects, as the magnitude of the GxE RR increases, the GxE OR diverges from the GxE RR . At a constant GxE RR , the divergence increases as the baseline risk of disease increases and becomes appreciable as the baseline risk of disease approaches 10%. Thus when there are no independent effects, the relationship between the GxE OR and the GxE RR is precisely the same as the relationship between the OR and the RR used to estimate main effects. When there are independent effects, the relationship between the GxE OR and GxE RR is more complex. Figure 3 illustrates the situation in which the RR for each independent effect is 2. As shown in this graph, the GxE OR is a good approximation of the GxE RR when the disease is very rare [p(D|ge) = 1%], regardless of the magnitude of the GxE RR . However, as the baseline risk of disease approaches 5%, the GxE OR begins to appreciably overestimate the GxE RR . In general, the larger the baseline risk of disease, the worse the approximation, even at moderate strengths of interaction. In fact, when the risk of the disease is 20%, the GxE OR indicates a strong interaction when the RRs do not provide evidence for interaction. Figure 3 The effects of the baseline risk of disease [p(D|ge)] and the magnitude of the multiplicative interaction estimate based on risk ratios (GxE RR ) on the multiplicative interaction estimate based on odds ratios (GxE OR ) when the RR for each independent effect is 2 (RR Ge = RR gE = 2). Figure 4 highlights the situations in which a researcher would reasonably conclude that there is no evidence for a substantial multiplicative interaction based on RRs (i.e. the GxE RR ranges from 1.0 to 1.5). In this circumstance, the GxE OR is a fair approximation to the GxE RR when the disease is very rare [p(D|ge) = 1%]. That is, a researcher using the GxE OR would appropriately conclude that there is no evidence for a multiplicative interaction. Here, as the baseline risk of disease approaches 3%, the GxE OR begins to appreciably overestimate the GxE RR . For example, when the GxE RR is 1.3, the GxE OR is 2. Although this is not a large overestimation it has important implications for interpretation. A researcher may not find a GxE RR of 1.3 indicative of a substantial multiplicative interaction, but may conclude otherwise when the estimate is a 2. Again, the larger the baseline risk of disease, the worse the approximation. When at least one of the independent effects is strong and the risk of the disease is greater than 1%, the GxE OR is a poor approximation to the GxE RR even when the GxE RR is weak. Figure 4 The effects of the baseline risk of disease [p(D|ge)] and the magnitude of the multiplicative interaction estimate based on risk ratios (GxE RR ) on the multiplicative interaction estimate based on odds ratios (GxE OR ) when the RR for the independent effect of G is 6 (RR Ge = 6) and the RR for the independent effect of E is 2 (RR gE = 2). Implications for Assessment of Additive Interaction The analyses we have shown so far indicate that using ORs to assess multiplicative interaction can lead to interpretational difficulties. We have focused on multiplicative interaction because this is the most common scale on which interaction is assessed when ORs are used as a measure of effect. However, the appropriate scale on which to assess interaction has been a controversial topic in epidemiology. Darroch [ 9 ] and Rothman and Greenland [ 10 ] have mounted a persuasive argument for the use of the additive scale to assess the presence of synergistic effects. As they note, additive interaction can be expressed in terms of RRs through the interaction contrast ratio. In Equations 4 and 5 below, we refer to the interaction contrast ratio calculated with RRs and ORs as the ICRR and ICOR, respectively. Eq.4 ICRR = RR GE - RR Ge - RR gE + 1 Eq.5 ICOR = OR GE - OR Ge - OR eE + 1 As seen in the assessment of multiplicative interaction (equation 2), the three component ORs of the ICOR formula can be replaced with the corresponding RR-OR equivalence formula (equation 5). Consequently, the factors that determine the relationship between the GxE RR and the GxE OR are the same factors that determine the relationship between the ICRR and the ICOR. Again, these factors are the magnitude of the GxE RR , the baseline risk of disease and the strength of the independent effects. The ICOR can result in the appearance of additive interaction when none would be observed using the ICRR, or can substantially overestimate the magnitude of additive interaction given by the ICRR (see figures 5 and 6 ). Figure 5 The effects of the baseline risk of disease [p(D|ge)] and the magnitude of the additive interaction estimate based on risk ratios (ICRR) on the additive interaction estimate based on odds ratios (ICOR) when the RR for each independent effect is 2 (RR Ge = RR gE = 2). Figure 6 The effects of the baseline risk of disease [p(D|ge)] and the magnitude of the additive interaction estimate based on risk ratios (ICRR) on the additive interaction estimate based on odds ratios (ICOR) when the RR for the independent effect of G is 6 (RR Ge = 6) and the RR for the independent effect of E is 2 (RR gE = 2). For illustration, we use the scenario depicted in figure 6 , where the RR Ge is 6, the RR gE is 2, and the baseline risk of disease is 6%. In the absence of additive interaction, the RR GE would be 7. The ICRR would reflect this absence of additive interaction (equation 6). Eq. 6 ICRR = RR GE - RR Ge - RR gE + 1 = 7 - 6 - 2 + 1 = 0 However, as shown in equation 7, the ICOR would not equal 0 and would give the appearance of interaction. Eq. 7 ICOR = OR GE - OR Ge - OR gE + 1 = 11.3 - 8.8 - 2.1 + 1.0 = 1.4 In this instance, one would reasonably interpret the results from equation 7 as evidence of additive interaction even though the same data using RRs (equation 6) would give no indication of additive interaction. Therefore, the problem of the OR overestimating the RR applies to interaction on the additive scale, in addition to the multiplicative scale. An example of distributional interaction from the literature The discrepancy between incidence odds ratio- and risk ratio-based estimates of interaction was noted by Li et al. in an occupational cohort study of mutant p53 positivity, a biomarker of genetic damage [ 11 ]. The authors reported estimates of the multiplicative interaction between a polymorphism in a DNA repair gene, XRCC1, and exposure to vinyl chloride. They found a multiplicative interaction estimate based on ORs of 2.91. However, the interaction estimate based on RRs was only 1.24 (see table 2 ). In a reexamination of their data, we found that the ORs substantially overestimated the additive interaction based on risk ratios as well. The ICRR was 0.96 (RR GE = 3.20, RR Ge = 1.82, RR gE = 1.42) and the ICOR was 8.85 (OR GE = 12.00, OR Ge = 2.50, OR gE = 1.65). In this circumstance, the overestimation can be attributed to the high baseline risk of the outcome among this cohort of vinyl chloride workers, 25%. These data highlight the potential for distributional interaction. Table 2 Example from Li et al: "A common polymorphism in XRCC1 as a biomarker of susceptibility for chemically induced genetic damage" [11] Individuals with XRCC1 high-risk genotype ( Gln – Gln ) Individuals with XRCC1 low-risk genotype ( Arg – Arg ) p53+ p53- p53+ p53- High vinyl chloride exposure (>4000 ppm-yrs) 8 2 11 20 Low vinyl chloride exposure (≤1000 ppm-yrs) 5 6 6 18 Risk Ratio 1.76 1.42 Odds Ratio 4.80 1.65 Conclusion The incidence odds ratio is a very convenient measure of effect with many appealing statistical properties including estimability in a case-control study. However, when assessing interaction, as when assessing main effects, interpreting the incidence odds ratio as if it were a risk ratio can be misleading. In particular, when the interaction based on risk ratios is reasonably high and the probability of disease among the unexposed is non-negligible, the interaction based on incidence odds ratios may diverge appreciably from the interaction based on risk ratios. Even when the disease is rare (e.g.,. disease risk is less than 5%), the incidence odds ratios can give the appearance of interaction, on either the additive or multiplicative scale, when the risk ratios would indicate the absence of interaction. In most situations where incidence odds ratios are used to assess interaction, the corresponding interaction based on risk ratios, the baseline risk of disease, and the strength of the independent effects are unknown. Therefore, the divergence of the interaction based on incidence odds ratios from the interaction based on risk ratios is unknown. The GxE RR can be estimated using equation A1 (Appendix 1) if the odds ratios for the joint and independents effects (OR GE , OR Ge , OR gE ) are known and a valid estimate of the baseline risk of disease [p(D|ge)] is available. However, even when these factors can be estimated, seemingly small errors in these estimates at critical thresholds can create a false sense of security. For example, there are circumstances as illustrated above, where a difference of as little as 2 percentage points in the probability of disease differentiates a valid from an invalid assessment of interaction using incidence odds ratios. Such small differences can easily be the result of measurement or sampling error. This paper provides guidelines for assessing the conditions under which there may be interpretational difficulties when using incidence odds ratios to assess interaction. The first guideline, that caution must be used when the outcome under investigation is relatively common, is consistent with the well known "rare disease assumption". Based on these analyses, there are many situations where the baseline risks are likely to be high enough and the interactions based on risk ratios strong enough to warrant caution. For example, in epidemiologic studies of psychiatric disorders, disease risk is often above 10%. In cancer epidemiology, when intermediate endpoints or biomarkers are of interest, outcome risks are often in the range of 30 to 50%. Under such circumstances, the equations and figures provided in this paper could be used to estimate the divergence between the interaction estimates based on incidence odds ratios and the underlying risk ratios. Regardless of design, in studies of interaction for common outcomes, incidence odds ratios should be interpreted with caution. The second guideline is less in line with conventional epidemiologic wisdom. Even when the outcome is rare, there are circumstances in which the incidence odds ratios can still be quite misleading when assessing interaction (e.g. the independent effect of the gene is strong). When assessing either multiplicative or additive interaction using incidence odds ratios, consideration should always be given to the validity of interpreting the odds ratios as risk ratios. The danger of distributional interaction, the appearance of interaction when using incidence odds ratios that would not exist if risk ratios were used or the exaggeration of the magnitude of the interaction based on risk ratios, should always be considered. Abbreviations RR, risk ratio • OR, incidence odds ratio • G, genetic factor • E, environmental factor • D, disease • GxE RR , multiplicative gene-environment interaction estimate based on risk ratios • GxE OR , multiplicative gene-environment interaction estimate based on odds ratios • p(D), probability of disease • p(D|G-E-), probability of disease among people without G and E (baseline risk of disease) • RR GE , risk ratio for joint effect of G and E • RR Ge , risk ratio for independent effect of G • RR gE , risk ratio for independent effect of E • OR GE , odds ratio for joint effect of G and E • OR Ge , odds ratio for independent effect of G • OR gE , odds ratio for independent effect of E • ICRR, interaction contrast ratio calculated with risk ratios • ICOR, interaction contrast ratio calculated with odds ratios Endnotes 1: This type of odds ratio might also be called the cumulative incidence odds ratio, risk relative odds ratio, probability relative odds ratio or Cornfield's odds ratio [ 10 , 12 ]. The discussion in this paper only applies to this type of odds ratio. It does not apply to odds ratios that result from case-control studies in which controls are sampled from everyone at risk (as in a case-cohort design). In such designs, the odds are a constant multiple of the risk, and their ratio is identical to the RR regardless of the rarity of the disease. Similarly, the OR calculated from case-control studies using incidence density sampling is equivalent to the rate ratio regardless of disease rarity. 2: Treating the OR as an approximation of the rate ratio, rather than the risk ratio may be preferable in many contexts. But the interpretation of the OR as the risk ratio is most common in practice. 3: We were introduced to the term "distributional interaction" by Dr. Stephen Ng when he was teaching epidemiology at Columbia University. However, we have not been able to locate any articles or books that use the term, although we suspect that it may derive from Miettinen's discussion of collapsibility. We prefer it to "interaction fallacy" because a) it avoids the implication that the interaction is statistically incorrect and b) it is descriptive of the source of the interaction. It arises from the different distribution of disease across strata. Appendix 1 If the odds ratios for the joint and independents effects (OR GE , OR Ge , OR gE ) are known and data on the baseline risk of disease [p(D|ge)] are available, the GxE RR may be estimated. Below, we provide a reformulation of equation 2 to estimate the GxE RR based upon these inputs (equation A1).

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1079912

What's in a virus? Folk understandings of hepatitis C infection and infectiousness among injecting drug users in Kings Cross, Sydney

Background To explore folk understandings of blood borne virus infection and infectiousness among injecting drug users in Kings Cross, Sydney. Methods Observational fieldwork was conducted in Kings Cross over a four month period. In-depth interviews with 24 current injectors and 4 key informants recruited from King Cross were undertaken. Results Hepatitis C (HCV) generated different meanings from HIV. HIV was considered "the dreaded" and generated fear of infection and dire disease progression. Whereas HCV was considered non-desirable but less threatening than HIV. The risks of transmitting HCV through sharing injecting paraphernalia was poorly understood. Some believed HCV infection was linked to poor hygiene and dirty water. Jaundice was mistakenly thought to indicate HCV infection and was used to gauge infectiousness. Many were confused about their current hepatitis C serostatus. Some participants thought they had a "dormant antibody" or that they had a "mild case" of infection. Participants were unsure what this meant for their own health or for their potential to infect others. Conclusion Participants displayed confusion about transmission risks for hepatitis C, conflating blood awareness and hygiene health promotion messages. Participants' reliance on the symptom of jaundice to gauge serostatus places them at risk of transmitting and contracting HCV. Participants were confused about what a positive HCV diagnosis meant for their own health and their ability to infect others. Education is needed to debunk misconceptions about jaundice and clarify medical terms such as 'antibody' at the time of diagnosis. Further clarification of messages about injecting hygiene and blood awareness are also required.

Introduction An estimated 210,000 people are living with the hepatitis C virus (HCV) in Australia [ 1 ]. HCV is transmitted mainly through the shared use of injecting equipment by injecting drug users (IDUs) [ 2 , 3 ]. HCV is the most prevalent blood-borne virus infection among Australian IDUs, varying from almost 90% to just under 50%, depending on the injecting population sampled [ 4 - 7 ]. HCV infection is a major public health concern. High prevalence combined with high rates of new infection, continue to result in a large number of chronically ill people, a percentage of whom will develop cirrhosis of the liver and hepatocellular carcinoma [ 8 , 9 ]. Moreover, the potential cumulative health care costs of HCV infection over the next 60 years will be approximately $4 billion [ 10 ]. Epidemiological and quantitative behavioural research on hepatitis C has concentrated on documenting risk behaviour and knowledge of hepatitis C transmission risks among injecting drug users [ 2 , 3 , 7 , 11 , 12 ]. Qualitative research has sought to contextualise risk behaviour, enhancing the findings from quantitative studies [ 13 - 15 ]. Few studies have sought to uncover the ways IDUs themselves make sense of the medical terms and clinical markers associated with hepatitis C [ 16 ]. This includes the way injectors differentiate between blood-borne viruses; folk interpretations of medical terms and the symptoms of disease; and the links between these lay understandings and risky injecting. This article explores, using qualitative data, folk understandings of the hepatitis C virus among a group of IDUs who live in or frequently visit Kings Cross, Sydney. The research utilised a socio-cultural approach to documenting and interpreting risky injecting practice [ 17 - 19 ]. It aimed to provide a window into the world of marginalised injecting drug users [ 20 ]. It sought not only to measure injectors' knowledge of hepatitis C against an expert standard of "right or wrong" fact, but also to reveal injectors' understandings of the virus as a real and threatening entity in their everyday lives. The significance of the approach lies in its ability to reveal interfaces between lay and expert knowledge: injectors take up the clinical language of medicine and health promotion and actively use it to make sense of living with HCV and to assess the likelihood of infecting others. The research therefore informs prevention efforts and education initiatives devised to inform people of the implications of their HCV status. Method Site selection The study took place in Kings Cross, Sydney, between July 2001 and February 2002. Kings Cross is home to a dynamic, open-air drug market and a "red light" district. The drug market offers opportunities for small and large scale drug dealing in heroin, and more recently, cocaine. A range of other drugs such as ecstasy, amphetamine and methamphetamine are also available. Kings Cross provides an interesting site for the study of injecting networks for several reasons. The dynamic, open-air nature of the drug market facilitates aspects of qualitative field work including observation of public injecting and a heightened awareness of issues among health workers (which in turn translates into research questions). Kings Cross is the home of numerous health and welfare services, many of which target IDUs. No other area in Australia has so many services targeting IDUs, coupled with the long history of health initiatives aimed at preventing blood-borne virus infection (BBVI) transmission since the arrival of HIV in the mid 1980s. This study posed questions regarding the continuation of risk practice in an environment where free, sterile injecting equipment is readily available and education about BBVI transmission is constantly being delivered by health care workers. Data sources and sample Three methods were used for data collection. Firstly, on-going contact with four key informants, including a local "guide" who was familiar with multiple injecting networks, provided continuous information on the state of the Kings Cross drug market and its impact on risk practice. Key informants were chosen because they were considered opinion leaders in their injecting network [ 21 ]. To facilitate field work, the research team asked service providers to nominate a guide [ 22 ]. The guide escorted the researcher through a variety of injecting locations, introduced the researcher to local identities and assisted in the selection of participants for in-depth interviews. The guide and other key informants helped to verify the accuracy of information as it emerged during the course of the study. Conversations with key informants were recorded in field note form. Secondly, observational fieldwork was conducted between November 2001-February 2002. Approximately 300 hours were spent in the field. Fieldwork involved mapping the range of public, semi-public and private injecting locations in Kings Cross. Mapping of physical locations was complemented by the identification of networks of injecting drug users who frequented these sites. Limited observations of in-situ injecting episodes also occurred. Observations were recorded in field note form. Thirdly, in-depth semi-structured interviews were conducted with IDUs living in or visiting the Kings Cross area. An interview schedule guided the process. The schedule was developed collaboratively by the investigators and key informants. The schedule comprised questions on drug use history, current injecting practice, BBVI knowledge and risk scenarios. Over three quarters of interview participants were recruited through the guide or by chain referral (snowballing). Interviews were conducted in a rented room at a welfare agency, in cafes and in public locations. Interviews lasted between thirty and ninety minutes. Ethics approval was obtained from the University of New South Wales Human Research Ethics Committee. Informed written consent was obtained for interviews. Participants received up to $30 (Aus) as reimbursement for their time. The guide was employed at casual research officer rates. In all, twenty four interviews were conducted, fourteen with men and ten with women. Ages ranged from 19 to 47 with most interviewees in their thirties. One participant was HIV positive. It was difficult to assess, on self-report, the HCV status of participants as there was confusion regarding current status, although most believed themselves to have been infected at some time. Participants primarily injected heroin, with most injecting cocaine when it was available. Few participants had stable housing, however most were homeless or itinerant, their lives characterised by social marginalisation, poverty and extreme poor health. Analysis In line with Smith [ 23 ] and Glaser and Strauss [ 24 ], field notes and interview data were analysed in an on-going manner. Interviews and field notes were coded for key words, themes, issues and events. These were compared, contrasted and synthesised to create a system of thematic classification. A number of processes were used to assess the validity of the analysis. First, the research team read all the transcripts and field notes and analysed these data for contexts for BBVIs transmission as identified in the literature; common concerns of and discourses used by participants; similarities and disparities in network membership and environmental location for injecting; and collective and individual patterns of social action that make up everyday life. Data were re-read for disconfirming evidence. A process of theoretical validity was undertaken to ensure that the units of classification (themes, issues, concepts) were sensitive to the accounts supplied by participants and to scholarly literature [ 25 ]. Theoretical validity extended to evaluating the internal logic of and relationships between units of analysis [ 26 ]. Validity involved the use of a collective process of checking, questioning and theorising [ 27 ]. Results Mixing messages: Folk understandings of 'Blood Awareness' and Hygiene Health Promotion messages For most participants, HCV generated significantly different meanings from HIV. HIV was colloquially referred to as "the dreaded", a term encapsulating fear of infection and dire disease progression. HIV elicited strong reactions, in particular the desire to prevent infection by avoiding certain types of injectors considered to be at high risk such as gay men, or those individuals 'known' to be infected with the virus. In contrast to HIV, the meanings attached to the hepatitis C virus were fairly diverse. While hepatitis C infection was not considered desirable, participants viewed HCV as less damaging to health and quality of life than HIV. However, there was little evidence of a forlorn fatalism in regard to HCV infection: it was not considered attractive or inevitable. Nor was it viewed as a 'badge' signifying the position of a 'real user' contrary to previous research [ 28 ]. Pragmatism rather than fatalism predominated: if participants could avoid sharing needles (needle-sharing being the focus of folk infection-control) then they would. In cases where circumstances were deemed to prevent safer use then risks would be taken. The concentration on preventing needle-sharing reflected the success of earlier HIV prevention campaigns based on the 'new fit for every hit' message. Some participants had good knowledge of 'blood awareness' health promotion messages, that is of the risks associated with transmitting HCV via the use of blood contaminated injecting paraphernalia and through touching others with bloody fingers etc. About half of the participants were less cognisant of these means of transmission. Indeed, a number of participants held the view that hepatitis C was transmitted "through dirt". In many cases participants were re-interpreting public health hygiene messages. These messages refer to the benefits of hand-washing and wiping surfaces after injecting to prevent the inadvertent spread of HCV via touch and environmental blood transmission. HCV transmission was associated as much with unhygienic practice and "dirty, desperate" people as it was with technical blood-to-blood transmission routes [ 29 ]. Some participants, like Bruce, provided explanations for contracting HCV which involved both hygiene and blood awareness messages: Well I'm hep C positive at the moment and have been for quite a long time and I think that came about when I wasn't using sterile injecting water. I was taking water out of the toilets and things like that and using that to inject and I think that's how I got hep C. [Interviewer: So it wasn't sharing needles with other people?] No. It could have been either that or finding somebody else's blood or something in one of the packets (containing a drug deal) or something and just continuing to have what was left of the packet or something like that that caused me to get it. Or it could have been just using a very, very old syringe where the blood had gone off or something. (Bruce, a 30 year old injector) . Bruce's account is indicative of the way participants associated HCV transmission with both unhygienic practice and scientifically known transmission routes. Bruce variously suggests he may have contracted HCV from toilet water; from blood left in an old deal packet (one assumes a small plastic bag); or from re-using a "very, very, old syringe". Bruce demonstrates good technical knowledge when he states that HCV can be transmitted through blood contamination in drug deal packets. He also exhibits a commonly held understanding that HCV transmission is related to unhygienic practice. In this case unhygienic practice involves the use of toilet water and blood that has "gone off" in a syringe of extreme age. The hygiene factor is also apparent in some participants' misconception that it is possible to infect ones-self with HCV through the re-use of one's own needle. In this case, the hepatitis C virus is not viewed as an agent which is external to the self, that is, it is caught from other people. Rather hepatitis C infection is thought to result from the "unclean" practice of re-using one's own needle. This misconception is based on the belief that one's own blood, once lodged in a used syringe, is capable of generating HCV as it changes or "goes off". According to this folk understanding, the health threat is endogenous to the drug user's body rather than exogenous or external [ 30 ]. Reading the jaundiced body Understandably, many participants were confused about the differences between hepatitis A, hepatitis B, and hepatitis C. The most obvious sign of this was the description participants gave of their own and others HCV seroconversion illness. Numerous participants stated that they "knew" they had contracted hepatitis C when they became jaundiced. Unlike hepatitis A and hepatitis B, jaundice is rarely associated with the acute phase of hepatitis C infection [ 31 ]. The commonly held assumption that hepatitis C status can reliably be determined by symptomatic jaundice has implications for prevention. In this case, infection and infectiousness is associated with a symptom that is much more likely to occur in the acute phase of hepatitis A and hepatitis B infection, not HCV infection. Participants actively 'read' their own bodies and the bodies of others, for jaundice. Jaundice was considered a reliable sign on which to assess hepatitis C status and the subsequent risks in lending or borrowing injecting equipment. Given the reliance some IDUs place on jaundice as a marker of HCV infection and infectiousness means that many may only seek HCV testing if they experience jaundice. Also some may well be basing their assumed negative status on the fact that they have not experienced jaundice. Michelle, a 35 year old injector, explains this logic: I mean, touch wood, I never got AIDS or anything like that, and how I was diagnosed I was staying in a half-way house, rehab type of thing...Once a week we'd do groups on women, health issues and things like that and this one week was about Hep C. And he (a doctor) said, 'Hands up the people that have got it' and everyone put their hand up except for me and I said, 'Well, I've not been tested...but I can't remember being yellow or anything like that."...He said 'You don't necessarily go yellow. Can you remember in the last five years having a really bad flu?' Everybody's antibodies: Folk understandings of clinical terms Reading the interview data as a whole, there is the distinct impression that many IDUs, at least half, were perplexed about the implications of a hepatitis C diagnosis. Most, at least two thirds, state that they are "carriers" who have "cleared" the virus or that the virus is currently "dormant". When asked about their current HCV status, over half of the participants spoke of having "antibodies" but were unsure what this meant in terms of their prognosis or their ability to infect others. Approximately ten participants recounted the fear and distress they felt after receiving a positive diagnosis. The lack of adequate hepatitis C pre and post test counselling has been documented in other research [ 16 , 32 ]. The following interview revealed the confusion and fear that can accompany a positive diagnosis: I haven't got AIDS and I haven't got Hep B but what I was told was that I carry the antibodies for (Hep) C, which I said 'What's that mean?'. Apparently it's that I might have a mild case of it and don't have it any more...They still want to follow it up now at the clinic and have a biopsy- cut a piece of my liver, test my liver...I should have went back (to the clinic) but that scared me. (Diane, a 29 year old injector) . Some participants, like Diane, spoke of having a "mild" or "small" case of HCV. Other used the clinical term 'antibodies' to describe having a resistance to HCV, somewhat like acquiring antibodies after having chicken pox. For example, Ken, a 22 year old injector, stated: " I've been tested for Hep C. Been cleared and then the doctor said you got antibodies against it and, then he said there's no sign of it in your system ." Another, Glen, a 30 year old who had shared needles in jail, thought he was " one of those strange people that might be able to fight it (hep C) ", thus perpetually avoiding infection. Phoebe, describes how proximity to someone else's hepatitis B and hepatitis C antibodies provided protection for her: I had a test...and they couldn't tell how me if I was a carrier or whether I had hepatitis A years ago. I had a hep B test and they said 'You're a carrier', and I said, 'Hang on, I've never had hepatitis in my life, never in my life.' 'Well', they said, 'you have been close to someone who has antibodies and that's a good thing'. I had a hep C test and they could only tell me the same thing. (Phoebe, a 42 year old injector) . Only a few participants understood that although they had spontaneously "cleared" the HCV, they were still open to reinfection. Similarly, only a couple of participants demonstrated knowledge of superinfection, that is infection with multiple genotypes of HCV. Confusion around diagnostic explanations, such as 'antibodies present' and 'virus cleared', left participants vulnerable to hepatitis C infection. These terms gave a false impression that injectors were protected from reinfection and superinfection because of the presence of antibodies. Discussion Marginalised IDUs experience a syndemic pattern of health concerns, that is, they are affected by a set of synergistic and mutually enhancing health and social problems [ 33 ]. Public health concerns about hepatitis C should be located within the syndemic nature of the health and welfare issues facing marginalised people, such as those who participated in this study. Within a syndemic context, "risk" has myriad meanings, including but not solely focused on, risks for the transmission of hepatitis C. While the majority of participants were concerned about contracting and transmitting hepatitis C, this concern was only one of many which permeated everyday life. Hepatitis C did not have the immediacy of "the dreaded" HIV. Infection with hepatitis C is common among IDUs' friends and acquaintances. In the minds of IDUs, hepatitis C infection offered an uncertain future, but unlike HIV, a future none-the-less. Indeed, hepatitis C was really the uncertain virus. For many participants there was uncertainty about modes of transmission other than through the sharing of needles. Some participants blended blood awareness messages with hygiene ones. This blending of messages created the misconception that "dirty" behaviour and "dirty" people were the cause of HCV infection. Participants were perplexed about the differences between the various hepatiti and the symptoms of their acute phases. Confusion accompanied hepatitis C diagnosis, with participants actively reinterpreting the medical language of their diagnostic experience. Lay people invariably read their bodies and the bodies of others for signs of infection and illness [ 29 , 33 ]. So too do they appropriate expert knowledge, including medical terms and clinical markers, making sense of these at individual and collective levels [ 34 ]. Sometimes medical terms and clinical markers are actively used in the assessment of risk practice [ 35 ]. Prevention efforts should continue to reiterate messages about the risks of sharing needles and other injecting paraphernalia. However prevention needs to be expanded to include education about the hepatitis C virus itself. Clarification, in accessible language, about the differences between the hepatiti is required, in particular the fact that jaundice is not a usual symptom of hepatitis C acute infection. The IDUs in our study were using the symptom of jaundice as a marker of infection and as a sign that they were capable of infecting others. IDUs should be informed that jaundice is not a reliable sign for gauging either infection or infectiousness. Similarly they should be advised that they should not wait for signs of jaundice before being tested for HCV. On the subject of HCV testing, the findings from this study support other research [ 16 , 32 ] which suggests improvements are needed in pre and post test counselling, and in the provision of long-term support and information mechanisms for people with HCV, particularly marginalised IDUs. Alleviating the fear surrounding the testing experience and a positive HCV diagnosis is vital if IDUs are to be encouraged to seek testing and treatment. The participants in this study all frequented the Kings Cross area where the range of drug services is the best in Australia, yet these IDUs were still engaging in risk behaviour and were unsure about a number of issues around hepatitis C. This suggests that even more support is required for these people to change their behaviour. One approach in Amsterdam has been the provision of case managers who arrange housing, methadone treatment and access to an injecting room for each client. Moreover, the complexity of hepatitis infection, that is, the possibility the virus can be spontaneously cleared and the prospect of reinfection and superinfection, requires explanation among this group. Professionally supported peer education might be an efficient means of 'spreading the word' about these matters [ 22 , 36 ]. Situating public health certainties about the hepatitis C virus within the context of marginalised injecting networks has proved a challenging task. Targeted and accessible education and health promotion messages are required to unravel the complexities of HCV and its implications for acute and chronic infection and infectiousness. On-going examination of folk understandings of medical terms and clinical markers is a must if prevention and support efforts are to be successful in controlling the HCV epidemic.

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1079913

Frequent detection of worm movements in onchocercal nodules by ultrasonography

Background Ultrasonography (USG) is known to be a suitable tool for diagnosis in lymphatic filariasis as the adult filarial nematode Wuchereria bancrofti in scrotal lymphatic vessels of infected men can be detected by the characteristic pattern of movement, the Filaria Dance Sign. In onchocerciasis, moving adult worms have not yet been demonstrated by USG. In addition the verification of drug effects on living adult Onchocerca volvulus filariae in trials is hampered by the lack of tools for longitudinal observation of alterations induced by potentially macrofilaricidal drugs in vivo . The present study was carried out to determine the frequency of detection of moving adult filariae of O. volvulus by USG. Methods In an endemic region for onchocerciasis in Ghana, 61 patients infected with onchocerciasis were recruited by palpation and onchocercomas examined by USG using an ultrasound system equipped with a 7.5 – 10 MHz linear transducer. Onchocercomas were recorded on videotape and evaluated with regard to location, number and size, as well as to movements of adult filariae. Results In the 61 patients 303 onchocercomas were found by palpation and 401 onchocercomas were detected by USG. In 18 out of 61 patients (29.5%), altogether 22 nodules with moving adult O. volvulus filariae were detected and are presented in animated ultrasound images as mp-4 videos. Conclusion Ultrasonographical examinations of onchocercomas where living adult filariae can be displayed may serve as a new tool for the longitudinal observation in vivo of patients with onchocerciasis undergoing treatment and as an adjunct to histological evaluation.

Background Onchocerciasis is endemic in 37 countries and about 18 million people are infected in Africa and Latin America. Health organisations are aiming to eliminate onchocerciasis as a major public health problem. The Onchocerciasis Control Programme (OCP) has successfully targeted transmission by control of the vector (black fly) but this programme ended in 2002. Within the scope of the African Programme for Onchocerciasis Control (APOC), annual mass treatment with the microfilaricidal drug ivermectin is carried out. However, elimination of transmission has proven more difficult than expected [ 2 ]. A recent conference concluded that eradication is not feasible with the present tools [ 4 ] and that new drugs are needed. Beside the indirect parameter of microfilariae count, surgical intervention (nodulectomies) is needed to verify effects of drugs on the vitality and fertility of adult Onchocerca volvulus after treatment. Thus, there is an urgent need for the development of more effective drugs that either kill or long-term sterilize the adult worms. In addition, it would be worthwhile to have a non-invasive tool to provide repeatable longitudinal observation of alterations in the vitality and motility of worms induced by potentially macrofilaricidal drugs. Different to regular detection and longitudinal observation of worm movements (Filaria Dance Sign-FDS) in lymphatic vessels in bancroftian filariasis [ 1 , 6 , 7 ] motile adult filariae of the species O. volvulus in onchocercoma tissue have not been displayed to date by ultrasonography. Therefore the usefulness of USG regarding frequency of detection of adult living O. volvulus filariae was evaluated in the present study. The non-invasive USG-method provides on one hand the opportunity for longitudinal observation in terms of number and size of onchocercomas and on the other hand repeatable examinations to observe alterations on motile adult O. volvulus filariae in vivo . Materials and Methods The study was part of a double blind, placebo controlled treatment study with doxycycline carried out in the Lower Denkyira District in the Central Region of Ghana. This study was approved by the Committee on Human Research and Ethics of the School of Medical Science at KNUST, Kumasi, Ghana. The study conformed to the principles of the Helsinki Declaration of 1975 (as revised1983 and 2000). Study site and patients After detailed explanation about the study in the Ghanaian language Twi, patients were pre-selected by palpation of worm nodules. The number and location of palpated onchocercomas was documented on patient forms. 61 participants, who had not receive antifilarial treatment within the past 2 years, were examined by ultrasonography before receiving doxycycline or placebo treatment. According to the study protocol, all patients also received ivermectin 150 μg ivermectin/kg body weight – 4 months after study start. To determine microfilaria load before treatment, two skin snips of the upper part of each buttock were taken with a corneoscleral (Holth) punch (Koch, Hamburg, Germany). Each skin snip was weighed with an analytical balance (Sartorius electronic balance, Göttingen, Germany). Microfilarial load was calculated per mg/skin. The skin snips were incubated at room temperature in 100 μl NaCl 0.9% for 6 – 20 hours in separate wells of a 96-well round bottom microtitre plate (Nunc, Roskilde, Denmark). Microfilariae were counted after incubation using 63-fold magnification of a microscope. Ultrasound examination Ultrasound examinations were performed before treatment started using a SonoSite 180 Plus ® (SonoSite Inc.; Washington, USA) system, equipped with an L38 mm 5 MHz – 10 MHz linear transducer. For the actual ultrasound examination of the onchocercomas, frequencies of 7.5 and 10 MHZ were used. A digital camcorder SONY DCR-PC120E PAL Handycam ® (Sony Corporation, Japan) was directly connected to the ultrasound machine to record real time videos on mini DV-tapes for documentation of the findings. Patients were examined in a supine position in order to avoid artefacts due to movements. The examiner observed each nodule in longitudinal and transverse scans to detect movements of adult filariae. First the transducer was positioned at the largest diameter of a nodule or at the largest echo-free area in case of cystic nodules. Thereafter, imaging was carried out in panorama-mode to provide more information. Every detected onchocercoma was measured in the two dimensional b-mode for length and width in the largest diameter the examiner could adjust. Nodules were measured in total – including the capsule consisting of connective tissue. The detection of all onchocercomas was recorded on videotape and later compared to the number of nodules detected by palpation. The video documentation was also used to evaluate nodules containing moving worms with regard to number, size, motility, echo-free areas in onchocercomas as sign for necrotic proceedings and acoustic shadowing, reflecting moving and static fragments of the worms. Results Study participants The study group consisted of 44 male and 17 female participants. Their age ranged from 19–61 years. All patients were tested for microfilarial load which ranged from 0 – 290 mf/mg skin (geom. mean 11.21 mf/mg). Altogether 356 onchocercomas were palpated, whereas 401 (geom. mean 5.140 nodules / per patient) onchocercomas were detected by USG in the participating 61 patients. In male patients a total of 272 (geom. mean 5.417) nodules were palpated while 303 (geom. mean 6.010) were detected by USG, in female patients 84 (geom. mean 4.487) onchocercomas were palpated and 98 (geom. mean 5.210) were detected by USG (table 1 ). Table 1 Number of nodules palpated vs. number of nodules detected by ultrasound (USG). Male patients N = 44 Female patients N = 17 Total N = 61 USG 303 98 401 Palpation 272 84 356 Correlation of palpation and USG Onchocercomas could be clearly differentiated from lymph nodes, where a sinus was visible with afferent and efferent lymphatic vessels, and from lipomas, which appear brighter, more echo dense and homogeneous than onchocercomas. Foreign bodies such as pieces of metal, stones or thorns could be differentiated from onchocercomas due to their sharp signals, which result from their well-defined borders and surrounding granuloma tissues. All palpated nodules could be detected by USG. In 23 patients the number of nodules detected by palpation matched with the number of onchocercomas detected by USG. In 26 patients more onchocercomas were detected by USG than by palpation. These onchocercomas were of a larger size and tightly packed in a surrounding capsule of connective tissue. By USG the examiner was able to differentiate more precisely the exact number of nodules, divided by septulae of connective tissue inside larger onchocercomas. USG of 12 patients showed less onchocercomas than palpated: 2 lipomas, 2 foreign bodies and 8 lymph nodes had been erroneously judged as onchocercomas by palpation. The 2 painful foreign bodies, which were seen by USG, were excised and verified. Reproducibility of USG findings 3 patients underwent USG examination on 3 consecutive days at the first time-point (onset of the study). All onchocercomas were detected at the same locations and judged as onchocercomas. 45 of the 61 study patients underwent a second confirmatory ultrasound examination 4 months after study start. All nodules judged as onchocercomas by USG at the beginning of the study could be rediscovered and still proved to be onchocercomas. 30 out of the 45 USG re-examined patients underwent nodulectomies at this time-point. After nodulectomies no more onchocercomas were visible by USG at locations known to be positive for these findings before. Measurement of onchocercomas The size of the nodules measured by USG varied from 0.18 cm 2 to 9.44 cm 2 (geom. mean 1.823, median 1.78). Evaluation of the total cross-sectional area (cm 2) of all nodules measured per patient showed that nodules were larger in older patients (figure 6 ). There was no significant relation between age and the occurrence of cystic nodules where worm movements could be documented by USG (Mann-Whitney U test: p = 0.1989). Volume measurements of the onchocercomas were not done since this would have been inaccurate with a 2-dimensional device. Therefore 3-dimensional ultrasound scanners are necessary. Patients with onchocercomas containing motile adult filariae In 18 out of 61 (14 male, 4 female) participants (table 2 ), 22 onchocercomas with moving adult filariae were observed by USG. 15 patients had one nodule, 2 patients had 2 nodules, and 1 patient had 3 nodules with living filariae. Anatomical sites where cystic nodules could be detected were thorax, trochanter, iliac crest, crena analis, knee and foot (heel). The frequency distribution of onchocercomas where worm movements could be observed is shown in table 3 . The living adult worms were visible as acoustic enhancement (bright echo) reflected from tissue moving in echo-free areas of onchocercomas ( additional file 1 , 3 ) and in one case in hypodense nodular tissue ( additional file 2 ). The echo-free parts of the nodule provided a so-called "acoustic window" where worm movements could easily be seen (figure 2A, 2B , 3B , 4A , additional file 1 , 3 ) in comparison to nodules where adult worms are packed tightly in a capsule of connective tissue (figure 1A, 1B , 3A , additional file 2 ). Within the echo-free areas moving adult filariae appeared as coiled and twisted structures moving around each other in the surrounding fluid. The echo-free areas differed from a very small part of the nodule to areas comprising the whole nodule (figure 2A, 2B , 3B , 4A, 4B additional file 1 , 3 ). Figure 1 1A : Longitudinal scan of the patient's right iliac crest. The b-mode image shows a small subcutaneous onchocercoma. Measurement is shown in the largest transverse and longitudinal section of the nodule. In this homogenous onchocercoma no worm movements were detected. 1B : Longitudinal scan of the crena analis. A medium sized homogenous subcutaneous onchocercoma can be seen. As in image 1A no worm movements were detected. A differentiation of worm centre, corresponding to a coil of worms, from the capsule was not possible in this nodule. A lateral shadow is visible on both sides of the onchocercoma (yellow arrowheads) Table 2 Total numbers of male and female patients with onchocercomas, in which motile or non-motile adult worms were detected by ultrasound (USG). Male patients Female patients Total Patients with onchocercomas where adult motile filariae could be detected by USG 14 4 18 (29.5%) Patients with onchocercomas where no adult motile filariae could be detected by USG 30 13 43 (71.5%) Total 44 17 61(100%) Table 3 Anatomical sites where onchocercomas with living adult filariae occurred: Anatomical site Crena analis Trochanter Iliac crest Thorax Knee Foot Total Number 7 5 3 3 3 1 22 (observed in 18 patients) Figure 2 2A : Longitudinal scan of the patient's right trochanter. The b-mode image shows a medium sized onchocercoma. The observer can easily differentiate between the worm centre containing a conglomerate of coiled adult filaria (e) and the capsule of the nodule. The worms are surrounded by cystic fluid. The yellow arrowheads are positioned where worm movements were detected. The conglomerate of worms causes a strong back wall reflection behind the onchocercoma. 2B : Transverse scan of the patient's right iliac crest. A large onchocercoma with a small cystic area (echo-free-black – zone) can be seen. The yellow arrowheads are positioned where worm movements were detected. Fragments of the worm body can be seen as a double layer membrane. The corresponding video image can be seen as Additional File 1 . Figure 3 3A : Longitudinal scan of the patient's right trochanter. A homogenous medium sized onchocercoma can be seen. Although there is no cystic, echo-free area seen, worm movements were detectable in the b-mode image (blue arrow). Lateral shadows and a back wall reflection are visible. The corresponding video image can be seen as Additional File 2 . 3B : Transverse scan of the patient's left iliac crest. The worm(s) is (are) surrounded by cystic fluid seen as echo-free areas (yellow arrowheads) and can be differentiated from the capsule of the onchocercoma. In this nodule no worm movements could be detected. Figure 4 4A : Transverse scan of the patient's left knee. A large cystic onchocercoma can be seen. Yellow arrowheads indicate at moving fragments of the worm(s), while the blue arrowheads indicates static fragments of the worm body (ies). The corresponding video image can be seen as Additional File 3 . 4B : Longitudinal scan of the patient's right trochanter. A medium sized cystic nodule where minimal movements were detected. The worm(s) is (are) surrounded by cystic fluid seen as echo-free areas (yellow arrowheads) and can be differentiated from the capsule of the onchocercoma. Beside motile adult filariae, all cystic nodules contained parts of probably degenerated fragments of adult worms, seen as particles where the ultrasound beam cannot pass through and is reflected and visible as double layers moving in the cystic fluid (figure 4A , additional file 3 ). The Pulse Wave Doppler Technique as described for the detection of adult filariae of Wuchereria bancrofti [ 8 , 14 ] was used to visualise movements of living worms of O. volvulus as a function of time and frequency, which however appears slow and rare in comparison to adult filariae in lymphatic vessels. Thus the relevance of the Pulse Wave Doppler technique appears less important for the observation and confirmation in onchocerciasis in comparison to examinations in bancroftian filariasis. Correlation to histology A preliminary analysis of onchocercomas by histology, confirmed the results on the proportion of live adult worms from an earlier study (85%)[ 10 ], which was based in the same endemic area. The data clearly show that most of non-motile worms are vital but that the adult worms are not detectable and do not appear as motile worms by USG. The reasons may be that i) worms are too tightly packed in host tissue to appear motile; ii) the connective tissue surrounding the onchocercomas is thick and thus reflects the ultrasound beam of the capsule but not of the worms inside. Out of the 18 patients presenting cystic nodules with motile worms, 8 patients were re-examined after 4 months before the above-mentioned nodulectomies. All cystic nodules could be rediscovered at the same location and still contained moving adult filariae. To correlate these findings in the histology 3 patients presenting nodules with visible movements underwent nodulectomies. In cystic onchocercomas adult filariae were found by histology (figure 5 A–C ). A nucleus is visible in the hypodermis of the worms and intact microfilariae are visible inside the uterus as sign of vitality at the time-point of nodulectomy. Figure 5 5A : Live adult female of O. volvulus of a placebo treated patient. A nucleus (nc) in the hypodermis of the body wall (lateral cord) is visible as sign of vitality of the worm. The intestine appears darker beside the two sections of the uterus. (Magnification × 40) 5B : Section of the same onchocercoma as seen in 5A. The nucleus (nc) is visible in the hypodermis. Intact microfilariae (mf) and pretzel stages (pz) are shown. (Magnification × 60) 5C : Part of the hypodermis and the cuticle of a female adult O. volvulus . The cuticle and the hypodermis appear intact and the nucleus (nc) as sign of vitality is clearly visible. (Magnification × 100). Figure 6 Regression plot of the total cross-sectional area of onchocercomas (detected by USG) in relation to the patient's age. The figure shows that nodules were larger in older patients. Discussion In the present study, onchocerciasis patients were examined by ultrasonography to determine the frequency of detection of living adult O. volvulus in onchocercomas. The current study is an improvement over previous attempts to detect worms in onchocercomas as it provides video documentation of worm movements and shows that, with the availability of higher resolution transducers, the frequency of detection of worms containing moving adult worms is surprisingly high. In 18 out of 61 (29.5%) of the examined patients, 22 onchocercomas with moving adult filariae were detected. Clinical trials to verify potentially macrofilaricidal effects of drugs currently lack methods for repeatable long-term observation of adult living filariae in onchocercomas in vivo . The use of ultrasonography to observe onchocercomas has been rare, since movements of adult worms embedded in nodules surrounded by connective tissue were difficult to image. In fact there is only one case listed in the literature where moving adult worms were detected [ 5 ]. Homeida et al. [ 11 ] first described the use of USG to detect onchocercomas in patients. Poltera and Zak [ 16 ] published in 1988 an ultrasound study on onchocercomas, where they described the ability to use USG to observe alterations in nodule tissue after treatment in vitro. Leichsenring et al. [ 12 ] evaluated in 1990 the use of ultrasound examinations on patients with regard to a suitable tool for longitudinal observation under treatment conditions, however did not show movements of worms. The higher sensitivity of nodule detection by USG in comparison to palpation and the ability to differentiate between onchocercomas and lipomas, lymph nodes and foreign bodies has been reported [ 12 ]. In 1994 Darge et al. [ 5 ] described that they observed distinct worm movements in 1 of 249 detected nodules of their patient group in Liberia. An animated documentation of moving adult filariae of O. volvulus by video has not been published so far. Due to the improved sensitivity of current ultrasound machines as well as digital video technique we were able to detect and record in almost 30% of the participants of the study 1 to 3 onchocercomas containing moving, adult filariae of O. volvulus . Although it has been observed (Prof. Buettner, Bernhard Nocht Institute of Tropical Medicine Hamburg, personal communication) that onchocercomas seem to become cystic over the course of time, and that worms die from their end of the body while the prosoma and the head of the worm keep moving in cystic fluid, we did not find a correlation between the occurrence of moving worms in nodules and age of the patients (18–61 years, Mann-Whitney U test: p = 0.1989). This is probably due to the fact that in our study area, transmission is still high despite the onset of ivermectin mass treatment and therefore, even younger patients already displayed a considerable number of cystic nodules. It might be possible that the number of cystic nodules is higher in areas where there have been several rounds of MDA resulting in reduced transmission and thus, over-aged nodules. Effects of drugs are currently verified by histological examinations of the nodules after minimal invasive surgery (nodulectomy). Worms can be evaluated with regard to number of male and female worms, size, embryogenesis and spermiogenesis [ 3 ]. Immunohistology is used to determine whether worms were alive before nodulectomy. To this, sections of nodules can be stained e.g. with antisera against O. volvulus GST1 (Glutathione-S-transferase) and Yersinia hsp 60 (heat shock protein), the latter cross reacting with mitochondrial hsp 60 in the worms [ 10 , 13 ]. GST1 and mitochondrial hsp 60 are only produced by living worms and appear as a strong staining in the hypodermis. The evaluation of treatment by the use of (immuno-) histology has the disadvantage that there is only one single time point for examination, which cannot be repeated and cannot be carried out at several different time points. In contrast, the use of USG offers a non-invasive, cost effective tool, which permits observation at many time points but has the disadvantage that adult worms of O. volvulus are coiled around each other and it seems not possible to determine their number and their sex, although the male worms are only 3–5 cm long in comparison to female filariae with a length of 30–80 cm. USG is not a surrogate for immunohistology. With USG it is impossible to evaluate embryogenesis and spermatogenesis of adult filariae. Furthermore, USG can provide qualitative, not quantitative information about worm number in nodules. Another disadvantage of USG is that only a fraction of worms that prove "vital" in histology is able to display movements either due to dense host tissue, or due to echo dense connective tissue surrounding the onchocercoma. Nevertheless, although the number of onchocercomas where motile worms could be detected appears to be few (5.5%), the fact that 29.5% of patients are carriers of cystic nodules with motile filariae offers the possibility for long-term observation of the vitality of worms in vivo in a reasonable number of infected humans. Therefore, information provided by USG may prove useful for the evaluation of potentially macrofilaricidal drugs and has the advantage of being non-invasive. The latter point is important, as the compliance of the patients is much better in cases where a painless method is used. Conclusion Ultrasonographical examination of cystic onchocercomas containing living adult filariae may serve as a new tool in the longitudinal observation of patients with onchocerciasis as an adjunct to histological evaluation. USG may also facilitate clinical trials in terms of observation of macrofilaricidal activities with regard to effects that may have been overlooked before (e.g. repeated high dose ivermectin treatment). Competing interests The author(s) declare that they have no competing interests. Authors' contributions S. Mand recruited patients, did the ultrasound examinations, compiled the data and wrote the paper draft. Y. Marfo-Debrekyei, A. Debrah and L Batsa carried out the skin snips and the microfilaridermia counting. They performed patient management during recruitment and ultrasound examinations. M. Buettner recruited patients, performed physical examinations, and carried out the treatment. K. Pfarr helped in manuscript preparation and offered constructive comments. O. Adjei did preparatory studies for proper selection of villages, negotiations with the village elders, and performed the ethical clearance. A. Hoerauf designed and supervised the study, participated with patient recruitment and ultrasound examinations, compiled the data and edited the final manuscript version. Supplementary Material Additional File 1 Transverse scan of the patient's right iliac crest. A large onchocercoma with a small cystic area (echo-free – black – zone) can be seen. Within this cystic area movements of the adult filaria(e) are visible. Fragments of the worm body can be seen as a double layer membrane. The corresponding image can be seen as figure 2B . Click here for file Additional File 2 Longitudinal scan of the patient's right trochanter. A homogenous medium sized onchocercoma can be seen. Although there is no cystic – echo-free area, worm movements are presented in the b-mode image (arrow). Lateral shadows and a back wall reflection are visible. The corresponding image can be seen as figure 3A . Click here for file Additional File 3 Transverse scan of the patient's left knee. A large cystic onchocercoma can be seen. Movements of a conglomerate of coiled adult filariae are displayed in the cystic fluid of the nodule. Static fragments of the worms are visible in the lower left part of the video image. The corresponding image can be seen as figure 4A . Click here for file

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1079914

Efficacy of acupuncture for cocaine dependence: a systematic review & meta-analysis

Background Acupuncture is a commonly used treatment option for the treatment of addictions such as alcohol, nicotine and drug dependence. We systematically reviewed and meta-analyzed the randomized controlled trials of acupuncture for the treatment of cocaine addiction. Methods Two reviewers independently searched 10 databases. Unpublished studies were sought using Clinicaltrials.gov , the UK National Research Register and contacting content experts. Eligible studies enrolled patients with the diagnosis of cocaine dependence of any duration or severity randomly allocated to either acupuncture or sham or other control. We excluded studies of acupuncture methods and trials enrolling patients with polysubstance use or dependence. We abstracted data on study methodology and outcomes. We pooled the studies providing biochemical confirmation of cocaine abstinence. Results Nine studies enrolling 1747 participants met inclusion criteria; 7 provided details for biochemical confirmation of cocaine abstinence. On average, trials lost 50% of enrolled participants (range 0–63%). The pooled odds ratio estimating the effect of acupuncture on cocaine abstinence at the last reported time-point was 0.76 (95% CI, 0.45 to 1.27, P = 0.30, I 2 = 30%, Heterogeneity P = 0.19). Conclusion This systematic review and meta-analysis does not support the use of acupuncture for the treatment of cocaine dependence. However, most trials were hampered by large loss to follow up and the strength of the inference is consequently weakened.

Introduction Recent research on cocaine abuse and its treatment are consistent in the estimates of the social, physical, emotional and financial costs attributed to this addiction [ 1 - 5 ]. Currently, there is no specific pharmacologic, behavioral, or psychosocial therapy that has consistently demonstrated treatment benefits[ 6 , 7 ]. Most current treatment approaches are extensions of treatments ordinarily applied to alcohol or opiate addiction[ 8 ]. The limited successes in treating cocaine addiction have led patients and clinicians to examine alternative approaches. Acupuncture is one such treatment option for addictions such as alcohol, nicotine and drug dependence[ 9 , 10 ]. Currently, more than 500 clinics in the United States, Canada and Europe, as well as several court-related programs, include acupuncture as a treatment option for drug dependence[ 11 ]. However, to date, the effectiveness of acupuncture as a primary treatment option or as an adjunct for cocaine dependence remains uncertain. The aim of our study was to conduct a systematic review and meta-analysis of randomized controlled trials assessing the impact of acupuncture on cocaine dependence. Methods Inclusion/Exclusion Criteria Eligible studies enrolled patients with the diagnosis of cocaine dependence of any duration or severity randomly allocated to either acupuncture, sham or other control. Acceptable outcomes measures included: self-reported frequency of cocaine use, self-reported amount of cocaine use, or biochemical confirmation of cocaine abstinence. Biochemical confirmation of cocaine abstinence is defined as the absence of the cocaine metabolite benzoylecognine in the urine. We excluded trials of acupuncture methods and trials enrolling patients with polysubstance use or dependence. Literature search Databases searched included: AMED (1985–November 2004), Campbell Collaboration (2001–January 2005), CINAHL (1982–January 2005), Cochrane Library (1998–January 2005), Cochrane Controlled Trials Registry (January 2005), E-Psyche (1993–January 2005), HTA (1988–January 2005), and MEDLINE (1966–January 2005). We additionally searched the Chinese literature through Wanfang (1997–January 2004) and the Chinese Hospital Knowledge Database (CHKD, 1994–2004). Unpublished studies were also sought using Clinicaltrials.gov and the UK National Research Register. We supplemented this search by hand-searching key journals and searching bibliographies of retrieved trials and reviews. We additionally contacted 5 authors to identify additional published or unpublished studies and to clarify methodological issues. There were no language restrictions. Two reviewers (EM, PW) working independently and in duplicate, reviewed the abstracts and full text versions of identified reports and adjudicated their inclusion. Data extraction Three reviewers (PW, EM, JG) working independently extracted data from the included studies using a standardized form which included: patient characteristics, treatment and control descriptions, types of outcomes measured, adverse events, and study results. Methodological reporting Two reviewers (EM, PW) working independently and in duplicate assessed the reporting quality of the reports. Items collected included randomization procedure, allocation concealment, blinding of patients, care providers, and outcome assessors, adequate description of loss to follow-up, and co-interventions. These items were treated as single items and were abstracted for the purpose of sensitivity analyses in our meta-analysis, but were not used as a weighting application in our analysis. Statistical analyses We measured chance-adjusted inter-rater agreement for eligibility using the kappa statistic (κ). Where reported, we extracted each trial's outcome data from the intention-to-treat analyses. If biochemical confirmation of cocaine abstinence was not reported, we contacted the authors for details. When a study had more than one control arm, we used the sham acupuncture arm as the control group. We pooled the rates of biochemically-confirmed cocaine abstinence in intervention and control groups across included trials using a random-effects meta-analysis and report the results using a forest plot describing the odds ratios (OR) and 95% confidence intervals (CI) at last reported time point. A priori explanations of between-study differences beyond chance included reporting of allocation concealment, use of intention-to-treat analyses, and large loss to follow-up (>50%). We tested for heterogeneity using the Zalen test and measured the proportion of between-study differences not attributable to chance with the I 2 statistic[ 12 ]. Our secondary analysis considered all drop outs as treatment failures. All statistics were performed using StatsDirect (Manchester, 2003). Results The search yielded 83 relevant abstracts (Figure 1 ). Of these, 20 were retrieved for potential inclusion, four studies were not randomized controlled trials [ 13 - 16 ], four studies investigated methodological issues in acupuncture trials [ 17 - 20 ], 2 included polysubstance abusers[ 13 , 21 ] and one investigated pharmacothearapy[ 22 ]. Table 1 describes the 9 trials included in the final analysis (See additional file 1 ). Chance-adjusted inter-rater agreement was high (κ = 0.96, 95% CI 0.91 to 1) [ 23 - 31 ]. Figure 1 Study identification for a Systematic Review of Acupuncture for Cocaine Dependence. Figure 2 Meta-analysis of 7 trials. Study characteristics The 9 RCTs were conducted in the USA and included 1747 participants: 488 participants in active groups and 821 assigned to control groups (One RCT did not describe group sizes[ 25 ]). One RCT included only crack cocaine users[ 27 ], 5 RCTs included samples with mixed forms of cocaine abuse (eg. intravenous, inhaled, or intranasal) and 3 RCTs did not describe the type of cocaine or the route of administration[ 26 , 29 , 31 ]. RCTs enrolled participants with different rates of anti-craving medication use and 3 RCTs included only patients using methadone in addition to cocaine[ 23 , 26 , 31 ]. Three RCTs enrolled some patients using methadone[ 24 , 27 , 28 ], 2 RCTs excluded patients who used methadone[ 25 , 29 ] and 1 did not report the use of anti-craving medication among enrolled subjects[ 30 ]. All 9 trials employed auricular acupuncture, 4 employed a specific auricular acupuncture regimen (National Acupuncture Detoxification Association: NADA) and 2 used a combination of auricular and body points. Five trials had more than 1 control group [ 24 - 26 , 28 , 31 ]or randomized subjects to receive methods including relaxation[ 26 , 28 , 31 ], anti-craving medication and brainwave modification[ 24 ], or psychosocial treatment[ 25 ]. Eight trials used urine assays for cocaine metabolites (benzoylecgonine) for biochemical confirmation of abstinence at follow-up; we were able to obtain results from 7 of them. Eight trials examined the likelihood of retaining patient participation in the trial, and 5 trials examined cocaine cravings; no trials reported participant follow-up or relapse. Reporting quality of manuscripts Although all trials were randomized, only 4 trials described the randomization technique[ 26 , 28 , 30 , 31 ]. Two trials employed restriction to balance the groups[ 26 , 28 ]. Allocation concealment was not adequately reported in any study. Information regarding acupuncture technique needle depth and needle type was present in 3 trials[ 23 , 26 , 29 ]. Methods of inserting a sham needle as a control were used in 8 trials[ 23 , 25 - 31 ]. Five trials reported more than 20% loss to follow-up (mean loss to follow-up across all trials was 50% [range, 0–63%])[ 25 , 26 , 28 - 30 ]. Only 4 trials described reasons for withdrawals[ 25 , 26 , 28 , 29 ]. Meta-analysis We pooled results from 7 trials that reported biochemical confirmation of cocaine abstinence. The pooled odds ratio estimating the effect of acupuncture on cocaine abstinence was 0.76 (95% CI 0.45–1.27, P = 0.3, I 2 = 30%, Heterogeneity P = 0.19) at the last reported time point (range 4–12 weeks) (see figure 2 ). Our a priori hypotheses failed to explain the heterogeneity that was present. That is, for each a priori explanation, the magnitude of the effect differed little irrespective of the level of the hypothesized explanatory factor. Our secondary analysis, considering all dropouts as treatment failures, resulted in a pooled odds ratio of 0.76 (95% CI, 0.54–1.08), P = 0.12, I 2 = 0%, Heterogeneity P = 0.5). Adverse effects reported included pain and fear of needles[ 23 , 26 , 28 , 29 , 31 ] No trials reported the proportion of participants suffering adverse effects. Discussion Statement of findings This systematic review and meta-analysis does not support the use of acupuncture for the treatment of cocaine dependence. However, most trials were hampered by large loss to follow up and the strength of the inference is consequently weakened. Strengths and weaknesses We minimized publication bias by conducting an extensive search through multiple databases, including Chinese-language databases. We successfully received original data from several authors. We also sought to minimize selection and ascertainment bias by including only randomized controlled trials with biochemical confirmation of abstinence in our meta-analysis. However, large loss to follow-up and unexplained inconsistency across the included trials weakens our inferences. The conduct of explanatory trials to ascertain the efficacy of acupuncture for cocaine addiction is challenging. We do not know whether there is a group of patients more likely to respond to this intervention who will agree to take part and stay enrolled in a clinical trial with adequate duration of follow-up. Pragmatic trials enrolling heterogeneous patients need to be large enough to detect small treatment effects and should be conducted according to the intention-to-treat principle to avoid large loss to follow-up. However, we recognize the difficulty of enrolling and maintaining patients who use or are dependent on cocaine in an RCT. Design strategies to maintain patients enrolled in trials of cocaine addiction treatment represent a research frontier. Blinding of participants in a clinical trial is important because knowledge of group assignment may influence responses to treatment[ 32 ]. Use of sham controls and blinding of patients may prevent bias. The sham acupuncture method has been an area of debate as it is difficult to determine if a needle inserted into the skin away from designated acupuncture points is inert[ 20 , 33 ]. In this systematic review, 8 trials applied sham acupuncture in the control groups[ 23 , 25 - 31 ], 3 of which used the NADA technique for acupuncture protocol and these trials found inconsistent results[ 26 , 28 , 31 ]. Three trials also used a similar 5-point acupuncture protocol and did not find a significant effect[ 25 , 27 , 29 ]. We identified one additional systematic review of acupuncture for cocaine addiction which is in the process of data collection for a Cochrane review, and worked collaboratively with the primary investigator [S. Gates, personal communication]. We found systematic reviews assessing the efficacy of acupuncture in patients abusing other substances (nicotine[ 10 ], alcohol and heroin[ 6 , 9 ]). Our findings are consistent with these other reviews in that they included trials with large loss to follow-up and were unable to draw strong inferences about the efficacy of acupuncture as a single intervention in the management of substance abuse. At present, no proven pharmacotherapies exist for cocaine dependence[ 8 , 34 , 35 ]. Current psychosocial strategies may only be effective in select patients with stable living conditions and social support and meta-analyses of these interventions are ongoing[ 6 , 36 ]. However, a lack of proof of efficacy does not indicate a lack of efficacy, and the data are dramatically hampered by dropouts. In treatment environments wishing to use multimodality therapy, acupuncture may be used for treatment of cocaine dependence since it is a relatively safe intervention and may be in line with patient values and cultural experiences. However, in resource-constricted environments, the use of acupuncture for cocaine dependence is not justified with the current evidence. Conclusion Our systematic review and meta-analysis yielded inconclusive data about the effect of acupuncture. The best estimate of effect is consistent with no treatment effect. Initiatives, such as court mandated programmes, which recommend acupuncture for cocaine dependence, are not supported by the available evidence. Authors' contributions Edward Mills developed the protocol, conducted the search and study selection, worked at data abstraction and quality rating and wrote the manuscript. Ping Wu conducted the search and study selection, worked on data abstraction and quality rating, and analyzed the data. Joel Gagnier worked at data abstraction and wrote the manuscript. Jon Ebbert provided critical revision and writing of the manuscript. We greatly appreciate the advice and contributions of Dr. Victor M. Montori Competing interests The author(s) declare that they have no competing interests. Supplementary Material Additional File 1 Table 1. Characteristics and findings of included studies Click here for file

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1079915

Measuring and reporting quality of life outcomes in clinical trials in cystic fibrosis: a critical review

Good quality clinical trials are essential to inform the best cystic fibrosis (CF) management and care, by determining and comparing the effectiveness of new and existing therapies and drug delivery systems. The formal inclusion of quality of life (QoL) as an outcome measure in CF clinical trials is becoming more common. Both an appropriate QoL measure and sound methodology are required in order to draw valid inferences about treatments and QoL. A review was undertaken of randomised controlled trials in cystic fibrosis where QoL was measured. EMBASE, MEDLINE and ISI Web of Science were searched to locate all full papers in the English language reporting randomised controlled trials in cystic fibrosis, published between January 1991 and December 2004. All Cochrane reviews published before December 2004 were hand searched. Papers were included if the authors had reported that they had measured QoL or well being in the trial. 16 trials were identified. The interventions investigated were: antibiotics (4); home versus hospital administration of antibiotics (1); steroids (1); mucolytic therapies (6); exercise (3) and pancreatic enzymes (1). Not one trial evaluated in this review provided conclusive results concerning QoL. This review highlights many of the pitfalls of QoL measurement in CF clinical trials and provides constructive information concerning the design and reporting of trials measuring QoL.

Review Cystic fibrosis and its management Cystic fibrosis (CF) is a life threatening, recessively inherited disease caused by defects in a single gene on chromosome 7 [ 1 , 2 ]. The faulty gene causes an increased production of thickened secretions in most organs of the body. In the respiratory tract this impairs the clearance of micoorganisms resulting in recurrent infections, inflammation, lung damage and eventually death from respiratory failure. In the pancreas, the pancreatic exocrine cells become blocked, leading to the failure of the pancreas to produce digestive enzymes causing the maldigestion and malabsorption of nutrients. Current median survival is more than 30 years [ 3 , 4 ], and half of children born in the 1990's are expected to survive to more than 40 years [ 5 ]. With increasing age, however, a high proportion of people develop diabetes mellitus, and some patients endure a variety of complications including pneumothorax, haemoptysis, chronic liver disease and osteoporosis. Cystic fibrosis has an extraordinarily demanding treatment regimen. The management of respiratory disease is directed at identifying and eradicating bacterial infection from the airways. Preventing chronic infection with Pseudomonas aeruginosa can slow down the deterioration in lung function and improve survival [ 6 , 7 ]. Respiratory disease is treated with antibiotics, mucolytics, bronchodilators and corticosteroids. Additionally, twice daily chest physiotherapy and an exercise regimen aid the clearance of respiratory secretions. The management of gastrointestinal symptoms hinges on the maintenance of body weight and, therefore, a high energy, high fat diet is prescribed. To aid the absorption of nutrients, oral pancreatic enzymes are taken with food. Malnutrition is managed with fat-soluble vitamins and oral feed supplements and/or where required, nocturnal enteral feeding. CF-related diabetes requires insulin, and further therapies are required to deal with other complications. The importance of clinical trials in CF Effective treatments, in particular those that are acceptable to patients, are crucial to slow down disease progression. Good quality clinical trials are essential to generate evidence to inform CF management, by determining and comparing the effectiveness of therapies and drug delivery systems. Often, the ultimate aim of treatment is to increase survival, but it is impractical for trials involving younger patients to have survival as a primary end-point. Instead investigators opt for proxy outcome measures. The progression of CF disease is usually evaluated by changes in lung function; forced expiratory volume in 1 second (FEV 1 ) being the typical primary outcome measure. It has been commonplace for researchers to conclude that because treatments had benefits for clinical outcomes there must also have been benefits for QoL. There are many such assertions in the literature without empirical evidence, however, the formal inclusion of QoL as an outcome measure in CF clinical trials is now increasing. The quality of clinical trials There has been considerable concern about the quality of reporting of randomised controlled trials (RCTs). Poorly-designed trials are likely to produce biased results and hence misinformed decision-making. Inadequate reporting means that readers are unable to assess the quality of the design of an RCT and are consequently unable to assess the usefulness of the conclusions. The CONSORT statement [ 8 ] was devised to improve the quality of reporting of RCTs. Both an appropriate QoL measure and sound methodology are required for valid inferences about treatments and QoL. With this in mind a review was undertaken of RCTs in cystic fibrosis where QoL was measured. The identification of papers EMBASE, MEDLINE and ISI Web of Science were searched to locate all full papers in the English language reporting RCTs in cystic fibrosis, published between January 1991 and December 2004. Papers were located that included 'cystic fibrosis' and 'quality of life' or 'health status' in the title, abstract or keywords, and these were hand-searched for all randomised controlled trials. Extra searches on 'well-being' were carried out in databases where this key word was accepted. Papers were included if authors reported that they had measured QoL or well-being. All Cochrane reviews [ 9 ] published before December 2004 were hand-searched. Only trials exclusively on people with cystic fibrosis were included i.e. trials were omitted where some of the subjects had cystic fibrosis. Papers were omitted which described a study on a subset of subjects in an RCT, but secondary analyses of RCTs where the primary findings had previously been published were included. Each author undertook searches and reviews independently. A summary sheet was used to record the following: rationale for measuring QoL; choice of QoL instrument, quality of scale description and scoring methods; sample size justification; quality of summary statistics for QoL and analysis; discussion of the clinical importance of QoL findings and general methodological quality in relation to QoL A total of 16 trials were identified. The interventions investigated were: antibiotics (4); home versus hospital administration of antibiotics (1); steroids (1); mucolytic therapies (6); exercise (3); and pancreatic enzymes (1). The trials, with patient characteristics, are summarised in Tables 1 and 2 . Table 1 presents a summary of RCTs evaluating antibiotics and steroids. Table 2 summarises the RCTs evaluating mucolytic therapies, exercise and pancreatic enzymes. Table 1 Summary of RCTs in CF measuring QoL: antibiotics and steroids Authors Brief description Sample QoL scale used Authors' main conclusion Authors' conclusion about QoL outcome ANTIBIOTICS Quittner et al. [14] Secondary analysis of Ramsey et al. [13] Secondary analysis of RCT. Tobramycin versus placebo in 3 cycles n = 520 (age >= 6 years) (n = 499 for QoL) mean age 21 years FEV 1 25% to 75% Non-validated 3-point scale (better/no change/worse) primary outcome Tobramaycin improved lung function (not reported here) Tobramycin associated with improved QoL Equi et al. [17] Azithromycin (250 gm or 500 gm dependent on weight) vs placebo; crossover design n = 41 (age 8–18 years) median FEV 1 = 61% (range 33% to 80%) QWB Significant improvement in FEV 1 compared with placebo, but not FVC or mid-expiratory flow No difference in QoL Wolter et al. [18] Azithromycin vs placebo; 2 parallel groups n = 59 (age 18–44 years; mean 27.9 years) mean FEV 1 = 56.5% CRQ Significant difference in FEV 1 and FVC favouring azythromycin Significant improvement in all domains of QoL Saiman et al. [19] Azithromicyn vs placebo; 2 parallel groups n = 185 (age >= 6 years) (n= 177 for QoL) mean age 20 years ≈ 60% had FEV 1 > 60% CFQ Significant difference in FEV 1 , lower risk of exacerbation, higher weight, but more side effects in treatment group Significant difference in physical functioning domain only HOME/HOSPITAL ANTIBIOTICS Wolter et al. [20] Home versus hospital IVs antibiotics; 2 parallel groups 17 adolescents and adults CRQ primary outcome No clinical compromise associated with home therapy Home IVs fared worse for fatigue and mastery, but better for personal, family, sleeping, eating and total disruption STEROIDS Balfour-Lynn et al. [24] Corticosteroids vs placebo; crossover n = 22 (age 7–17 years; mean 10.3 years) mean FEV 1 = 64% (range 21% to 102%) Ad hoc VAS scales No significant benefit in any of the outcomes No changes in well-being FEV 1 = forced expiratory volume in one second, expressed as percent predicted; FVC = forced vital capacity; QWB = Quality of Well-being Scale; CFQ = Cystic Fibrosis Questionnaire; CRQ = Chronic Disease Respiratory Questionnaire; ≈ = approximately. QoL is secondary outcome measure unless otherwise indicated. All authors refer to QoL in the title, abstract or paper except [24] who refer to well-being. Table 2 Summary of RCTs in CF measuring QoL: mucolytic therapies, exercise and pancreatic enzymes Authors Brief description Sample QoL scale used Authors' main conclusion Authors' conclusion about QoL outcome MUCOLYTIC THERAPIES Ranasinha et al. [27] DNase vs placebo; two parallel groups n = 71 (age = 16–55 years) mean FEV 1 ≈ 47% Ad hoc 9-item scale Significant improvement in FEV 1 but not in FVC DNase did not improve overall well-being but improvements in feeling, cough frequency and chest congestion Ramsey et al. [28] 3 doses of DNase vs placebo; 4 parallel groups n = 181 (age 8–65 years) mean FEV 1 between 58.6% and 84.6% for the 4 groups Ad hoc 9-item scale FEV 1 and FVC improved across all doses compared with placebo DNase associated with decreased dyspnoea and improved well-being Fuchs et al. [29] 2 doses of DNase vs placebo; 3 parallel groups n= 968 (age 5–54 years) mean FEV 1 ≈ 60% Ad hoc 9-item scale Improved lung function on DNase Increase in general well-being Wilmott et al. [30] 2.5 mg DNase or placebo twice daily n = 80 children and adults (age >5 years; mean ≈ 20) mean FEV 1 ≈ 40% Ad hoc scale No effect of drug on change in FEV 1 or FVC No differences on well-being scales Suri et al. [31-33] Open crossover study of DNase once daily 2.5 mg vs alternate day 2.5 mg and saline n = 48 (age 7–17 years) (n = 40 completed study) QWB-SA No evidence of differences between active treatments; daily treatment better than saline for FEV 1 No effects Eng et al. [34] 10 ml of either normal or hypertonic saline; parallel groups n = 58 (age 7–26 years) mean FEV 1 ≈ 52% Ad hoc VAS of perceived change Significant differential improvement from baseline in FEV 1 for hypertonic saline An improvement, but group difference did not reach statistical significance EXERCISE Selvadurai et al. [36] Comparison of aerobic/ resistance training and standard care; 3 parallel groups n = 66 (age 8–16 years) mean FEV 1 ≈ 57% QWB Aerobic training better for peak aerobic capacity. Resistance training better for weight gain, lung function and leg strength Aerobic training associated with better QoL Klijn et al. [37] Anaerobic training vs normal daily activity; 2 parallel groups n = 20 (age 9–18 years; mean 14 years) mean FEV 1 = 75.2% (exercise group); 82.1% (control group) Dutch CFQ Anaerobic and aerobic performance improved in training group, but not control group QoL improved in training group but not in control group Orenstein et al. [38] Aerobic versus upper-body strength training n = 62 (age 8–18 years) Analysis on 53 cases of complete data QWB Strength and aerobic training may increase upper-body strength, and physical work capacity No significant effects PANCREATIC ENZYMES Gan et al. [39] 4 versus 1 capsule daily crossover design n = 13 (age 19–46 years; mean 28 years) mean BMI = 21 Symptoms and general well-being on 10-point scale No difference between treatments No significant changes in scores for well-being FEV 1 = forced expiratory volume in one second, expressed as percent predicted; FVC = forced vital capacity; QWB = Quality of Well-being Scale; CFQ = Cystic Fibrosis Questionnaire; BMI= body mass index, ≈ = approximately. QoL is secondary outcome measure unless otherwise indicated. All authors refer to QoL in the title, abstract or paper except [34] [39] who refer to well-being. Review of CF trials that have measured QoL Antibiotic therapy One of the most important therapies for CF is antibiotics aimed at preventing, eradicating and controlling respiratory infection. Pseudomonas aeruginosa (PA) is the most prevalent infection [ 10 ], and the prognosis of chronically infected patients is considerably worse [ 11 ]. It is also known that pulmonary exacerbations have a strong negative impact on QoL in CF [ 12 ], therefore an effective antibiotic should be able to demonstrate improvements in QoL. Of the four antibiotic trials that reported measuring QoL in CF (as a secondary outcome) one evaluated tobramycin and the other three assessed azythromycin. Tobramycin's mode of delivery is appealing to patients as it is nebulised, delivering high concentrations of the drug to the site of infection; making the treatment less complex and time-consuming than by intravenous administration. Ramsey et al. [ 13 ] conducted a placebo-controlled RCT of inhaled tobramycin and concluded that the drug improved lung function. Quittner et al. [ 14 ] reported on QoL from a secondary analysis of this work. Patients were assigned to receive either tobramycin (300 mg twice daily) or placebo for three treatment cycles, with each cycle consisting of 28 days on the drug followed by 28 days off the drug. QoL was assessed only at the end of each treatment period (28 days on the drug) using a non-validated, three point, uni-dimensional global rate of change questionnaire. Patients (or parents of some children) and physicians reported whether the patient's condition remained unchanged, improved or deteriorated. The authors reported that a greater number of patients receiving tobramycin felt better at the end of each treatment cycle. There is evidence that patients taking the drug were more likely to report an improvement in their condition after each period of treatment. However, how 'feeling better or worse' would impact on different aspects of a person's QoL is unknown and the authors themselves suggest that evaluation with CF-specific QoL scales would be beneficial. It is also unclear how many children's ratings or parental proxy ratings were included in the analysis. Pre-treatment data were not collected so it is unknown what happened in the periods off treatment. Without this information, and the magnitude of all changes, it is impossible to deduce what the overall changes would have been if QoL had been measured at baseline and at 6 months. Macrolide antibiotics are unable to kill PA but it is thought that they may reduce the activity of the bacteria [ 15 ]. Azythromycin has become a focus of interest in CF because it is possible that it reduces sputum viscosity and airway adhesion of PA [ 16 ]. If a drug can reduce bacterial activity and inflammation, and enable sputum to be expectorated more easily, it may be expected to improve QoL. If it can also be administered orally, rather than being given intravenously or via a nebuliser, its potential impact on QoL may be considerable. Equi et al. [ 17 ] undertook a 15 month, randomised, double blind, placebo-controlled crossover trial. Patients received either azythromycin (bodyweight <40 kg = 250 mg daily; >40 kg = 500 mg daily) or placebo for six months. This was followed by a two month washout period prior to the treatments being crossed over. Lung function was the primary outcome measure. QoL was one of several secondary outcomes and was measured using the Quality of Well Being Scale (QWB). The scale is not described in the paper but it is a utility instrument, typically administered by interview. It generates a single score by summing the scores of the three subscales: mobility, physical activity and social activity. The authors reported an improvement in FEV 1 for the drug compared with placebo. The only results given for QoL were: 'The median difference in the visual analogue score (range 0–100) for well being between the end of the azythromycin and placebo treatment periods was 0, as was the change in the total quality of well being score'. It cannot be ascertained whether the use of a CF specific scale would have detected treatment differences. Wolter et al. [ 18 ] reported data from a parallel design RCT comparing azythromycin with placebo. Patients took azythromycin 250 mg/day or placebo for three months and were assessed at baseline and each subsequent month for lung function, weight and QoL. Quality of life was measured using the Chronic Respiratory Disease Questionnaire (CRQ). The CRQ has four subscales: fatigue, mastery, emotion and dyspnoea which can be summed to provide a total QoL score. The authors reported that treatment with azythromycin significantly reduced the rate of decline in lung function over time. Improvements in the domains of mastery, emotion, dyspnoea and total score were greater for those who received azythromycin. Improved fatigue scores were only seen in the azythromycin group. Although means and standard deviations of QoL scores are presented for each treatment group, there are a high proportion of missing values, and in the absence of confidence intervals it is difficult to assess the clinical importance of the results. Similarly, Saiman et al. [ 19 ] described a parallel designed, placebo-controlled RCT to 'determine any association between azythromycin and lung function in CF'. Treatment was prescribed (bodyweight <40 kg = 250 mg; >40 kg = 500 mg) on three days each week for six months. QoL was one of several secondary outcome measures, and was evaluated using the USA Cystic Fibrosis Questionnaire (CFQ). Unfortunately, the scale, which is a CF specific QoL measure, was not described in the paper. The CFQ child measure is a 33 item self report instrument that includes three broad domains of QoL: physical symptoms, emotional functioning and social functioning and there are five domains that are specific to CF; body image, eating disturbances, treatment burden, respiratory symptoms and digestive symptoms. The adult version has 48 Items across 12 domains: physical, role, vitality, emotion, social, body, eating, treatment, health, weight, respiratory and digestion. The authors presented the QoL results as 'three broad factors' (physical, psychosocial, and body image) plus a total score. Presumably, these factors were an amalgamation of domains, although no explanation is provided as to how they were derived. The authors also appear to have combined the child and adult versions of the CFQ but provide no rationale or account of how this was done. A differential improvement in pulmonary function and nutritional status was reported for the azythromycin group, but for QoL the only statistically significant difference reported was for the 'physical factor'. The CFQ scales are usually scored from 0–100. If this is the case, the observed difference of 2.7 in the 'physical factor' change score is unlikely to have clinical importance for the patient. Even a difference of 5.3, which is the extreme of the stated 95% confidence interval, is likely to be only marginally clinically important. Moreover, the small difference reported can be largely accounted for by an average deterioration of 1.9 in the placebo group. The problems of interpretation are exacerbated by a lack of baseline data on QoL scores. Home intravenous antibiotic therapy Home intravenous (IV) therapy is a popular form of treatment. It has the advantages of cost-saving by freeing up hospital beds and avoiding cross infection; and the patient and family are able to continue their normal activities. However, a crucial question is whether patients would adhere to their treatment sufficiently to ensure that the home and hospital environments would provide equivalent outcomes. Wolter et al. [ 20 ] conducted a parallel designed RCT of home compared with hospital IVs. The aim was 'to determine the equivalence of home and hospital care... so that if no difference was detected between the two modes of treatment, this could be stated with confidence'. Those randomised to home therapy spent 2–4 days in hospital being taught how to prepare and administer their own antibiotics. Data were obtained from 17 adolescent and adult patients for 31 admissions of respiratory exacerbation. Therapy consisted of ceftazidime, 2 g 12 hourly and tobramycin 4–6 mg/kg daily as a single bolus. QoL was the primary outcome, measured using the Chronic Respiratory Disease Questionnaire (CRQ). Patients were assessed on days 0, 10 (cessation of drug) and 21. On day 21 they were asked to score the degree of disruption to family, personal, sleeping and eating aspects of their life on a 7-point scale. The median duration of treatments was similar for both home and hospital groups. The authors reported no differences concerning dyspnoea and emotional functioning, but the fatigue, mastery and total CRQ scores were poorer for home patients. However, improved QoL was reported in the areas of personal, family, sleeping, eating and total disruption for home, compared with hospital admissions. There were no reported statistical differences in clinical outcome. QoL scores were the main outcome, but the study was only powered on the dyspnoea QoL scale. The sample size calculation, carried out during the study design, was based on 95% power to detect 'differences of 5 or more units in the dyspnoea score'. The authors reported that 'a 5 units difference... was hypothesised as an important change'. Yet after the data had been collected it was clear that the power calculation was invalid, and the sample too small. The estimated difference in dyspnoea change scores was 2.5, but no confidence intervals were presented. It is likely that a 95% confidence interval for this difference would contain values greater than 5. Anti-inflammatory therapy Lung inflammation can occur very early in life [ 21 ] and corticosteroids have the potential to reduce lung damage arising from inflammation. These drugs are among the most potent anti-inflammatory agents available and there is widespread prescribing of inhaled steroids in CF [ 22 ]. Oral corticosteroids are associated with several adverse effects, although the adverse effects of inhaled steroids are fewer. However, a Canadian trial was stopped prematurely because of the increased frequency of PA [ 23 ]. Because there are perceived and potential benefits and harms of steroid treatment, QoL measurement is very useful. One such trial was located. Balfour-Lynn et al. [ 24 ] conducted a double blind, placebo-controlled, randomised crossover trial in which fluticasone propionate (400 ug daily) was given as a dry powder inhaler. The drug was inhaled for six weeks with a four-week washout period before crossover. There were several biochemical and clinical outcome measures. At the beginning and end of each treatment period, scores for general well-being and appetite were recorded on a 10 cm visual analogue scale, in order to establish symptom severity. The authors concluded that there were no changes in respiratory symptoms, well-being or appetite scores. However, there is no discussion of the clinical importance of the confidence intervals in what the authors acknowledge is a small and possibly underpowered study. Mucolytic therapies Mucolytic agents enable respiratory secretions to be expectorated more easily. Dornase alfa (DNase) is a nebulised treatment intended for administration prior to chest physiotherapy to maximise chest clearance. Nebulised hypertonic saline is also a potential mucolytic therapy for CF. This review identified six RCTs of mucolytic therapies that measured QoL as a secondary outcome. Four of these were placebo-controlled trials of DNase, one compared DNase to hypertonic saline and one was a placebo-controlled trial of hypertonic saline. Early studies evaluating the effective dosage, biochemical efficacy and safety of DNase in CF adults, employed a non-validated ad hoc measure of QoL [ 25 , 26 ]. This comprised five questions concerning general well-being (feeling, energy, physical activity, appetite, sleep) and four CF-related symptoms (cough frequency, cough severity, ease of sputum expectoration and chest congestion). Patients reported these items on a five-point Likert response scale. Additionally, the magnitude of dyspnoea was rated on a visual analogue scale. Even though there were no data describing the validity, reliability or sensitivity of the measure, it was subsequently used in several studies of DNase. These included the four randomised controlled trials of DNase reported here. Ranasinha et al. [ 27 ] conducted a double-blind, placebo-controlled RCT in which patients received either 2.5 mg DNase twice daily or placebo for 10 days. The authors reported an improvement in lung function following DNase therapy. QoL was measured on seven occasions over the study period of 47 days (including pre-trial and follow-up). Only baseline QoL scores are reported but the authors infer that although there was no improvement in overall well-being or dyspnoea there were improvements in 'feelings', cough frequency and chest congestion. The total absence of any QoL change data makes it impossible for the reader to judge or interpret. In a double blind placebo-controlled RCT performed by Ramsey et al. [ 28 ] children and adults were recruited. The trial consisted of four parallel groups (three different doses of DNase and placebo). The study period was 42 days with medication administered for the first ten days.Mean percentage change in lung function from baseline for each treatment group were provided at days 3, 10, 21 and 42. An improvement in lung function was observed during the administration of DNase, with values declining towards baseline following the treatment period. The authors reported a decreased perception of dyspnoea and an improved perception of well-being in the DNase groups compared to controls. Voice alteration and sore throat were more frequent among patients receiving DNase. However, QoL was not measured past day 10 so it is unclear whether QoL would have demonstrated similar trends to those of lung function. No baseline values are presented for QoL, but mean changes (from baseline to Day 10) are given for each group, without any measures of variability. There is no discussion of any possible dose-response (as is presented for FEV 1 ), or of the clinical importance of the observed differences. These differences appear to be too small to be clinically important, and it is difficult to detect any consistent trends in the dose-response. Problems in interpretation are exacerbated by the lack of baseline data on QoL. It is impossible to judge if patients had scope for improvement, whether the scales were sensitive, or whether any particular dose is to be preferred with respect to QoL. Fuchs et al. [ 29 ] conducted a double-blind, placebo-controlled RCT with three parallel groups (2.5 mg DNase once or twice daily and placebo). Children and adults were treated over a six-month period. Both doses of DNase resulted in slightly improved lung function. The authors report an increase in general well-being and a decrease in CF symptoms, although information is not available concerning specific symptoms of the measure (e.g. cough frequency). This was a large study and although the authors reported statistically significant differences these do not appear to be clinically important. There were ceiling effects in QoL measures at baseline: for each group the mean score was 3.9, with a maximum score of 5 (ceiling). Although the authors reported that the average change in well-being score for the once-daily group was significantly greater than for placebo, this change is extremely small, and is not replicated in the twice-daily group. There is therefore little convincing evidence about the effects of the treatments on QoL. Wilmott et al. [ 30 ] undertook a double blind, placebo-controlled RCT with two parallel groups. Patients received either 2.5 mg DNase or placebo twice per day for 14 days. Clinical and QoL data were recorded on days 1, 8 and 15. Similar changes in lung function occurred in both groups. The authors reported no differences between the groups for any of the well-being or CF symptom items, but they do not provide enough information to enable the reader to interpret the data. There was evidence of greater improvement in dyspnoea (measured on the VAS) in the treatment group. This was statistically significant at day 8, but not at day 15, although the difference may have still been clinically important at day 15. This possible treatment effect was not reported in the QoL results, where, for all scales (including dyspnoea at different levels of activity) they report 'no difference between the groups'. DNase is a comparatively expensive treatment for CF and not all patients can benefit from it. Hypertonic saline is an inexpensive potential alternative. A comparison of DNase and hypertonic saline was undertaken by Suri et al [[ 31 - 33 ] – publications of same trial]. They conducted an open, randomised crossover trial. Children were randomised to once daily DNase (2.5 mg), alternate day DNase (2.5 mg) or twice daily 5 mL 7% hypertonic saline in blocks of 12 weeks' duration with a two week washout period between treatments. In addition to the efficacy of the three treatments the study aimed to compare cost-effectiveness and therefore a utility measure of QoL, the Quality of Well-being Scale-Self Administered (QWB-SA), was chosen. The QWB-SA contains five domains: acute and chronic generic symptoms, self care, mobility, physical activity and performance of usual activities. These domains are combined to produce a well-being score of between 0 (death) and 1 (symptom-free full function). Parent and child completed the questionnaire together. The authors reported no difference in improved lung function between daily and alternate day DNase (16% and 14% improvement in FEV 1 % respectively). A mean FEV 1 % predicted improvement of only 3% was observed for the hypertonic saline condition and a statistically significant difference was reported between daily DNase and hypertonic saline. There were no significant changes from baseline for any treatment on the QWB-SA scale; neither were there differences between the treatment change scores. The trial was not powered for QoL and in one paper [ 31 ] the authors included confidence intervals. An improvement in reported well-being may be expected to accompany these relatively large changes in lung function in the DNase groups, yet the authors do not discuss possible reasons for these negative findings. Eng et al. [ 34 ] conducted an open-label, placebo-controlled, parallel trial. Patients were randomly allocated to receive 10 ml of either normal saline (0.9% NaCl) or hypertonic saline (6.0%NaCl) twice daily (nebulised prior to physiotherapy) for two weeks. Change in lung function was the primary outcome and patient-perceived CF related symptoms were a secondary outcome. Patients rated perceived change of dyspnoea, fatigue, appetite, exercise tolerance, sleep and general well-being on a 10 cm visual analogue scale (VAS) on days 14 and 28. They also rated the effectiveness of sputum clearance on a similar VAS based on their diary information. Hypertonic saline improved lung function (at day 14) compared with normal saline; the mean increase in FEV 1 % predicted from baseline was 15% for the hypertonic saline compared with 2.8% for normal saline. The authors reported that the administration of hypertonic saline significantly improved exercise and the quality of sleep. For the other symptoms and general well-being there were no statistically significant differences between the groups. However, it is unlikely that the study was adequately powered for symptoms and well-being scores. While some of the estimated differences may only appear marginally important, it is probable the 95% confidence intervals for most of the symptoms and well-being scores would contain clinically important values. Given this problem, together with a total lack of information about QoL and symptoms at baseline, the QoL results are inconclusive. Exercise Lung function and exercise tolerance decrease as CF disease progresses. Exercise training aims to preserve and improve fitness levels and enable CF patients to perform everyday activities. Patients with high levels of aerobic fitness have better survival than those with low fitness levels [ 35 ]. Aerobic training aims to improve cardiovascular function whereas anaerobic or resistance training aims to improve muscle strength. Three RCTs of exercise measuring QoL as a secondary outcome were located. Selvadurai et al. [ 36 ] conducted an RCT to compare aerobic and resistance training. Children, admitted to hospital for a pulmonary exacerbation, were randomised to one of three parallel groups; aerobic training, resistance training and a control group. The mean duration of hospitalisation was around 19 days for each group. The exercise groups received five training sessions each week. All groups received in-patient standard care – intravenous antibiotics, chest physiotherapy and nutritional supplements. Peak aerobic capacity and lung function appear to be primary outcomes, measured on admission to hospital and at discharge. Quality of life, measured by the QWB scale, was assessed at baseline and one month following discharge. The authors do not describe the scale, its scoring method, or what the tabulated values represent. They report that aerobic training was associated with better peak aerobic capacity, activity levels and QoL, whereas children who received resistance training had better weight gain, lung function and leg strength. Klijn et al. [ 37 ] investigated the effects of anaerobic training in non-hospitalised children who were randomly assigned to either a training group or control group. The exercise group trained for 30–45 minutes, twice each week for twelve weeks. There were several clinical and physiological outcomes. QoL was measured by the Dutch version of the Cystic Fibrosis Questionnaire (CFQ). A 47 item teen/adult scale and a 35 item child scale of the CFQ were employed but there was no description of the psychometric properties of this recently-translated scale. The domains of the scales were not described and neither was the instrument's scoring system. All outcome measures were evaluated at baseline, at the end of the training period and at 12 weeks' follow-up. The authors reported that by the end of the training period the children in the exercise group had improved their anaerobic and aerobic performance and QoL. At twelve weeks follow-up anaerobic performance and QoL remained higher than pre-training values. However, for QoL scores there are no direct statistical tests comparing the groups, and no confidence intervals. It also appears that data from the CFQ child and teen/adult scales were incorporated into the same analysis and it is unclear how this was achieved. There was inadequate reporting of summary statistics to enable the reader to interpret the findings, and selective reporting of QoL data. The CFQ scales consist of numerous domains but only the physical functioning domain was statistically significant. Even so, the authors conclude that 'anaerobic training has measurable effects on QoL'. Furthermore, the interpretation of the physical function data is unreliable because baseline imbalances in this score were not considered (training group mean 70.3; control group mean 83.2). Orenstein et al. [ 38 ] conducted a one-year randomised trial to compare the effects of a home-based, semi-supervised, upper-body strength training regimen with a similarly structured aerobic training regimen. Sixty-two children participated in the trial although analysis was undertaken on 53 completed cases. Participants in both exercise conditions were visited at home once per week for the first eight weeks then monthly for the remainder of the study. All patients were encouraged to exercise at least three times per week for a year. Aerobic fitness, pulmonary function, strength and QoL were measured at baseline, 6 and 12 months. QoL was measured using the interview format of the QWB scale: for children younger than 12 years a parent responded. The authors concluded that strength and aerobic training may increase upper body strength and that both types of training may increase physical work capacity. There were no statistically significant differences for either within or between group analyses for QoL. Interpretation of results rests on p-values, with no discussion of clinical importance, although the reported differences in total well-being score appear to be too small to be of clinical importance. Pancreatic enzyme therapy The majority of CF patients develop pancreatic insufficiency which leads to the maldigestion of dietary lipid, protein and carbohydrate. Pancreatic enzyme replacement therapy is used to manage maldigestion with patients taking numerous capsules each day. Gan et al. [ 39 ] conducted a double-blind, randomised crossover trial to compare a high lipase pancreatic enzyme preparation (Pancrease-HL) with regular Pancrease capsules. The rationale was that if the same strength of pancrease could be put into 1 capsule instead of 4, adherence to treatment may improve. Thirteen adults participated in two study periods of 14 days. During each period patients took 5 capsules 3 times per day; either 4 of Pancrease and 1 of placebo or 1 of Pancrease-HL and 4 placebo. The primary outcome measures appear to be mean fat and nitrogen excretion. General well-being was measured daily on a ten point scale. The authors reported no differences between the groups for any outcome. It is unclear whether the scores presented are baseline or change scores, or if the daily ratings were combined in some way to calculate the well-being scores. The authors' conclusion is that one capsule of the new preparation appears to be 'equivalent' to four capsules of the regular preparation, which suggests that the design should have been an equivalence study. However, in the absence of a clear hypothesis for the study, or a sample size calculation it is difficult to be sure. Whatever the hypothesis, the possible lack of power means that confidence intervals are essential for valid interpretation of results. Discussion of QoL data from CF trials The value of the evidence from a trial depends on the quality of its design. Much of the data presented here was difficult to interpret. There are several reasons for this, and these are presented in this discussion. Choice of QoL measure Only a few papers provided a rationale for measuring QoL [ 14 , 20 , 30 , 31 ] or a rationale for the choice of QoL instrument [ 18 , 20 , 30 , 31 ]. A variety of scales, reported to measure QoL, were employed: a uni-dimensional global rate of change questionnaire [ 14 ],'ad hoc' CF rating scales [ 24 , 27 - 30 , 34 , 39 ], the Quality of Well-being Scale [ 17 , 31 , 36 , 38 ], the Chronic Respiratory Disease Questionnaire [ 18 , 20 ], and the American [ 19 ] and Dutch [ 37 ] versions of the Cystic Fibrosis Questionnaire. The fact that a scale has been previously used on a CF population does not mean that it has been appropriately validated for use in CF studies. Apart from CF specific QoL instruments [ 40 , 41 ] only the SF-36 [ 42 , 43 ] and the Chronic Respiratory Disease Questionnaire (CRQ) [ 44 ] have undergone appropriate psychometric testing in CF. A chosen scale should be relevant to the participants, disease and intervention. It is also important that a scale is sensitive to change, otherwise a negative finding could be an artefact of the insensitivity of the scale and not of the real effect of the treatment on QoL. It may be more ethical not to measure QoL at all than to use an inappropriate scale which could give misleading results. Quality of reported data In order for readers to assess the importance of RCT findings, it is essential to have adequate descriptions of the outcomes and data. CONSORT recommends: 'Authors should give full details of how the primary and secondary outcomes were measured' and 'for each primary and secondary outcome, a summary of results for each group and the estimated effect size and its precision (e.g. 95% confidence interval)'. Description of scale and scoring methods Some papers gave neither a full description of the scale nor an explanation of the scoring method [ 17 , 19 , 30 , 36 , 37 , 39 ]. Several studies included children but it is not always clear whether the child, parent or a combination responded. Irrespective of the difficulties encountered with the interpretation of a combination of patient and patient proxy data, it is useful to know from whom the data have been obtained. A comprehensive description of the scale and scoring methods is particularly important in cases where scales have been merged across child and adult versions [ 19 , 37 ] or the scale transformed [ 38 ]. One paper stated that two scales with different numbers of items and domains had been combined but did not justify this or describe how it had been done [ 37 ]. While the statistical power is likely to be increased by such pooling, there is no guarantee that it is clinically meaningful. Baseline data QoL outcomes are often secondary, and a RCT may not have been powered on them. It is therefore important that adequate summary information is provided. A table of baseline characteristics, by treatment group, allows readers to judge the success of the randomisation. There may be clinically important baseline differences between the groups, or ceiling or floor effects. In several papers it was impossible to assess the validity of the conclusions, as there was incomplete summary information about QoL baseline values [ 17 , 19 , 20 , 28 , 34 , 37 ]. Statistical versus clinical significance It is important to distinguish between statistical significance and clinical significance. This is addressed in the CONSORT statement as follows: 'The difference between statistical significance and clinical importance should always be borne in mind. Authors should particularly avoid the common error of interpreting a non-significant result as indicating equivalence of interventions. The confidence interval provides valuable insight into whether the trial result is compatible with a clinically important effect, regardless of the p-value'. Most statistical tests will give a confidence interval for the difference, as well as a p-value. A 95% confidence interval gives a range of values, which we can be 95% sure contains the true difference. When the p-value is greater than 0.05, and the 95% confidence interval contains no values of any clinical importance, it is reasonable to conclude that any real difference between treatments is too small to be of any interest. If a study is too small then the observed difference may be too small to reach statistical significance, even in the presence of a genuine difference in treatments. Conversely, in large studies, small changes in both FEV 1 and QoL scores may be statistically significant as determined by a p-value but the observed differences may be too small to be clinically relevant. In this review there was a tendency for QoL results to be interpreted in terms of statistical significance. Of the two papers where QoL was the main outcome, Quittner [ 14 ] provided a full description of the data, whereas Wolter [ 20 ] gave no confidence intervals or measures of variability. Only three studies in which QoL was a secondary outcome reported confidence intervals [ 19 , 24 , 31 ] even though the studies were not powered on the QoL outcome. Most reported standard deviations or standard errors but three gave no description of variability [ 17 , 27 , 39 ]. Overall, there was little or no discussion of QoL results when they were statistically non-significant. Neither was there adequate discussion of the relationship between QoL results and those for the primary outcomes. Types of trials, sample size and QoL Superiority trials It is important that an RCT has adequate power. The majority of RCTs are superiority trials, where the aim is to test whether one treatment is better than another (or than placebo). CONSORT states: 'Ideally a study should be large enough to have a high probability (power) of detecting a statistically significant or clinically important difference of a given size if such a difference exists'... 'Reports of studies with small samples frequently include the erroneous conclusion that the intervention groups do not differ, when too few patients were studied to make such a claim'. In this review some papers did not report a formal sample size calculation for any outcome [ 24 , 28 , 38 , 39 ]. One study [ 24 ] reported a retrospective power calculation based on the observed data, which is of little value [ 8 ]. No study had an a priori sample size calculation for QoL when this was a secondary outcome. Crossover trials In some circumstances using a crossover design can alleviate the problem of requiring a large sample. Four crossover trials were located [ 17 , 24 , 31 , 39 ]. Crossover trials are useful where the effect of a treatment can be switched on and off, and it is ethical to do this. When feasible they can be more efficient than parallel-group designs because each participant acts as his/her own control and a smaller sample is required for the same statistical power. However, although a great deal might be known about the pharmacological effect of a drug in the body and its consequent effect on lung function, much less is understood about possible carryover effects on QoL. This is potentially compounded in studies where there are more than two treatment periods. Non-inferiority trials There are instances in which the aim is not to demonstrate superiority of one treatment over another, but to show therapeutic equivalence i.e. that one treatment is not inferior to another. The methods used for superiority trials are then of no use; failure to find a statistically significant difference is not the same as establishing equivalence. In fact, it is impossible to show 'exact' equivalence, so researchers have to define the range of values, near zero, that are of no clinical interest. This value of 'no difference' must, logically, be less than the minimal clinically important difference (MCID). For example, if the MCID for a QoL scale is 10 points a difference of 3 units between groups would probably be classed as 'equivalent'; it may be less clear how to view a difference of 7 units. This is a clinical decision, not a statistical one, and the values need to be established before the sample size calculation is done. For the two cases in this review where equivalence designs were apparently employed [ 20 , 39 ] there was either a lack of precision in the aim of the trial, or an inappropriate analysis. In the one trial where QoL was the primary outcome [ 20 ], the sample size calculation was not presented in terms of equivalence, and was revised after data were collected. This resulted in a retrospective adjustment to the definition of what had been deemed to be a clinically important difference. There was no sample size calculation in the other study [ 39 ]. Missing values Many RCTs suffer problems with missing values. These can be sporadic (e.g. where a patient misses an appointment or omits to return one questionnaire) or because of dropout, where data are completely missing after a certain time. This can be a common problem in QoL measurement and researchers have to decide how to deal with incomplete data. One way is to omit cases with missing values. If values are missing 'completely at random', then analysis on the complete data can be valid, although it is against the principle of 'intention to treat'. However, missingness is more often related to a change in condition [ 45 ]. In practice it is very difficult to be sure that values are missing completely at random, simply because the values are unknown. The implications of missing data are potentially serious if about 15% or more of the values are missing, or if there are different patterns of missingness between treatment groups. When there is a significant amount of missing data it is highly desirable to check the sensitivity of the results by using more than one statistical method. There are a variety of methods that can handle incomplete data. Different methods give potentially different results, because they make different assumptions about the reasons for missingness. Some methods are relatively simple (e.g. use the last recorded value) but p-values and confidence intervals should really be adjusted. Methods like t-tests and repeated measures ANOVA require complete data. However, there are more sophisticated methods like Generalised Estimating Equations (GEEs) that can be used to analyse longitudinal data with different numbers of observations per participant. Nonetheless, the fact that the analysis is possible does not mean that it is valid. Researchers should compare participants with and without missing data to check for potential systematic differences. Most papers reported missing observations for the QoL outcomes, some of which were substantial [ 18 , 31 , 38 ]. Only Suri [ 31 ] gave any description of dropouts and checked that they were not the most severely affected participants. Orenstein [ 38 ] omitted dropouts from the analysis because of potential bias, but did not explore the nature of the dropouts and the possible bias resulting from their exclusion. Some researchers used GEEs for their analysis [ 18 , 31 , 38 ] but gave incomplete details of the chosen options. Conclusion Authors tend to provide definite statements about QoL, but no trial in this review provided conclusive data. Even well-designed clinical trials can be difficult to manage, and high-quality reporting is essential. In the papers in this review the treatments and patient characteristics were generally well described. Difficulties arose when QoL scales were not described and summary information was not provided. Journal editors place word restrictions on papers so full descriptions of all secondary outcomes are not always possible. However, interpretation was further confounded when the emphasis was purely based on statistical significance ignoring clinical relevance. Although the CONSORT statement is an excellent guide for RCTs, it was not available when some of the trials were reported, and it does not specifically address QoL assessment and psychometric validity [ 46 ]. Properly validated disease-specific QoL measures are most appropriate for the evaluation of CF therapies as they include the relevant items that are likely to be sensitive to change [ 47 ]. Without suitable guidelines it is difficult for authors to employ QoL scales appropriately. This review highlights many of the pitfalls of QoL measurement in CF clinical trials and provides constructive information concerning QoL in trial design and the reporting of QoL data. Authors' contributions Both authors planned the study, independently located and reviewed the trials, drafted the paper and agreed the final version

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1079916

The French Aging Males' Symptoms (AMS) scale: Methodological review

Background The AMS is an internationally used health-related quality of life (HRQoL) scale. The aim of this paper is to provide evidence that the French AMS scale measures HRQoL are as valid as other language versions. We also intend to show whether the application of AMS is really limited to aging males only or not. More generally, we like to demonstrate that the AMS scale is a relevant, validated, sensitive instrument to measure HRQoL and change of symptoms in France. Methods We performed a representative survey in France to get data AMS scale data. The French data were compared with existing data from other European countries. Only community-based data were used for this comparison. Results and Discussion Reliability (here consistency, Cronbach' s alpha) was found to be good and almost identical with other countries. Validity: the internal structure of the AMS (factorial analysis) was sufficiently comparable with the comparison group of other countries in Europe to conclude that the scale really measures the same phenomenon. The sub-scores and total score correlations (Pearson) were high (r = 0.8–0.9) but only somewhat lower among the sub-scales (r = 0.5–0.7). This suggests that the domains are correlated. The comparison of the French AMS with the generic quality-of-life scale SF-12 showed a good correlation (Pearson r = 0.48 – 0.51) as reported from other countries. We observed also a good correlation between the AMS scale and the depression scale HAD (Pearson r = 0.62). The analysis of the AMS structure across age groups showed sufficient similarity to suggest that the AMS is also useful for younger age groups. Conclusion The French AMS scale is a standardized HRQoL scale with good psychometric characteristics (reliability, validity) as shown for other international versions. We suggest that the AMS scale could be also used in age groups under 40 years to measure and compare HRQoL in males. Since the application of the AMS in younger age was not investigated before, confirmation in future studies is needed.

Background The Aging Males' Symptoms (AMS) scale was originally developed as symptoms profile scale in Germany to evaluate health-related quality of life (HRQoL) [ 1 ]. It is a self-administered scale to assess symptoms of aging, to compare the severity of complaints, and to measure therapeutic interventions [ 1 - 3 ]. The AMS scale is an internationally used scale. Currently, translations into 17 languages are available following methodological recommendations for linguistic and cultural adaptation. These versions are available in a published form [ 2 , 4 , 5 ] and can be downloaded from the Internet . Norm values of the standardized AMS scores (total score and three domain scores) however were only published for Germany until now. In addition, little is published yet about the comparability of the scale across countries, except one recent paper that provided promising impressions, based however on small numbers in most of the countries [ 6 ]. It is important to analyze if the scale measures similarly in different countries or whether one could pool results of clinical studies across countries. The aim of this paper is to provide evidence that the French AMS scale measures similarly (similar reliability and validity) compared with other language versions. We also intend to show whether the application of AMS is really limited to aging males only or not. More generally, we would like to demonstrate that the AMS scale is a relevant, validated, sensitive instrument to measure HRQoL and change of symptoms in France. Methods The French data are based on representative national sample of French males aged 15 and more years: 963 males underwent a computer-assisted telephone interview that included the French AMS scale [ 7 ]. This approach is in full agreement with national ethical regulations and data privacy rules. AMS scores were available for 903 men. The sample was constructed using quotas method by stratification on sex, age, householder's profession, geographic region and population density. We compared the French data with existing data from other European AMS data built the comparator for this methods paper. Results of this database were published recently [ 6 ] as methodological paper of the AMS scale and kindly provided by L.A.J. Heinemann for looking at possible similarities or differences of the French AMS scale. Only community-based European data were used for this comparison: men aged 40 years and more. The bulk of the data came from Germany (96%), UK (2%), and only very small samples (n < 40) from Spain, Portugal, Italy, and Sweden [ 6 ]. These samples were combined to build the dataset called "Europe, others" in the following text and tables. The important difference with the French database is the absence of males under 40 years in the "Europe, others" data. Using these two databases, we were able to review the most fundamental psychometric characteristics of the French AMS, i.e., particularly reliability and some aspects of validity. It is the first quality requirement for a new scale – here the French AMS – to provide evidence that reliability or replicability is sufficiently good and not different from the same scale in other language regions. We used only the Cronbach's alpha measure. In contrast to systematic and random variation, reliability gives an estimate of method-related measurement error that should be low – what translates in a high consistency coefficient "alpha". A second basic requirement is validity: whereas reliability can be determined easily with a relatively simple indicator, the validity is almost always a continuous process (construct validation). As a first step, we will analyse the internal structure of the French AMS and analyze the total scale and sub-scales by means of factorial analysis. If the scale is similar to the scales used in other countries in Europe (particularly in Germany), the same domains should be identified and similar factor loadings should be found. The analyses were done with SPSS Windows 10.0 and STATA 6.0. Results and Discussion Reliability Table 1 shows the internal consistency measured with Cronbach's Alpha. The consistency coefficients "alpha" fell between 0.7 and 0.9 in France. The values are not materially different from the alpha values in the combined European sample, i.e. for the AMS total score as well the three subscales. This is indicative for a very acceptable consistency of the French AMS scale – and – compatible with Europe outside France. Table 1 Internal consistency coefficients (alpha) for the AMS scale in France compared with rest of Europe: total score and scores for the psychological, somatic, and sexual sub-scale. Mean and SD of the scale and sub-scales. Community samples. France (N= 903) Europe * (N = 5907) Mean (SD) alpha Mean (SD) alpha Total score 27.7 (9.3) 0.89 28.0 (9.4) 0.90 Psychological score 7.6 (2.9) 0.75 7.4 (3.1) 0.82 Somatic score 12.5 (4.7) 0.81 12.6 (4.7) 0.81 Sexual score 7.6 (3.1) 0.76 7.9 (3.4) 0.80 * Database is identical with the methodological review of international data published earlier [6] and kindly provided by L.A. J. Heinemann In addition, the very close mean values (SD) of the scale's total score and domain scores in the two comparison groups contribute to evidence that the scale does not work differently in France compared to the rest of Europe. Test-retest correlation coefficients (Pearson's correlation) in a very small sample in France were earlier published [ 6 ] and support the above conclusion: Cronbach's alpha for total scale and domains ranged from 0.7 to 0.9 (details in [ 6 ]). Validity The first step of validation is usually to multivariately demonstrate the internal structure ("dimensions") of a given scale through factor analysis. Table 2 shows the comparison of the factorial structure of the French AMS scale and "Europe, others". The structure of the French AMS is not materially different from "Europe, others", i.e. the same three domains were found in the French scale as were described initially for the German original scale. In addition, the proportion of explained variance by the AMS scale is also very similar: 52% and 56% in the French and European analysis (52% in the original German scale in 1999). The loadings of the 17 items on the 3 factors/domains of the French AMS are astonishingly similar with those from rest of "Europe, others". Table 2 Internal structure and domains of the AMS scale. Comparison of community samples of France and other countries of Europe [6] N FRANCE 903 EUROPE, other 5907 Item Nr. Psych Somat Sex Psych Somat Sex Burned out 13 0.3 0.6 0.7 Depressive, more 11 0.5 0.6 0.8 Irritability, increased 6 0.7 0.6 Anxious, more 8 0.6 0.7 Nervousness, more 7 0.7 0.6 Joint complaints, more 2 0.6 0.7 Sweating, increased 3 0.5 0.4 0.5 Sleep, need for more 5 0.7 0.6 Well-being, impaired 1 0.7 0.7 Sleep disturbances, more 4 0.6 0.6 Muscular weakness 10 0.6 0.6 Physical exhaustion 9 0.6 0.6 Sexual potency, impaired 15 0.8 0.9 Morning erections, less 16 0.8 0.9 Libido, disturbed 17 0.8 0.8 Passed peak 12 0.5 0.5 0.4 Decrease of beard growth 14 0.2 0.4 0.3 Factor loadings only depicted if 0.5 or more # in the sub-scales: psychological (psych), somatic (somat), and sexual sub-scale (sex). # Except if conflicting loadings /not in original domain (italic) Thus, the French scale does obviously not differ from other versions, and particular the German original, i.e. from this perspective. However, as reported in a previous publication [ 6 ], there are two items that are not particularly helpful in forming the sexual factor: The statement that the beard growth decreased and the feeling to have "passed the peak of life". The loading were (very) low, or showed up in two different domains. Thus, also the French AMS supports the conclusion of an earlier publication [ 6 ], " that item 12 ....14 could be eliminated if a new standardization is planned". This however seems not to be feasible in the near future. Three other items from the psychological domain (burned out, depressive, sweating) have low loadings on more than one domain (somatic domain) in the French AMS or even slightly higher loadings in the "wrong domain". Nevertheless, similarity with the original German AMS scale [ 6 ] and "Europe, others" is dominating some differences (including those mentioned above). At least, this seems not to undermine the quality of the French AMS scale. Moreover, it is not likely that these weaknesses influence the use of the scale in practice since individual items are not used – only average scores of the total scale or the three domains. Moreover, it is unlikely to have negative impact on multinational studies, because intra-individual comparisons over time (before/after treatment) are the main criterion that might not be affected very much. Sub-scores and total score correlations The relations among the sub-scales and the aggregate total scale are patterns that are important in the methodological assessment of a scale. In theory, the correlations between subscales (supposed to be independent) should be much lower than the correlations with the total score to which all sub-scales should significantly contribute – the domains are really independent from each other (as suggested by the factorial analysis with orthogonal factors). However, table 4 shows only a somewhat lower Pearson's correlation among sub-scales (r = 0.5–0.7) as compared with correlation of sub-scales with the total score (r = 0.8–0.9). Table 4 Domain score – total score correlations of the AMS scale. Comparison between French and other European community samples. DOMAINS Psychological score Somatic score Sexual score France (n = 903) Total score 0.8 0.9 0.8 Psychological score -- 0.7 0.5 Somatic score -- -- 0.6 Other Europe (n = 5907) Total score 0.8 0.9 0.8 Psychological score -- 0.7 0.5 Somatic score -- -- 0.5 This suggests that the sub-scales are not as independent from each other as one would expect them to be according to the applied model. However, this is again identical for the French AMS and the results obtained in "Europe, others". This however means, that the results of subscales should no be seen in isolation but in the context of other scales, or the result of the total score. French AMS and other scales A sufficient correlation with an internationally well-accepted QoL scale, like SF 12 [ 7 ], should be demonstrated also for the French AMS – to check the claim that the AMS scale is a health-related QoL scale. We applied both scales to all participants of the second half of the French survey (n = 461). High Pearson's correlation coefficients were observed between the total AMS score and the subscale PCS-12 (r = 0.48; p < 0.0001) as well as with MSC-12 or the SF-12 (r = 0.51; p < 0.0001). For comparison, other authors [ 3 ] reported as concurrent validity high correlations of the German AMS scale with the SF-36 for an older population sample (40–70 years): Similar to our results with SF-12, the AMS total score was statistically significant correlated with SF-36: r = 0.49 (p < 0.0001). Similarly, we observed a good Pearson's correlation with the HAD depression scale [ 8 ] and the AMS total score (r = 0.62; p < 0.0001). We conclude that the French AMS is as able to measure HRQoL as observed for the German version. In addition the AMS result can reflect the degree of depression as measured with the HAD scale. AMS applicable for all age groups The utility of the AMS scale for younger age groups is another question that was often raised but not answered yet due to lack of data. The representative French study included also younger age groups (from 15 years upward). Table 3 (see Additional file) shows the dimensions/domains of the French and the "Europe, others" results in two broad age groups (under/equal 50 vs. over 50 years). The factor loadings of the three domains differ not systematically between the two age groups. It should be kept in mind that the French sample included males from 15 years upward whereas the sample "Europe, others" started with 40 years. Table 3 Internal structure and domains of the AMS scale. Comparison of community samples of France and other countries of Europe [6] in 2 age groups. N France 903 Europe, other 5907 Age Under = 50 (n = 617)* Over 50 (n = 346) Under = 50 (n = 459)* Over 50 (5448) Item Nr. Psych Somat Sex Psych Somat Sex Psych Somat Sex Psych Somat Sex Burned out 13 0.2 0.6 0.4 0.6 0.7 0.7 Depressive, more 11 0.6 0.7 0.5 0.5 0.8 Irritability, increased 6 0.6 0.6 0.6 0.6 Anxious, more 8 0.6 0.7 0.7 0.7 Nervousness, more 7 0.7 0.7 0.7 0.7 Joint complaints, more 2 0.6 0.6 0.7 0.8 Sweating, increased 3 0.6 0.2 0.4 0.2 0.6 0.5 Sleep, need for more 5 0.7 0.7 0.6 0.6 Well-being, impaired 1 0.7 0.6 0.6 0.7 Sleep disturbances, more 4 0.6 0.6 0.6 0.5 Muscular weakness 10 0.6 0.7 0.5 0.5 0.6 Physical exhaustion 9 0.7 0.6 0.6 0.6 Sexual potency, impaired 15 0.7 0.8 0.9 0.9 Morning erections, less 16 0.7 0.8 0.8 0.9 Libido, disturbed 17 0.7 0.8 0.8 0.8 Passed peak 12 0.5 0.3 0.4 0.4 0.3 0.6 0.4 0.1 0.5 0.4 Decrease of beard growth 14 0.4 0.4 0.7 0.3 0.4 0.3 Factor loadings only depicted if 0.5 or more # in the sub-scales: psychological (psych), somatic (somat), and sexual sub-scale (sex). # Except if conflicting loadings /not in original domain (italic). *Age range different: France 15–50, Europe 40–50 To answer the question if the AMS measures different phenomena in groups under 40 than in "aging males" over 40, we further stratified the French sample and repeated the factor analysis for 10-year age groups (Table 5 [see Additional file]): There is clear evidence from this analysis that the AMS scale measures a similar phenomenon (HRQoL) in all age groups. Of course, the proportion of persons with impaired HRQoL increases with age, i.e. the mean total and domain scores of the AMS increases with 10-year age groups. The mean values of the AMS total score were in the French study: 24.6, 24.7, 27.3, 30.3, and 32.4 scoring points for the age groups under 30, 30–39, 40–49, 50–59, and 60+ years old, respectively. Similarly, the mean scoring points of the three domains increase with age – particularly so in the psychological and somatic domain (data not shown). Table 5 Internal structure and domains of the AMS scale in 10-year age groups. Comparison across age in the French community sample (n = 903). Total scale: % variance explained 49.6 46.2 52.5 56.7 52.2 Domains: % variance explained 13.5 20.2 15.9 17.6 18.3 10.3 12.2 23.1 17.2 10.3 28.9 17.4 17.7 20.3 14.1 Age group (years) Under 30 (n = 197) 30–39 (n = 203) 40–49 (n = 169) 50–59 (n = 154) 60+ (n = 180) Item Nr. Psych Somat Sex Psych Somat Sex Psych Somat Sex Psych Somat Sex Psych Somat Sex Burned out 13 0.2 0.5 0.1 0.6 0.3 0.5 0.2 0.7 0.5 0.5 Depressive, more 11 0.6 0.8 0.5 0.5 0.2 0.8 0.6 Irritability, increased 6 0.5 0.6 0.7 0.8 0.5 Anxious, more 8 0.6 0.7 0.7 0.3 0.6 0.8 Nervousness, more 7 0.4 0.6 0.3 0.5 0.3 0.7 0.6 0.7 Joint complaints, more 2 0.7 0.6 0.5 0.5 0.6 Sweating, increased 3 0.5 0.5 0.1 0.6 0.2 0.5 0.6 0.2 Sleep, need for more 5 0.6 0.6 0.6 0.8 0.6 Well-being, impaired 1 0.6 0.5 0.6 0.8 0.8 Sleep disturbances, more 4 0.6 0.4 0.4 0.6 0.6 0.5 Muscular weakness 10 0.6 0.5 0.5 0.6 0.7 Physical exhaustion 9 0.6 0.6 0.6 0.6 0.6 Sexual potency, impaired 15 0.8 0.6 0.2 0.8 0.7 0.8 Morning erections, less 16 0.7 0.6 0.3 0.7 0.8 0.8 Libido, disturbed 17 0.7 0.5 0.7 0.8 0.8 Passed peak 12 0.7 0.2 0.5 0.4 0.5 0.4 0.3 0.3 0.4 0.4 Decrease of beard growth 14 0.7 0.8 0.7 0.7 0.2 0.5 0.4 Factor loadings only depicted if 0.5 or more # in the sub-scales: psychological (psych), somatic (somat), and sexual sub-scale (sex). # Except if conflicting loadings /not loading in the original domain (italic ) Table 5 (see Additional file) shows a tendency that the scale explains an increasing percentage of the total variance with increasing age, although the numbers are in the same ballpark (ranging from 46% to 56%). The factor loading in the somatic and sexual domain seems to be quite stable across age groups. The psychological domain seems to vary more but not clearly associated with increasing age. As discussed above, some items of the scale are more unstable in their association to only one factor and have low loadings in the French study: item 12 and 14 of the sexual domain ("passed peak", beard growth"), item 11 and 13 of the psychological domain ("depressive, "burned out"), and item 3 of somatic domain ("sweating"). In general, the internal structure of the scale is remarkably similar across age groups. From this result we suggest that the name of the Aging Males' Symptoms scale might be somewhat misleading: The AMS could be applicable in all age groups to measure HRQoL and not only in "aging males", if the results of the French study can be confirmed in other studies. Thus, we could also calculate reference values across age with the French survey. This will be published in a joint review paper together with reference values from other countries than Germany that became available in recent time. This paper is under preparation. Conclusion The French AMS scale is a standardized HRQoL scale with good psychometric characteristics (reliability, validity) as shown for other international versions. We suggest that the AMS scale should be also used in age groups under 40 to measure and compare HRQoL in males. Since the application of the AMS in younger age was not investigated before, confirmation in future studies is needed. Competing interests We like to declare that the authors EM, NM, CT are working in a pharmaceutical company. But we don't see a conflict of interest in this methodological study. Authors' contributions EM: involved in the co-ordination of the project, contributed to writing the paper; NM: involved in the data analysis and contributed to writing the paper, CT: responsible for the project, contributed to writing the paper;

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1079917

The measurement of response shift in patients with advanced prostate cancer and their partners

Background There is increasing evidence to support the phenomenon of response shift (RS) in quality of life (QoL) studies, with many current QoL measures failing to allow for this. If significant response shift occurs amongst prostate cancer patients, it will be necessary to allow for this in the design of future clinical research and to reassess the conclusions of previous studies that have not allowed for this source of bias. This study therefore aimed to assess the presence of RS and psychosocial morbidity in patients with advanced prostate cancer and their partners. Methods 55 consecutive advanced prostate cancer patients and their partners completed the Prostate Cancer Patient & Partner questionnaire (PPP), shortly after diagnosis and again at 3 months and 6 months. At the follow-up visits, both patients and partners also completed a then-test in order to assess RS. Results Partners consistently showed greater psychological morbidity than patients in relation to the prostate cancer. This was most marked on the General Cancer Distress (GCD) subscale (p < 0.001, paired t-test), and regarding worries about treatment (p = 0.01). Significant RS was identified in partners and patients by the use of the then-test technique, particularly on the GCD subscale, the concerns about treatment and the concerns about urinary symptoms items. Conclusion These results suggest the presence of RS in patients with advanced prostate cancer and their partners, with higher levels of psychosocial morbidity noted amongst partners. This is the first study to identify RS in partners and calls into question the interpretation of all studies assessing changes in QoL that fail to allow for this phenomenon.

Background Quality of life (QoL) assessment is increasingly used as a major outcome parameter in health care, both for clinical decisions and policy making. It is vital therefore, that the measurement of QoL accurately reflects differences between patient groups, and changes in QoL over time. A defining characteristic of human beings, however, is the way in which we adapt to changing circumstances and one of the implications of this is the phenomenon of 'response shift' (RS) in self-ratings. The concept of RS originated in the 1970's in the field of educational measurement [ 1 , 2 ]. GS Howard and colleagues argued that, in using self-report instruments, researchers assume that individuals evaluating themselves have an internalised standard for judging their level of functioning with regard to a given dimension, and that this internalised standard will not differ between experimental and control groups or change from one testing to the next ( pre-test to post-test ). However, if the individual's standard of measurement should change between the pre-test and post-test (due to some form of intervention or change), the two ratings would reflect this difference in addition to any actual change taking place. Consequently, comparison of the two ratings will be invalid. Since longitudinal QoL measurement traditionally relies on the comparison of pre- and post-test questionnaires, usually in patients adapting to some form of change, this problem calls into question the interpretation of all data acquired in this manner [ 3 ]. Response shift, when applied to the area of QoL, is defined as a change in the meaning of one's self-evaluation of QoL as a result of: (a) change in the respondent's internal standards of measurement ( recalibration ); (b) change in the respondent's values ; or (c) redefinition of life quality ( reconceptualization )[ 4 ]. There are many techniques suggested for evaluating RS [ 5 , 6 ] the most established of these being the retrospective pre-test design proposed by G. S. Howard [ 1 , 2 ]. The design consists of: Pre-test Assessment at beginning of study, i.e. before intervention / change Post-test Subsequent assessment, i.e. after intervention / change Then-test Retrospective assessment of first evaluation This retrospective re-evaluation assumes that the subject uses the same criteria for the conventional post-test and the then-rating, and thus allows comparison between the 2 assessments: (see Figure 1 ) Figure 1 The then-test approach to measuring response shift (from Sprangers et al 1999) [27]. Traditional Change Difference between pre-test and post-test scores, as would be measured by standard QoL assessment techniques, i.e. reported treatment effect. Response Shift Difference between pre-test and then-test scores. Actual Change Difference between then-test and post-test score, reflecting the change in QoL having allowed for response shift. The concept of RS is still in its early stages and recent research has also highlighted a need to focus on a subject's 'appraisal' of QoL, which may be influenced by personality, culture or situation. Different appraisal parameters can be measured in relation to the types of RS outlined above: 1) Change in frame of reference (relate to reconceptualization ), 2) Change in recall or sampling of experiences (relate to reprioritization ) and 3) Change in standards of comparison to appraise experience (relate to recalibration ). Indeed a QoL Appraisal Profile has been proposed for potential use alongside other QoL measures [ 7 ]. Whilst differences in appraisal will undoubtedly exist if formally measured, their interpretation within the context of modern psychometric models means that these need not be viewed as sources of error, but instead as processes integral to the assessment of QoL [ 8 ]. Defining the impact of chronic illness on the partners of patients is a relatively recent endeavour [ 9 ]. The ageing population, changes in medical practice leading to shorter inpatient hospital stay and prolonged survival, have resulted in a significantly increased burden being placed on carers, the majority of whom are partners [ 10 ]. The morbidity of partners of those with chronic illness has been well described, particularly through qualitative research [ 11 , 12 ] and has consistently found that the QoL of the partner is often worse than that of the patient [ 13 - 16 ]. The first published work on the QoL of spouses of prostate cancer patients, however, was by Kornblith et al in 1994 [ 17 ]. Problems reported by patients and partners were of a similar nature, but spouses reported significantly greater psychological distress than patients, in keeping with the studies mentioned previously. As a result of these findings, the Prostate Cancer Patient and Partner Questionnaire (PPP) was developed to help to identify psychosocial morbidity within both patients and partners [ 18 ]. As part of the validation process, the PPP was administered to a sequential sample of 135 couples with any stage of prostate cancer, attending outpatient clinics[ 19 ]. The questionnaire is unique in that it was developed and validated simultaneously in patients and partners, i.e. the partner component is not an adapted patient questionnaire. The aim of this study was to determine whether incorporating a then-test into the PPP would detect a significant RS in patients with advanced prostate cancer and/or their partners. Whilst RS had previously been identified by Rees et al. in the assessment of lower urinary tract symptoms amongst a cohort of patients with advanced prostate cancer [ 20 ], it was not known how such patients would assess other factors relating to their diagnosis, or how their partners would rate QoL. Two hypotheses were therefore proposed. First, that patients would initially under-report the impact of both their urinary symptoms (where present) and diagnosis of prostate cancer on their QoL, then with adaptation and symptomatic improvement, retrospectively lower their initial QoL scores. Second, that there would not be a significant RS in partners, predicting that the higher levels of psychosocial morbidity in previous partner studies may relate to a lack of adaptation, with the disease process itself perhaps acting as a catalyst for response shift in patients. However, with no previous work on RS in partners, these hypotheses were purely speculative. Methods For the purpose of the study, patients were classified as having locally advanced disease if they had 2 out of 3 from the following: (a) PSA > 10 ng/ml, (b) Gleason Grade > 7, (c) Clinical Stage ≥ T3 [ 21 ]. Patients were excluded from the study if unable to complete the questionnaires, either through lack of understanding, poor literacy or limited life expectancy (<6 months). It was also decided that concurrent illness (e.g. another malignancy or severe cardio-respiratory disease) or concurrent social or emotional problems would also affect the results of the study, and patients with these problems should not be included. Patients were recruited from outpatient clinics in 3 hospitals in the South-West of England (Taunton and Somerset Hospital, Southmead Hospital and Bristol Royal Infirmary). Patients and partners were seen in their own homes within a week of the patient's initial diagnosis, and at the first visit completed the PPP (Appendix [see additional file 1 ]). Subsequent visits took place 3 months and 6 months later, and following the post-tests at these visits a then-test was incorporated, with both groups asked to re-evaluate their QoL at the time of the previous assessment using the PPP. It was emphasised that the then-test was not asking patients / partners to recall their previous answers but instead to provide a renewed judgment. Patients and partners filled in the questionnaires simultaneously, in the presence of the interviewer, and were asked not to compare their answers in order to remove any bias that this might cause. Results Between 20 th July 2000 and 19 th July 2001, 55 patients and 43 partners gave informed consent to take part in the study. Despite excluding patients from the study with a predicted life expectancy less than 6 months, 2 patients died shortly after their diagnosis, and thus 53 patients and 41 partners completed all 3 assessments. The mean age of the patients was 72.9 years (SD 8.5, Range 54 – 92 years). Prostate specific antigen (PSA) levels at presentation ranged from 4.4 to 5050 ng/ml, with a median of 57.2. Histological diagnosis was made in 46 patients, with 1 well differentiated tumour, 27 moderately differentiated and 18 poorly differentiated, according to the Gleason grading system. Nine patients were treated as a result of a clinical diagnosis alone. Distant metastases were identified in 13 patients and the remaining 42 patients were therefore considered to have locally advanced disease. 44 patients received treatment with hormonal therapy alone (LHRH analogue), whilst 8 patients received radical radiotherapy in addition to their neo-adjuvant hormonal therapy. Three patients did not receive treatment during the study, but were placed on an active surveillance or 'watchful waiting' regime. General Cancer Distress (GCD) Subscale Partners showed significantly higher cancer-related distress (p < 0.001) than patients at all 3 visits (Table 1 ). Table 2 examines the use of the then-test, and the impact of this on what has been termed the 'traditional change' and 'actual change'. Using a paired t-test, a statistically significant difference between the pre-test and then-test scores was identified at both visits 2 and 3. A significant change was identified in both patients and partners between visits 1 and 2 using conventional pre and post-testing (traditional change), but this change was increased by the incorporation of the then-test methodology, to a p value <0.001 for both groups. The traditional change between visits 2 and 3 was of borderline significance in both groups, but the addition of the then-test again led to highly significant actual change (p < 0.001 in both groups). These changes are depicted graphically in Figure 2 . Table 1 Mean scores on GCD Subscale for patients and partners Visit 1 Visit 2 Visit 2 – Then-test Visit 3 Visit 3 – Then-test Patients Mean (n) 4.4 (55) 3.2 (53) 5.4 (53) 2.6 (53) 4.8 (53) S.D. 2.7 2.5 2.7 2.0 2.5 Partners Mean (n) 7.8 (43) 6.5 (41) 9.2 (41) 7.2 (38) 9.0 (38) S.D. 2.4 2.3 2.6 2.3 2.4 Difference* 3.2 3.1 3.7 4.5 4.1 95% C.I. 2.3 – 4.0 2.3 – 4.0 2.6 – 4.7 3.5 – 5.5 3.1 – 5.1 P value ** <0.001 <0.001 <0.001 <0.001 <0.001 *Difference in mean scores for the pairs at each visit, i.e. 43 at visit 1 ** Paired t-test Table 2 Changes in scores on the GCD Subscale for patients and partners Months 0 – 3 Months 3 – 6 Pre-test – Then-test ('Response Shift') (p*) Patients (n = 53) 1.0 (0.004) Pre-test – Then-test ('Response Shift') (p*) Patients (n = 53) 1.6 (<0.001) 95% C.I. 0.4 – 1.7 95% C.I. 1.0 – 2.2 Partners (n = 41) 1.4 (0.001) Partners (n = 38) 2.6 (<0.001) 95% C.I. 0.6 – 2.3 95% C.I. 1.8 – 3.3 Pre-test – Post-test ('Traditional Change') (p*) Patients (n = 53) 1.2 (<0.001) Pre-test – Post-test ('Traditional Change') (p*) Patients (n = 53) 0.6 (0.05) 95% C.I. 0.6 – 1.7 95% C.I. 0.0 – 1.1 Partners (n = 41) 1.3 (0.002) Partners (n = 38) -0.7 (0.06) 95% C.I. 0.5 – 2.1 95% C.I. -1.5 – 0.0 Then-test – Post-test ('Actual Change') (p*) Patients (n = 53) 2.2 (<0.001) Then-test – Post-test ('Actual Change') (p*) Patients (n = 53) 2.2 (<0.001) 95% C.I. 1.6 – 2.8 95% C.I. 1.6 – 2.8 Partners (n = 41) 2.7 (<0.001) Partners (n = 38) 1.8 (<0.001) 95% C.I. 2.0 – 3.4 95% C.I. 1.0 – 2.6 *Paired t-test Figure 2 Changes in mean scores on General Cancer Distress Subscale in patients and partners. The results showed improvement in 'general cancer distress' as identified by this questionnaire over the first 6 months following diagnosis (Table 1 ). In addition to showing greater degree of worry/concern at all assessments, partners were also seen to undergo a larger RS than patients. Using a paired t-test this difference between patients and partners in magnitude of 'response shift' did not reach statistical significance during the first 3 months (p = 0.3); however, the difference was statistically significant between the second and third assessments (p = 0.02). Social subscale Scores on the social subscale were relatively low overall and no significant difference in mean scores was seen between patients and partners at visit 1, 2 or the visit 2 then-test. A statistically significant difference was seen, however, at visit 3, and the then-test at this time also showed a highly significant difference between patients and partners (mean score 0.8 versus 2.3 respectively, p < 0.001). This subscale showed no statistically significant change with the use of the then-test, except for partners between visit 2 and 3 (Table 3 ). However, the additive effect of the then-test made the actual change on this subscale highly statistically significant for partners, both between visits 1 and 2, and visits 2 and 3. No significant changes were seen in patients using this subscale. The greater RS in partners than patients between visits 2 and 3 was highly statistically significant (p < 0.001, paired t-test). Table 3 Changes in scores on the Social Subscale for patients and partners Months 0 – 3 Months 3 – 6 Pre-test – Then-test ('Response Shift') (p*) Patients (n = 53) 0.4 (0.1) Pre-test – Then-test ('Response Shift') (p*) Patients (n = 53) 0.2 (0.1) 95% C.I. -0.1 – 0.8 95% C.I. -0.5 – 0.6 Partners (n = 41) 0.3 (0.3) Partners (n = 38) 1.6 (<0.001) 95% C.I. -0.3 – 0.8 95% C.I. 1.0 – 2.2 Pre-test – Post-test ('Traditional Change') (p*) Patients (n = 53) 0.04 (0.8) Pre-test – Post-test ('Traditional Change') (p*) Patients (n = 53) -0.1 (0.6) 95% C.I. -0.3 – 0.3 95% C.I. -0.4 – 0.2 Partners (n = 41) 0.5 (0.02) Partners (n = 38) -0.7 (0.004) 95% C.I. 0.06 – 0.9 95% C.I. -1.2 – 0.2 Then-test – Post-test ('Actual Change') (p*) Patients (n = 53) 0.4 (0.1) Then-test – Post-test ('Actual Change') (p*) Patients (n = 53) 0.1 (0.3) 95% C.I. -0.04 – 0.8 95% C.I. -0.3 – 0.1 Partners (n = 41) 0.8 (0.001) Partners (n = 38) 0.9 (0.001) 95% C.I. 0.3 – 1.2 95% C.I. 0.4 – 1.4 *Paired t-test Worries about treatment Partners were statistically significantly more worried than patients for those receiving treatment, except at the visit 2 then-test (which was based on a very small sample since few patients had commenced treatment by visit 1). Concerns about treatment would appear not to decline with traditional pre/post-testing, but the results at the visit 3 then-test were significantly different from the pre-test at visit 2 in both patients and partners, suggesting a significant improvement in treatment related concerns. This difference was greater in the partners than in the patients (p = 0.01, paired t-test). The patients (and their partners) receiving no treatment at the time of the initial visit showed higher degrees of worry/concern than those already established on treatment (0.8 versus 0.6 for patients, 2.1 versus 1.6 for partners). Otherwise, the results for this question are difficult to interpret, due to small numbers at subsequent visits. Worries about pain The low number of patients with significant pain limits the interpretation of these results. Overall, partners appeared to be more concerned about pain than their husbands, but these differences only reach statistical significance with the then-tests. Traditional pre/post-testing revealed no significant change in either group, but the incorporation of the then-test resulted in a significant 'actual change' for both patients and partners between visits 1 and 2 (p = 0.03 for patients, p = 0.02 for partners, paired t-test). The difference in then/pre- test scores ('response shift') between visits 2 and 3 also reaches statistical significance (p = 0.05 patients, p = 0.04 partners), but otherwise no statistically significant results were found. Worries about urinary symptoms Partners showed higher levels of concern about urinary symptoms than the patients. These differences reached statistical significance at visits 2 and 3, and the visit 2 then-test. The traditional change in worry/concern, as measured by the pre/post-test design, was non-significant over both the first and second three-month periods. However, incorporating the then-test, the actual change in the first 3 months was significant in both patients and partners. Patients had a greater actual decrease in concerns than their partners. In the second 3-month period, traditional measurement would suggest that both patients and partners became more concerned about urinary symptoms. However, using the then-test methodology, the actual change in concern was a decrease in both groups. This decrease did not, however, reach statistical significance. Worries about physical limitation The low proportion of patients with physical limitation due to their prostate cancer means that although partners were consistently more concerned, according to their responses to this question, this did not reach statistical significance. No significant differences were identified on analysis of then-test results. Worries about sexual function The results for this question show that only 33% of patients were sexually active at the beginning of this study. No significant differences were seen between patients and partners at each assessment, although patients tended to have higher scores on this question. The only significant change using the then-test occurs in the patient group, between visits 2 and 3 (p = 0.01). Discussion This study revealed that patients with advanced prostate cancer retrospectively lowered their QoL scores at both the 3 and 6-month visits. Whilst this was consistent with our initial hypothesis, it was also noted that partners in some cases demonstrated significantly worse QoL scores than the patients (particularly on the General Cancer Distress scale). In addition, tentative to our second hypothesis, we observed evidence of RS in the partners of the patients. Although this was no less than in the patients, partners in some cases (e.g. Social subscale between visits 2 & 3) showed a significant RS where patients did not. These findings confirm the previous evidence in prostate cancer of higher psychological morbidity amongst partners compared to the patients. The consistency of this finding in prostate cancer studies and those looking at other conditions suggests that this is a genuine phenomenon. Although this could merely represent a difference in the way QoL questions are answered by men and women, the literature suggests that male partners are just as likely to have high levels of psychological morbidity as female partners. Indeed one study of couples with colon cancer found that the adjustment of husbands was far worse than that of wives of cancer patients [ 22 ]. The then-test results were statistically significantly different from pre-test results on several scales, particularly the GCD subscale, concerns about treatment and concerns about urinary symptoms items. Whilst this may provide an estimate of the magnitude and direction of a RS effect, the fundamental question is whether these differences really represent RS or some other confounding factor(s). Social desirability may play a part in the retrospective worsening of QoL scores, in which the subject feels their QoL should have improved with treatment and therefore they will place lower scores on then-test evaluation. It is also possible that the patients and partners may feel an implicit pressure to 'please the doctor', again leading them to lower their retrospective QoL scores. Although the theory of RS was not explained to the subjects, it is likely that many will have understood the purpose of then-testing. In addition, the issue of recall bias and memory must also be considered in relation to the then-test. Differences in prospective versus retrospective health assessments may be due to recall bias [ 5 ]. In a study by Ahmed et al assessing patients' QoL following stroke, a memory checklist was incorporated to provide an objective measure of their functional ability and so determine the effects of recall bias on the final results [ 23 ]. Whilst patients with 'good' memory demonstrated large response shifts and those with 'poor' memory greater variability, a significantly lower then-test rating was noted in those with stroke, with no such effect amongst the control group. This implies that the changes could not be wholly attributed to recall bias and previous research would support this [ 24 ]. Furthermore, in this study an attempt was made to minimise the effects of recall bias and memory by using relatively small time differences between assessments (3 months). If the then-test is performed too close to the pre-test, it is possible that subjects will remember their previous answers and score the questionnaire accordingly. Conversely, if the then-test is performed at a later time-interval, it is possible that memory effects may have an increasing role. The effect of changes in timing of the then-test is an interesting area for future research. It would therefore appear safe to conclude that a substantial part of the difference seen between pre and then-test scores can be explained by RS. As mentioned above, the GCD subscale, concerns about treatment and urinary symptoms items showed statistically significant change, for both patients and partners, suggesting that a RS was occurring in these areas in both groups. Incorporating the then-test tended to increase the magnitude of the changes measured using the pre / post-test method, and in many cases led these changes to become statistically significant. No formal adjustment was made for multiple testing and it is therefore possible that some results were artefactual. However, most of the results reported as significant involved low probability values suggesting that chance was an unlikely explanation. As highlighted earlier, in some cases partners demonstrated a significant RS, where patients did not. However, this could represent a phenomenon unique to the PPP questionnaire, and it would be interesting to study this effect using other validated QoL questionnaires. It is possible that the wording of the PPP, concentrating on worries and concerns, is actually looking at anxiety levels in both patients and partners, an organic condition in itself, thus giving both groups a catalyst for a RS as measured by the questionnaire. Previous studies have shown that self-reported QoL of seriously ill patients is higher (i.e. better) than the patients' proxies (e.g. their partners) believe them to be [ 25 ]. This finding has been explained with reference to RS. The findings of this study raise the question as to whether partners in this study, whilst undergoing a response shift in terms of their own levels of psychological morbidity, would also recognise a RS on assessing their partners QoL? The identification of significant RS in both groups taking part in this study calls into question the interpretation of studies that do not allow for its presence. Particularly in clinical trials, the measurement of differences in QoL across treatment arms may be jeopardised when RS affects the treatment groups differently [ 26 ]. Since this is a relatively new concept from a methodological viewpoint, it remains unclear to what extent response shift occurs in different settings, but research in the area is emerging [ 27 - 29 ]. Conclusion This study highlights the presence of RS in both patients with advanced prostate cancer and their partners, with higher levels of psychosocial morbidity seen amongst the partners. This is the first study to identify RS in partners and not only calls into question the interpretation of studies assessing changes in QoL, but also raises the question as to whether RS of this type could actually be identified as a therapeutic goal, both in chronically ill patients and their partners. Authors' Contributions JR was involved in the design of the study, acquisition of data and subsequent analysis and interpretation. MC participated in the study coordination and drafting the manuscript. DW participated in study design and data interpretation. CO'B helped conceive & design study in addition to data interpretation. PE performed statistical analysis. RM helped conceive, design and coordinate study. All authors approved the final manuscript. Supplementary Material Additional File 1 The Prostate Cancer Patient and Partner Questionnaire (PPP) Click here for file

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1079918

Measurement properties of the UK-English version of the Pediatric Quality of Life Inventory™ 4.0 (PedsQL™) generic core scales

Background Health related quality of life (HRQL) has been recognised as an important paediatric outcome measurement. One of the more promising measures to emerge in recent years is the Pediatric Quality Of Life Inventory (PedsQL™), developed in the US. Advantages of the PedsQL™ include brevity, availability of age appropriate versions and parallel forms for child and parent. This study developed a UK-English version of PedsQL™ generic module and assessed its performance in a group of UK children and their parents. Methods PedsQL™ was translated to UK-English. The psychometric properties of the UK version were then tested following administration to 1399 children and 970 of their parents. The sample included healthy children, children diagnosed with asthma, diabetes or inflammatory bowel disease and children in remission from cancer. Results Psychometric properties were similar to those reported for the original PedsQL™. Internal reliability exceeded 0.70 for all proxy and self-report sub-scales. Discriminant validity was established for proxy and self-report with higher HRQL being reported for healthy children than those with health problems. Sex differences were noted on the emotional functioning subscale, with females reporting lower HRQL than males. Proxy and self-report correlation was higher for children with health problems than for healthy children. Conclusion The UK-English version of PedsQL™ performed as well as the original PedsQL™ and is recommended for assessment of paediatric HRQL in the UK.

Background Advances in medical research have changed the emphasis in healthcare from diagnosis and management of infectious disease to prevention and control of chronic conditions. While there have been major advances in treatment of previously life threatening conditions (e.g. cancer, cystic fibrosis), treatments can be aggressive and associated with both acute, and long-term morbidity. Recognition of this has led to a shift from measuring efficacy of treatment purely in terms of survival, to one that also takes into account the quality of the resulting life. As a consequence, a number of measures of health related quality of life (HRQL) have been published. Many are based on the definition of HRQL described by the World Health Organisation (WHO), and include separate measurement of physical, emotional and social functioning. A recent systematic review [ 1 ] concluded that one of the more promising measures for children was the PedsQL™ [ 2 ]. Developed in the US, the advantages of PedsQL™ include brevity, availability of age appropriate versions and parallel forms for child and parent. PedsQL™ integrates generic core and disease specific modules into one measurement system. PedsQL™ 1.0 [ 2 ] was described as a generic instrument. This was developed from work with children with cancer but designed for use as a non-categorical instrument. Subsequent publications have reported several refinements to the generic measure. PedsQL™ 2.0 and 3.0 included additional constructs and items, a more sensitive rating scale and a broader age range. PedsQL™ 4.0 included further core dimensions to match those described by WHO. Recent reports confirm the reliability and validity of this generic measure [ 3 , 4 ]. The success of PedsQL™ can be seen in its wide use in research and translation into many European and other international languages. In this paper we report the performance of the UK-English version of PedsQL™ 4.0 generic core module in a sample of healthy children and children with chronic health conditions. Methods Measures PedsQL™ includes parallel child self-reports (age range 5–18 years) and parent/carer proxy-reports (age range 2–18 years). Items on self and proxy-report are virtually identical, differing only in developmentally appropriate language and first or third person tense. Instructions ask how much of a problem each item has been during the past month and responses are made on a five-point scale ranging from 0 (never a problem) to 4 (almost always a problem). The generic module comprises twenty-three items that contribute to four subscales: Physical Functioning, Emotional Functioning, Social Functioning and School Functioning. It has also been shown that Physical Functioning can be viewed as a distinctive scale, while the remaining subscales can be more parsimoniously viewed as a single Psychosocial Health Summary Scale [ 3 ]. A Total Scale score can also be calculated. We employed self-report forms for ages 8–18 years and parallel proxy forms. Translation followed recommended guidelines [ 5 , 6 ]. Preliminary changes to the original questionnaires were made by three experienced psychologists and reviewed by Dr Varni, who recommended further modifications. The revised questionnaires were administered to 13 parents and 22 children and cognitive interview techniques [ 6 ] were used to obtain feedback about the interpretation and understanding of items and response ratings. Further changes were made to the questionnaires in response to feedback from parents and children. Dr Varni reviewed the revised measure and authorised all changes. Procedure Healthy children and their parents were recruited through 23 schools in South Wales. Written information was sent to parents who completed questionnaires at home, returning them to school by a specified date, along with signed consent for their child's participation. Children were given verbal and written information before completing questionnaires in class, under the supervision of a researcher. Children with either asthma, diabetes, inflammatory bowel disease (IBD) or in long-term remission from cancer were identified through patient information systems. Families were informed about the study by post and arrangements made for those who consented, to complete questionnaires either in clinic or at home under the supervision of a researcher. In addition to PedsQL™ all families completed a brief questionnaire concerning demographic information and child health. Based on this, children with any chronic health problems were excluded from the schools sample, ensuring this group was healthy. Analysis Items on PedsQL™ were reverse scored and linearly transformed to a 0–100 scale, with higher scores indicating better HRQL.[ 3 ] Scale scores were created by dividing the sum of responses by the number of items answered (to account for missing data). Internal reliability was assessed using Cronbach's Alpha [ 7 ] and range of measurement was determined based on the percentage of scores at extremes of the scaling range [ 8 ]. Discriminant validity was evaluated through a comparison of healthy children and those with chronic health conditions. A multivariate analysis of variance (MANOVA) was undertaken in order to determine differences in sub-scale ratings depending on child age, sex or health status. Pillai's Trace was calculated as this is robust to departures from normality. The source of significant variance was then located by Analysis of Variance (ANOVA). Finally the relationship between self and proxy-report was assessed by correlation. Ethics The Welsh Multi-centre Research Ethics Committee gave ethical approval to this work. Results Sample From 2002 families approached, a total of 1399 were recruited to the study (response rate = 69.88%), with 1034 families being recruited from schools and 365 from clinics. The remaining 603 families either failed to return questionnaires to schools by the cut-off date (N = 349) or did not reply to letters from clinics inviting participation in the study (N = 254). The sample was homogenous in ethnic background with 90% having been born in the UK and describing themselves as British. All participants either had English as their first language (96%), or were bilingual in English and Welsh (4%). 34% of mothers had left school at 16 (15% with no formal qualifications, 19% with some GCSEs) 39% had completed further education and 22% had qualifications from higher education. Only 5% of the sample did not provide this information. All children were aged from 8–18 years (mean age for self-report = 12.58, sd = 2.6; mean age for proxy-report = 11.86, sd = 2.3). Self-report forms were completed by 684 males and 715 females and proxy-reports were completed by 459 parents of males and 504 parents of females. A complete breakdown of the sample is given in table 1 . Table 1 Summary of recruitment and questionnaire completion by child health Healthy Diabetes Asthma IBD Cancer survivor Total Number of self- reports completed 1034 124 99 76 66 1399 Number of proxy-reports completed 665 103 74 67 61 970 The difference in self and proxy-report completion shown in table 1 , resulted from parents (N = 429) who gave consent for their child to complete PedsQL™, but did not complete a questionnaire themselves. The majority of proxy-reports were completed by mothers (84%), the remaining forms being completed by fathers (14%) or other carers such as stepparents and grandparents (2%). Internal reliability All self and proxy-report sub-scales exceeded the minimum standard of 0.70, whilst the total score exceeded 0.90. Range of measurement The full range of 0–100 was used for all four proxy-report subscales and the majority (3/4) of self-report subscales. A range of 10–100 was used for Emotional Functioning on the self-report, with nobody scoring at the lowest end of this sub-scale. Table 2 presents scale means and percentage of scores at the floor and ceiling for the original PedsQL™ [ 3 ] and the UK-English version. No floor effects were seen on the UK-English self or proxy-report for healthy children or those with known health conditions as no scale had more than 0.3% scoring at the minimum. However, ceiling effects existed for the healthy sample and ranged from minimal (e.g. 3.2% and 3.6% for self and proxy-report, respectively for Total Score) to moderate (e.g. 41.4% and 37.4% for self and proxy-report, respectively for Social Functioning). Ceiling effects also existed for those with known health conditions; as for the healthy sample the largest effect was for Social Functioning (35.3% and 26.3% for self and proxy-report). Healthy children and their parents reported more ceiling effects than those with health problems. As table 2 demonstrates, patterns of ceiling and floor effects are similar, to those reported for the original PedsQL™ [ 3 ], although ceiling effects are smaller in the UK population. Scale means are also similar on both versions of the measure. Table 2 Comparison of scale statistics for UK-English and original PedsQL™ [3] 4.0 self and proxy-report Scale Scale statistics Mean (SD) Total UK Sample Mean (SD) Total US Sample Percentage floor chronic health condition/healthy (UK Sample) Percentage floor chronic health condition/healthy (US Sample) Percentage ceiling chronic health condition/healthy (UK Sample) Percentage ceiling chronic health condition/healthy (US Sample) Self-report Total score 82.25 (13.09) 79.62 (15.26) 0.0/0.0 0.0/0.0 1.4/3.2 1.9/7.2 Physical health 86.08 (14.06) 80.19 (19.30) 0.3/0.0 0.0/0.0 12.1/20.5 13.1/25.8 Psychosocial health 80.50 (14.06) 79.37 (15.70) 0.0/0.0 0.0/0.0 2.5/4.1 5.2/12.0 Emotional functioning 76.99 (18.43) 78.10 (20.66) 0.0/0.0 0.3/0.8 4.9/15.6 22.4/29.8 Social functioning 86.85 (16.86) 84.09 (18.50) 0.3/0.2 0.0/0.0 35.3/41.4 33.2/47.1 School functioning 77.29 (16.92) 75.87 (19.71) 0.0/0.1 0.3/0.5 8.2/11.1 13.0/23.1 Proxy-report Total score 81.12 (13.85) 80.87 (16.73) 0.0/0.0 0.2/0.0 0.7/3.6 4.1/10.3 Physical health 84.99 (16.08) 81.38 (23.18) 0.0/0.1 2.3/0.0 7.4/26.7 18.5/39.6 Psychosocial health 79.00 (14.70) 80.53 (16.52) 0.0/0.0 0.2/0.0 1.7/4.6 5.6/13.8 Emotional functioning 74.67 (17.67) 77.95 (20.67) 0.0/0.1 1.4/0.1 6.1/12.1 19.5/29.5 Social functioning 84.62 (17.24) 85.38 (19.17) 0.3/0.0 0.5/0.0 26.3/37.6 34.4/58.1 School functioning 77.72 (18.50) 77.80 (22.00) 0.3/0.0 1.7/0.3 8.5/17.9 15.5/34.5 Discriminant validity There were significant differences in reported HRQL between males and females (Pillai's trace = 0.012, p = 0.003) and across the chronic health conditions (Pillai's trace = 0.107, p = 0.000) for self-report. Age group was not significant (Pillai's trace = 0.006, p = 0.082). For proxy-report, no difference in reporting was detected between parents of males and females (Pillai's trace = 0.007, p = 0.188) or of different ages (Pillai's trace = 0.008, p = 0.094). Significant differences in reporting were confirmed across chronic health conditions (Pillai's trace = 0.219, p = 0.000). No interactions were found between any combination of these three factors for either self or proxy-report. Thus one-way ANOVAs were undertaken comparing the four chronic health conditions and healthy children for self and proxy-report (see table 3 ) and comparing males and females for self-report only. Table 3 One-way ANOVA comparing chronically ill and healthy children: self and proxy-report Scale Self-report Proxy-report N Mean (sd) df F N Mean (sd) df F Total Score 4,1393 23.84 4,965 41.07 Asthma 99 75.31(16.90) *** 74 71.79(17.53)*** Diabetes 124 82.46(12.76) 103 77.54(12.21) *** Cancer 66 75.68(15.40) *** 61 70.96(17.06) *** IBD 76 74.18(14.66) *** 67 72.65(17.62) *** Healthy 1033 83.89(11.84) 665 84.61(11.19) Physical Health 4,1392 41.60 4,964 46.87 Asthma 99 76.14(19.10)*** 75 73.36(20.60) *** Diabetes 124 84.75(13.65)** 103 82.97(13.67) *** Cancer 66 78.10(17.64) *** 61 75.04(18.79) *** IBD 76 75.08(18.21) *** 67 71.54(21.98) *** Healthy 1032 88.51(11.62) 665 89.06(12.27) Psychosocial Health 4,1393 14.53 4,964 28.16 Asthma 99 74.9(17.48) *** 74 71.20(17.95) *** Diabetes 124 81.24(13.77) 103 74.62(13.27) *** Cancer 66 74.37(15.85) *** 61 68.83(17.92) *** IBD 76 73.64(14.35) *** 67 73.21(17.43) *** Healthy 1033 81.84(13.21) 664 82.21(12.67) Emotional functioning 4,1393 9.85 4,962 23.24 Asthma 99 70.66(20.06) *** 74 67.23(21.20) *** Diabetes 124 78.85(18.28) 102 66.01(17.80) *** Cancer 66 73.56(18.39) * 61 68.36(18.04) *** IBD 76 68.11(18.90) *** 67 67.26(21.41) *** Healthy 1033 78.49(17.94) 663 78.28(15.54) Social functioning 4,1393 5.89 4,964 14.09 Asthma 99 81.76(21.35) *** 74 76.96(21.69) *** Diabetes 124 89.15(13.91) 103 85.28(15.98) Cancer 66 81.27(18.36) ** 61 73.28(22.93) *** IBD 76 83.82(16.61) 67 82.12(18.09) * Healthy 1033 87.65(16.46) 664 86.82(15.42) School functioning 4,1386 14.12 4,960 26.90 Asthma 99 72.37(19.62) *** 74 69.02(22.55) *** Diabetes 124 77.70(17.39) 103 72.62(17.64) *** Cancer 63 67.38(20.36) *** 58 63.45(21.71) *** IBD 73 69.52(17.28) *** 66 70.46(20.95) *** Healthy 1032 78.87(15.89) 664 81.52(16.09) * Denotes difference from healthy children at p < 0.05 * *Denotes difference from healthy children at p < 0.01 * **Denotes difference from healthy children at p < 0.001 Scores for children with a chronic health condition were lower than those for healthy children on all proxy-report scales, with most differences reaching significance (see table 3 ). For self-report, children with asthma, IBD and cancer survivors showed lower scores than healthy children on all scales, with most differences reaching significance (see table 3 ). In contrast, children with diabetes did not report lower HRQL than healthy children for all domains; for this group scores were higher than those of healthy children for emotional and social functioning, although this was not significant. The only sub-scale on the self-report measure to show significant sex differences was Emotional Functioning, with females reporting lower HRQL than males (F (1,1396) = 29.66; p = 0.001). Although the mean score for female respondents at 74.39 (sd = 19.32) was lower than the male mean score of 79.71(sd = 17.04), these scores are still at the high end of the scale. The differences were however big enough to be reflected in both the composite psychosocial summary score (mean score: females = 79.65 (sd = 14.38), males = 81.39(sd = 13.68), F (1,1396) = 5.35; p = 0.021) and the total score (mean score: females = 81.32 (sd = 13.24), males = 83.21(sd = 12.89), F (1,1396) = 7.30; p = 0.007). Table 4 shows the correlation between self and proxy-report. Moderate correlation was shown between the two forms on the same subscales, although correlations were higher for children with a chronic health condition than for healthy children. Table 4 Correlation between self-report and proxy forms Scale: Total Sample Healthy children Children with chronic health condition Total score 0.56* 0.32* 0.67* Physical health 0.53* 0.20* 0.61* Psychosocial health 0.51* 0.34* 0.63* Emotional functioning 0.41* 0.27* 0.51* Social functioning 0.50* 0.42* 0.60* School functioning 0.49* 0.32* 0.56* * Correlation is significant at P < 0. 001 Discussion The performance of the UK-English PedsQL™ (age range 8–18 years) was found to be similar to that reported for the original PedsQL™ [ 3 ]. We found excellent internal reliability of 0.90 for both self and proxy-report total scales, indicating the suitability of the total scale scores for individual patient analysis [ 9 ]. All subscale and summary scores exceeded 0.70, making them acceptable for group comparisons. This is comparable to the reliabilities reported for the original PedsQL™ of 0.88 and 0.90 for self and proxy-report total scales respectively, with all subscale and summary scores also exceeding 0.70[ 3 ]. As with the original PedsQL™, although no floor effects were found the existence of ceiling effects should be noted [ 3 ]. Thus whilst the full range of scoring options is used for the majority of subscales, responses tend to be skewed towards the top end of the scale for all subscales, for both self and proxy-report. However, it has been suggested ceiling and floor effects are to be expected in generic HRQL instruments, simply because they aim to be applicable to a wide range of populations [ 10 ]. It is possible that the health conditions of the children who took part in the study were well controlled, leading to better HRQL ratings. This issue should be explored further through the administration of PedsQL™ to children with a wider range of health issues including those experiencing acute health problems. PedsQL™ performed as hypothesized using the known-groups method. There were differences in HRQL between healthy children and those with chronic health conditions for both proxy and self-report. However, children with diabetes scored significantly lower than healthy children on only one dimension – physical functioning. Indeed, they reported better HRQL than children with other chronic health problems and on social and emotional functioning rated their HRQL as better than healthy children, although this did not reach significance. The similarity in HRQL between children with diabetes and their healthy peers has been noted elsewhere [ 11 ]. Furthermore, a study into the impact of diabetes screening on adult HRQL reported similarities in HRQL of adults with and without diabetes – both before and after diagnosis [ 12 ]. This suggests that the findings of our study are neither atypical nor indicative of a problem with PedsQL™ measurement, but rather represent a meaningful difference in the HRQL of children with diabetes and those with other chronic health problems. Whether this is due to good disease management, the positive support of the diabetes care team or other factors remains unclear. What is apparent however, is that this issue merits further investigation. Varni et al [ 3 ] did not report any differences in parental or child reporting of HRQL either by age or sex of the child. Whilst this study also found no differences in reporting for the proxy-report, a significant difference in male and female reporting of HRQL was found on the emotional functioning sub-scale of the self-report, with females reporting significantly lower levels of emotional functioning than males. The difference between males and females reflected in our data is consistent with much of the psychological literature concerning gender differences in emotional health [ 13 , 14 ]. In addition to suggesting that females are more likely to suffer more emotional health problems such as anxiety and depression than males, studies have also proposed that this gender difference is rooted in adolescence [ 15 , 16 ]. Furthermore, differences in male and female responses to illness have also been suggested, with females more likely to suffer depression following traumatic injury [ 17 ] and to display greater anxiety about chronic illness [ 18 , 19 ]. Thus the difference in emotional functioning we report here, would seem to reflect a genuine disparity between males and females and so offer further evidence for the validity of PedsQL™ as a sensitive measure of the emotional functioning of children and young people. Moderate correlation was found between self and proxy-report. The pattern of parent-child correlation for the total sample is similar to that reported for the original PedsQL™, where better correlation was found for physical than for psychological and social functioning [ 3 ]. Yet, it should be noted that correlation is better between parents and children where the child has a chronic health condition. Indeed the most marked difference in correlation is on the physical health scale, suggesting that parents and children are more likely to share information about an issue if it is perceived as a problem (in this case physical health). Thus, whilstprevious research has found that parents and children agree more about physical problems, rather than internalising problems such as anxiety or sadness [ 20 ] this may depend in part on whether or not the child has a health problem. Furthermore, it is likely that proxy-reports reflect parental anxiety about their child; in this study parents of children with chronic health problems consistently underestimated their child's HRQL. The limited correlation observed between self and proxy-reports confirms the need to measure both child and parent perspectives when evaluating paediatric HRQL. Furthermore, in situations when the child is unable or unwilling to complete the self-report making it necessary to use a proxy-report to estimate HRQL, the knowledge that this estimate maybe inaccurate should be considered. A potential limitation of this study is that retest reliability and responsiveness was not conducted. However, it has been argued that test-retest reliability may be less useful than internal consistency reliability in HRQL instrument development [ 21 ]. Internal consistency is suggested as a more valuable assessment of the reliability of a measure because of the likelihood of short-term fluctuations in health conditions such as those employed in this study, in which external factors such as disease and treatment variables are known to influence functioning. Conclusion We have shown that the UK-English PedsQL™ is valid and reliable, replicating some of the previous findings for the generic PedsQL™ [ 3 ] for the first time with a UK population. The UK-English measure will be a valuable tool for assessing the HRQL of school-aged children in the UK, providing a useful outcome measure in both a research and clinical setting. Authors' contributions PU made substantial contributions the acquisition of data, analysis and interpretation of data and the drafting of the article. CE made substantial contributions to the design of the study, the acquisition of data and interpretation of the data, drafting and revising the article. IC, MJ, AM, IR & JW all made substantial contributions to conception and design of the study and have been involved in revising the paper for important intellectual content. All authors have given final approval of the version to be published. Competing interests The author(s) declare that they have no competing interests.

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1079919

Current and future developments in managed care in the United States and implications for Europe

The paper reviews and evaluates current and future approaches to cost containment in the United States. Managed care was once seen as an effective approach to supporting health care quality while containing costs in the USA. In recent years payors started to look in other directions, since prospects for limiting expenses faded. Nowadays consumer driven health plans seem to be on the rise. The reasons for the decline of managed care, the growing popularity of the consumer driven health plans and the implications for Europe are discussed.

Introduction On an international basis, the development of health care policy is increasingly being influenced by cost considerations. Advances in health science and the delivery of care continue to expand the capabilities of treatments. The ability of nations and communities to pay for this care from available resources is a major subject of debate. One focus of this debate has been research comparing health care utilization in Europe and the United States. Research has frequently demonstrated that, while Europe has greater capacity and higher utilization of services than the United States, Americans are paying more for these services. This discussion has intensified as nations on both sides of the Atlantic struggle to provide health and other programs while maintaining economic stability [ 1 , 2 ]. Frustrating as they may be for health care providers and payors, this situation presents important opportunities for research concerning health care policy. In particular, the experience of the United States during recent years, contains significant economic challenges with direct implications for health care financing and delivery. After eliminating federal budget deficits during the 1990s, America has generated a new round of shortfalls (exceeding $400 billion) annually through a combination of international military involvements and an economic downturn. This situation may impact Medicare, the major source of health care funding for the elderly and the largest single payor for these services [ 3 , 4 ]. Resource limitations are also challenging other major health care payors in the United States. Private insurance has carried the burden of cost shifting from public payors and has also suffered from the impact of the economic recession [ 4 ]. The Medicaid program, which funds health services for the indigent and elderly, has been challenged by budget difficulties of many state governments [ 5 , 6 ]. The pressures that are being exerted by these developments on the health care system of the United States promise to create opportunities for policy making concerning cost containment during the immediate future. The Bush Administration is already rediscovering Medicare managed care [ 7 ]. This type of activity could spill over to into other areas of the economy as the administration struggles to reconcile the costs of international commitments and domestic programs with its recent tax reductions. Beyond these circumstances, current and future approaches to cost containment in the United States need to be viewed in a broader context. Managed care for a variety of payors was once seen as an effective approach to supporting health care quality while containing costs. During the past decade, the attractiveness of this approach to many employers has faded and prospects for limiting health care expenses have become confused [ 8 ]. These developments caused payors in America to look in other directions for approaches to containing health care expenses. In this context, the recent experience of the United States with respect to health care and its economic impact may have valuable implications for that nation and for the rest of the world. In order to understand these implications, the development of managed care and other approaches to health care policy must be reviewed and evaluated. To provide a broad view of these changes, this information is presented as a review of existing sources and data rather than the development of completely new information. Development of Managed Care in the United States Historically, managed care evolved in the United States to influence the use of medical care by improving outcomes and efficiency. The past three decades of experience with this approach suggests that at least some of these objectives have been realized. During the second half of the twentieth century, managed care developed in the United States as a mechanism for constraining the growth of health care costs by controlling the delivery system. This approach originated in the western United States in the form of staff model plans such as Kaiser Permanente which employed physicians and other caregivers directly. In the private health insurance industry, managed care plans controlled costs and the delivery of care by restricting hospital utilization, such as admissions and lengths of stay, by limiting access to specialists, and by encouraging healthful behaviors among subscribers [ 9 ]. The popularity of managed care in the private sector of the American economy encouraged its adoption by public payors. The size of the Medicare program and the growth of health care expenses for the elderly stimulated the federal government to offer Medicare managed care options during the 1970s and 80s [ 10 ]. State governments, burdened with health care expenses for the indigent and elderly, and lacking the ability to run operating deficits, became even more interested in this approach [ 11 ]. During the 1980s and 90s, managed care expanded rapidly in the United States. In private health insurance, a major shift occurred from traditional indemnity insurance to managed care plans in many markets. This development was stimulated by an increase in the numbers of businesses offering managed care as an option to employees. The proportion of employees in large firms (those with more than 200 employees) enrolled in managed care plans grew from 5 percent in 1984 to 50 percent in 1993 [ 12 ]. As the use of managed care spread, interest in traditional indemnity plans declined. By 1998, only 14 percent of employees in large firms were enrolled in indemnity insurance plans [ 13 ]. During the 1980s and 90s, increased enrollment in private sector managed care in the United States was spurred by the ability of this approach to contain health care costs. The data in Table 1 demonstrate that, by 1997, average monthly insurance premiums for these plans were lower than for private health insurance. The data also indicate that the differences between premiums were greatest in areas where managed care penetration was lowest ($52 lower than non managed care plans) and in areas where managed care penetration was highest ($35 lower). This information suggests that managed care was successful in controlling costs in a variety of settings. Table 1 Average monthly insurance premiums by type of insurance plan, 1997. Average Family Premiums ($) All Plans Managed Care Non Managed Care HMO Penetration Less than 25 Percent 439 401 453 25 – 35 Percent 417 408 423 35 – 45 Percent 400 400 401 45 Percent or More 394 380 415 Source: 1997 Robert Wood Johnson Foundation Employer Health Insurance Survey. The expansion of managed care in the private sector of the United States was paralleled by increased adoption of this approach by public payors. Historically, the level of financial risk generated by the elderly and indigent, the major populations covered by Medicare and Medicaid, had limited government interest in privatizing these plans and the participation level of insurance companies in this area. The failure of the Clinton administration to enact national health care reform did not impede the development of Medicare managed care. This approach expanded rapidly during the 1990's, fueled in part by the introduction of flexible plans. Enrollment in Medicare managed care, which had remained close to 1,000,000 between 1985 and 1991, increased to more than 6,000,000 by 1999 [ 14 ]. In the same period, State governments turned to managed care as a means of constraining health care costs. With fewer resources than the federal government, States had more incentives to use this approach. Between 1997 and 2001, enrollment in full risk Medicaid managed care plans increased by 40.6 percent [ 15 ]. The development of managed care in the United States was stimulated by interest in improving both outcomes and efficiency of health care. During the 1980s and 90s, the proliferation of this approach was related to pressure for efficiency and cost containment. In this role, managed care performed well throughout most of the two decades. As the use of this approach by all payors increased, the annual per cent change in per capita health spending in the nation declined from 5.0 – 6.9 between 1991 and 1993 to 2.0 – 2.2 between 1994 and 1996. Among private health insurance plans, where managed care penetration was highest, annual per capita changes in premiums declined from 12.5 percent in 1988 to 4.8 percent in 1997 [ 16 ]. Available evidence indicates that managed care was able to reduce health care expenses in the United States through constraints on utilization of service. From its beginnings, traditional managed care controlled utilization of hospital care, a major source of health care costs, through physician gate keeping and pre authorization mechanisms. In the western part of the nation, where use of this approach was highest, it supported hospital admission rates and length of stay that were lower than those in other areas. During 2002, the hospital admission rate for the western region of the United States (955.4 per 10,000 population), was 18.6 percent below the national average (1,174.6 per 10,000). During the same period, the mean hospital stay in the west (4.4 days) was 8.9 percent below the national average (4.9 days) [ 17 ]. Reductions in health care utilization brought about by managed care in the western United States have been adopted in other areas of the nation and the world. Physician profiling and the development of preferred provider arrangements with long term care providers have been employed by providers and payors to constrain hospital utilization and related to costs [ 18 ]. The Decline of Managed Care In retrospect, it appears that the success of managed care in the United States during the last decades of the twentieth century also led to its undoing. Health care is a dynamic sector of many national economies. Upon review of the American experience, it seems that a number of factors contributed to the decline of this approach. The initial wave of opposition to managed care appeared as challenges to control of health care utilization, such as choice of providers. Physicians were never reconciled to allowing insurance plans to choose practitioners and hospitals. During the 1990's, a wave of class action litigation was directed at the ability of plans to direct referrals for health services. The plaintiffs in these cases, including consumers, physicians, and other providers, sought financial damages and injunctions against managed care business practices. Although courts in the United States were reluctant to declare the costs control methods of plans illegal, the full range of litigation supported negative perceptions of managed care among consumers and businesses [ 19 ]. During the late 1990s, the legal reaction against managed care in the United States was accompanied by deteriorating relationships between plans and health care providers. Increased consumer dissatisfaction with the business practices of plans, including apparent arbitrary denials of service and failure to pay claims promptly, added fuel to provider complaints about low payment rates. Providers also objected to the terms of risk contracting agreements which required them to carry a significant burden of pharmaceutical costs and other expenses. All of these developments generated a wave of opposition to managed care by hospitals and physician groups. These activities frequently resulted in the termination of contracts and network instability [ 20 , 21 ]. During the second half of the 1990's, consumer litigation and provider reactions against managed care caused these many of these plans to change their traditional business approaches. In order to regain the favor of consumers and providers, plans in many communities loosened controls on provider utilization. Plans relaxed the use of physicians as gate keepers and allowed consumers direct access to specialists. Requirements for referrals to other types of services were loosened. Stringent authorization procedures for use of hospital emergency departments and for certain surgical procedures were also relaxed. This process amounted to a substantial movement away from the aggressive health care management approaches which had supported the rise of this approach [ 22 ]. To replace these utilization control mechanisms, many managed care plans introduced mechanisms that included more interaction with subscribers, such as case management and electronic information exchange. Subscribers were encouraged to modify health related behaviors by visiting web sites and obtaining additional information concerning preventive care. In many communities, managed care plans placed increased emphasis on wellness programs and disease management. The industry was shifting its focus from controlling utilization to influencing it. The retreat from direct management of health care utilization toward softer approaches led to a general weakening of accountability to consumers. With fewer direct controls in place and greater reliance on indirect mechanisms the assessment of performance of individual plans by businesses and government became more difficult [ 8 ]. Related to these approaches, some managed care plans adopted business strategies which placed greater emphasis on profitability. Chief among these was a movement away from contracting for high risk populations. Facing increased dissatisfaction from consumers and greater competition from traditional insurance in the market place, private plans in many areas increasingly focused on lower risk populations as a means of improving profitability. As a result, many private managed care plans withdrew from participation in public managed care programs with high risk populations such as the elderly (Medicare) and the indigent (Medicaid). Between 1998 and 2000, the number of plans serving Medicare patients declined by 20 percent. By 2002, total Medicare managed care enrollment was lower than it had been in 1997. Between 1998 and 2000, the number of insurance plans participating in Medicaid declined by 15 percent. The development had a major impact on State governments because more than half of all Medicaid recipients were enrolled in managed care [ 23 ]. Increased efforts by managed care plans to compete in the United States health care marketplace also led to the adoption of other business strategies characteristic of traditional insurance. These included reduced a greater emphasis of raising premiums to support profitability. This approach effectively passed along a higher proportion of health care expenses to subscribers. Managed care plans also catered more to consumer preferences in order to expand market share [ 24 ]. All of these activities effectively changed managed care from its traditional structure in the United States. As the focus on continuity of care and regulation of utilization diminished, managed care plans became more like other types of insurance such as indemnity plans and preferred provider organizations. As choice of providers was substituted for gate keeping, physicians and increased premiums took the place of utilization controls, the line between managed non managed care became blurred. This entire process amounted to a decline of managed care shortly after the approach had reached its highest level of acceptance. This decline was reflected in a decreased ability of managed care to restrain costs, a major reason for the increased use of this form by payor during the 1980s and 90s. As a result of this development, a major barrier to health care spending was removed and per capita expenditures began to rise. This situation is illustrated by changes in annual per capita health care spending for all payors in the United States, summarized in Figure 1 . Figure 1 Annual percent change per capita health care spending United States 1991 – 2002. This information identifies both the zenith and the decline of managed care. Between 1994 and 1996, the impact of this approach reached a high point, as annual increases in per capita spending declined to only about two percent per year. The decline of managed care during the late 1990s produced a rapid erosion of this position. Annual increases in per capita spending nearly quadrupled to almost eight percent by 2000 and kept increasing after the turn of the century. These developments were paralleled by in annual increases of changes in per capita spending for private health insurance, the area of the economy in which managed care developed prior to the 1970s. After declining to less than five percent in 1997, annual increases in per capita expenses for private health insurance rapidly recovered to over 10 percent after the turn of the century. These developments are summarized in Figure 2 . Figure 2 Annual percent change per capita spending for private health insurance United States 1988 – 2002. The increases in pre capita health expenditures identified in these tables occurred because of a retreat from traditional cost controls by managed care plans and because of the transfer of many expenses to consumers. As previously noted, changes in the behavior of these plans in some metropolitan areas included less reluctance to raise premiums, rather than contain costs, in order to avoid operating losses. Many of the 'soft' programs that were added in order to compete with traditional health insurance were offered at an additional cost. This information concerning the impact of managed care on health care expenses does not identify the impact of the decline of the approach on continuity of care and other outcomes, for which quantitative data are not available. It might be assumed that, with increased emphasis on consumer choice and less attention to control of referrals and provider participation, the integrity of care across health systems also deteriorated during this period. All of these developments effectively moved managed care plans in the United States closer to traditional insurance plans in their behavior and impact on health care expenditures. They undermined the ability of this approach to differentiate itself from traditional health insurance as a mechanism which actively managed care and contained costs. They made it considerably more difficult for the approach to exert a direct impact on health care outcomes. Ironically, these characteristics had been the major selling points of the approach since its inception. It should be noted that, during this period, traditional insurance plans have also moved toward managed care by adopting features of health maintenance organizations such as utilization controls. As a result, the border between these two types of insurance is now almost non existent. The change in the character of managed care also had profound implications for the future of health care cost containment in the United States. It deprived government and the private sector of one of their most powerful weapons in restraining expenditures. It also signaled an important change in the direction off public and private sector health policy. The movement of managed care away from utilization controls and toward higher premiums shifted the burden of health policy toward the consumer. It suggested that payors would support consumers in shaping health care and the organizations that provide it, rather than having the payors assume leadership. This change would lead to important developments in the policy environment of this sector. The Rise of Consumer Driven Health Care The decline of managed care as the major driver of health care policy and reimbursement within the United States has opened the way for new forces to shape this area. The nature of these forces became visible in the late 1990s as managed care plans shifted responsibility for health care decision making to consumers. The resulting annual increases in health care expenditures, summarized in Figure 1 and 2 , were also effectively shifted to consumers through higher premiums, deductibles, and copayments. For example, in preferred provider organizations, the most widely used health plans, single coverage deductibles increased more than 50 percent between 2002 and 2003. More employers began offering high deductible plans. The rate of increase of out of pocket spending has increased every year between 2000 and 2003 [ 25 , 26 ]. The rise of increased consumer involvement in health care in the United States has developed on several fronts. At the broadest level, it has taken the form of a greater consumer role in decision making concerning treatment. In the late 1990's the use of television, newspapers, and electronic media to market health care to consumers became pervasive. Drug companies became major users of this approach. They have conducted enormous media campaigns to promote the use of sexual stimulants, allergy medicines, and cosmetic treatments. Access to large marketing budgets has made it possible for these companies to reach millions of consumers with their messages. Local and regional diagnostic firms have also marketed magnetic resonance imaging and whole body scanning through similar campaigns [ 27 ]. These efforts have been successful because they gone directly to users of health care. They have circumvented insurance companies, managed care, and even physicians. Listeners are urged to 'ask' or 'tell' their doctor to prescribe any number of medications or tests. The clear message of all of these initiatives is for individual consumers to take a greater role in health care decision making. Greater user involvement in health care that was stimulated by the decline of managed care, as well as media initiatives of the pharmaceutical industry and other groups, have led directly to the development of a new type of health insurance in the United States, the consumer driven health plan. These mechanisms complete the change initiated by the decline of managed care by directly assigning health care decision-making to consumers. Consumer driven health plans are designed to address the major objectives of managed care, development of healthful behaviors and containment of health care costs. Components of consumer driven plans usually include the following. - High deductibles as incentives for greater consumer participation in the cost of care - Catastrophic coverage for high cost services such as inpatient hospitalization - Consumer savings accounts for funding of prevention and screening services - Procedures for roll over of unused savings account balances to future time periods - Support for consumer decision making through availability of internet based information concerning health care risk factors and provider outcomes - Tracking of employee health expenses through the system [ 28 , 29 ]. These components have been designed to replace structures of managed care plans which addressed the same objectives. Encouragement of healthful behaviors and health status objectives are addressed through consumer savings accounts, rollover provisions, and internet based information. These provisions have effectively transferred responsibility for the management of care from the primary care physician gatekeepers employed by managed care plans to the consumer. This transfer has been supported by a combination of financial incentives and electronic data [ 28 , 30 ]. Consumer driven health plans also contain provisions for cost containment. High deductibles and catastrophic coverage are intended to limit expenditures by payors. These provisions effectively reduce expenses for many of the pharmaceutical and ambulatory care expenses currently being pushed by media advertising. By excluding payor reimbursement for them, these plans are transferring these expenses to the consumers, or providing incentives to eliminate the purchases altogether. This amounts to a cost containment approach very different from the utilization controls employed by managed care plans [ 31 ]. The implementation of consumer driven health plans has generated extensive controversy in the American health care system. Supporters of this approach have argued that it is logical because it places major responsibility for health care decision making in the hands of the party who will be most influenced by those decisions, the consumer. They have emphasized that, under these plans, positive health care behaviors and decision making are rewarded by fewer out of pocket expenses. They have also noted that consumers are provided with extensive electronic information needed to support effective decision-making [ 30 , 32 ]. Supporters of consumer driven plans also have suggested that these approaches include realistic mechanisms for health care cost containment. They have pointed out that managed care relied on limitations on the utilization of care to limit spending, while consumer driven plans assign these decisions to users of care. They have suggested that the new approach can satisfy both the payor and the consumer, by limiting insurance expenses for care and allowing individuals to purchase additional services [ 28 ]. Opponents of consumer driven health care have argued that this approach is inferior to managed care, that it is a mechanism for employers and payors to abdicate their responsibilities. They have suggested that, rather than supporting consumers with the advice of physicians and other health care providers, it turns them loose to make decisions on their own. They have indicated that a set of financial incentives and internet based information is no substitute for the relationship between a patient and a caregiver [ 29 ]. Opponents of consumer driven plans also argued that the real purpose of these approaches is financial, that they are better identified as defined contribution plans. Opponents of these plans suggested that they are really covers for the abdication of financial responsibility by payors. They have argued that the major purpose of these plans is to shift health care costs from employers to employees. They have contended that high deductibles and catastrophic coverage are the real solution for employers and payors seeking to reduce premiums, regardless of the impact on subscribers [ 30 , 31 ]. The debate concerning consumer driven health care is probably only beginning in the United States. It bears similarities to the discussion surrounding the rise of managed care in the 1970s and 80s. One important difference between these situations may be the economic background. The ascendancy of managed care developed against the background of an upturn. Consumer driven care is developing at a time of economic instability and limited resources. Indeed, these conditions may be supporting the rise of the new approach. A review of the current status on consumer driven plans suggests that they are still evolving. Many of these plans are still developing their own provider networks. Others are partnering with existing plans in order to expedite the process. Some plans are developing generic fee schedules for services and allowing consumers to develop their own networks. Consistent with this approach, consumers cover additional health care costs from their own resources [ 31 ]. The development of information infrastructure has become an important part of the implementation of consumer driven plans. In order to involve consumers in a meaningful way, plans must make available extensive electronic data to support decision making. These data include a variety of online sources of information including many types of research. They also must include financial information concerning provider prices and discounts. In order to enable consumers to participate in health care decision making on a continuing basis, plans must also make available data concerning consumer accounts which identify the impact of choices on available funds. The plans which are entering the consumer driven market in a serious manner must have all of these information resources available. They require an extensive investment in data infrastructure [ 31 ]. The implementation of consumer driven health plans is proceeding rapidly in the United States. Because of the size of the American population, it still occupies a relatively small proportion of the health care market. The data in Figure 3 demonstrate that, by 2004 enrollment in consumer driven health plans is projected to reach 1,000,000. This enrollment is still dwarfed by existing traditional insurance and managed care populations. Figure 3 Participants in consumer drive health plans United States 2001 – 2004. The distribution of consumer driven health care enrollment across employers is summarized in Figure 4 . This data indicate that market penetration by these plans is still modest, but that it has established a foothold in a wide range of employers [ 28 ]. This suggests that interest in these plans is not limited by employer size. Figure 4 Percent of employees adopting consumer driven health plans United States 2003. It should be noted that these data reflect the growth of consumer driven plans among private insurance plans. This approach has not yet been tested with Medicare or Medicaid populations. These effort may require the development of new designs for plan components, especially those which involve consumer decision making, because of differences in health care mind sets and behaviors among the elderly and the indigent. The future of consumer driven health care in the United States is difficult to predict. Additional time and utilization data will be required to determine whether this approach generates sufficient enrollment to develop into a major force within the health care system of the United States and whether other nations adopt it. Implications for Europe The recent development of managed care and consumer driven health care in the United States has important implications. Rightly or wrongly, the American health care system has historically been a source of approaches for implementation in Europe and elsewhere. The amount of change and innovation that has marked this system in recent years has generated more than enough material for consideration by the international community. One of the most significant developments in the United States health care system in the past several years has been the decline of managed care. This development probably came as a surprise to health care policy makers elsewhere in the world. In its early days, managed care had great promise for addressing the two most important objectives of health care planning, improving patient outcomes while containing costs. During the 1980s, this approach seemed to be on its way toward meeting this objective in the American health care system. Managed care penetration of the private insurance, Medicare, and Medicaid markets were increasing substantially and previous annual increases in per capita health care expenditures were declining. The reasons for the decline of managed care probably emanate from conflicting forces within the American health care system and the environment surrounding it. These include the desire to support buyer preferences and the need to restrain increases in health care expenditures at the same time. In the American system during the late 1990s, cost containment lost out. The lesson that may emerge from this is one of realism. You cannot have your cake and eat it too. At the same time, this story is more complicated than a struggle between choice and cost containment. It is part of a rising interest in consumer choice within the wider health care environment and the economy of the United States. Pharmaceutical companies and other providers of health services have recognized this and begun direct marketing to consumers through the media. This development has involved bypassing physicians and going directly to users of care, just as consumer driven care involves bypassing managed care and its utilization controls. These developments suggest that consumer driven health care in the United States is not an isolated event, but part of a wider trend. These developments pose a major dilemma for health policy makers. The parameters of this dilemma may differ among nations. It can be suggested that, in the United States, advocates of managed care should not have 'bailed out' so quickly when consumer dissatisfaction began to increase. This assumes that purchasers of care will ultimately side with cost containment. The preceding information demonstrates that the following chain of events has occurred in the United States. Each of these events was related to wider developments in the American economy. - Managed care helps reduce health care cost increases - Increased managed care results generates popular dissatisfaction - Managed care plans reduce controls and increase prices - Health care costs increase substantially - Consumer driven plans provide a mechanism for government and employers to unload health care costs on consumers This important juncture is where Europe and the rest of the world may have an advantage over the United States. It is clear that Europe has the opportunity to invest in managed care and to use this approach to restrain costs and improve patient outcomes. In so doing, European nations would stay with the first step on the preceding chain of events and avoid the disadvantages of the remaining ones. But before we elaborate further on possible implications for Europe, we should emphasize that the stage for the health care actors in Europe is quite different from the one in the USA. Health care systems in Europe vary greatly among countries, but in general European health care systems have some characteristics that make them different from the system(s) across the Atlantic. First, Europe has a highly valued tradition of universal health care coverage and governmental administration. Furthermore, Europeans tend to entrust responsibility to the (central) government and not to private agencies. Finally, competition among providers and purchasers is restricted by highly structured conditions by the government. The above comes down to the point that in health care Europeans have difficulties getting market elements off the ground. They do not seem to trust the invisible hand (of the market). This has probably resulted in a slower rate of system innovation, but fewer policy development and implementation errors, in these countries [ 33 , 34 ]. Germany, Switzerland and The Netherlands are nowadays starting to adopt managed care (tools) with caution, focusing on support for patient outcomes and cost containment. At the same time, Sweden and the United Kingdom, implemented dramatic changes very rapidly. Obviously, Europe also offers the opportunity for a number of different approaches to this issue to develop simultaneously [ 35 , 36 ]. Any evaluation of the potential impact of consumer driven health care in Europe must address specific aspects of this approach. For example, what would be the implications of providing information concerning pharmaceuticals directly to consumers in Europe? It could also be conjectured that Europeans might prove to be more deliberate and conservative consumers of pharmaceuticals than Americans. It could be suggested that Europeans would make more extensive use of information before reacting to the first television commercial and asking their physicians for Viagra or Allegra, or some other drug. At the same time, however, the answers to these questions are not clear. They may vary substantially among European countries [ 36 , 37 ]. A more important question regarding consumer driven health care concerns the implications of exchanging free choice of providers for higher cost sharing by consumers. In societies with established medical networks which have become acclimated to managed care, the free choice option might have little impact. At the same time, the recent strength of the European economies and the euro might reduce the impact of higher cost sharing and make free choice of providers more affordable [ 38 , 39 ]. Another area of consumer driven health care that deserves evaluation is the potential impact of providing health information directly to consumers on the use of services and costs. In the United States, the effectiveness of this approach depends greatly on the extent to which consumers read, evaluate, and act on health information provided through websites and other electronic media. It is clear that Europeans probably make as much use of the internet as Americans, however, it remains to be seen what the impact of electronic medical information will be on consumer behaviors in these countries [ 40 ]. The answers to these specific questions could vary substantially across the European continent. The degree to which managed care is retained could be related to the commitment of individual governments, the influence of medical establishments, and the availability of disposable income. From a policy making standpoint, this issue probably requires evaluation on a nation by nation basis. From an international standpoint, the potential impact of the decline of managed care and the rise of consumer driven health care in the United States is a fascinating issue. It is still too early to determine what the impact of these developments will be on the rest of the world. One answer to this question is probably that changes in health care policy require adequate information on the effectiveness of the alternative being considered, as well as the status quo. It appears that the difficulties with managed care experienced by the United States during the 1990s have prompted a rapid movement away from this form, without a clear idea of the effectiveness of consumer driven care, or other alternatives. Health care has too much of an impact on large populations and on national spending to approach it by 'looking before leaping'. All of this suggests that Europe and the rest of the international community are in an excellent position to profit from the American experience without much risk. They can view what is going on with critical eyes and reach their own conclusions about whether the transition from managed to consumer driven care supports patient outcomes or not. They can monitor the data and determine which of these forms has greater potential for cost containment. The opportunities for learning may increase as financial pressures place greater pressures on the American system for cost containment.

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1079920

Correlates of motivation to prevent weight gain: a cross sectional survey

Background This study is an application of the theory of planned behaviour (TPB) with additional variables to predict the motivations to prevent weight gain. In addition, variations in measures across individuals classified into Precaution Adoption Process stages (PAPM-stages) of behaviour change were investigated. Methods A cross-sectional survey among 979 non-obese Dutch adults aged 25–35 years was conducted. Multiple binary logistic regression analysis was conducted to assess the associations of Body Mass Index (BMI), demographic factors and psychosocial variables from the TPB with the intention to prevent weight gain. Differences in BMI, demographic and psychosocial factors between PAPM-stages were explored using one-way analysis of variance and chi-square tests. Results Eighty-five percent of respondents intended to prevent weight gain. Age, attitudes and risk perceptions related to weight gain were the strongest correlates of intention (age: OR = 1.12, 95%CI: 1.04–1.20; attitude OR = 7.91, 95%CI: 5.33–11.74; risk perception OR = 1.24, 95%CI: 1.11–1.38). Significant differences were detected between the PAPM-stages in almost all variables. Notably, perceived behavioural control was lowest among people who had decided to prevent weight gain. Conclusion Messages to influence attitudes towards the prevention of weight gain and risk perception may affect people who are not yet motivated to prevent weight gain. Interventions increasing people's perceived behavioural control in overcoming barriers to prevent weight gain may help people to act on their intentions.

Introduction Prevalence of overweight and obesity in Western societies has increased rapidly [ 1 ]. This is a threat for public health by its link to chronic illness and disabilities and impaired quality of life [ 2 - 4 ]. Overweight is a result of a long-term positive imbalance between energy intake and energy expenditure produced by a relative excess in energy input (diet) and/or a deficit in energy output (physical activity) [ 5 ]. In The Netherlands, the prevalence of obesity has approximately doubled over the past 20 years [ 1 ]. The prevalence of overweight and obesity is about 45% for men and 35% for women, while 11% of the men and 12% of the women are obese [ 5 , 6 ]. To stop the increase in the prevalence of overweight and obesity, effective strategies for the management and prevention of overweight and obesity need to be developed. Current strategies aimed at treating obesity are not very successful in the long-term [ 7 , 8 ]. The behavioural changes necessary to achieve significant weight loss and maintain a healthy weight are difficult for most people. Therefore, intervention programs aimed to prevent weight gain by encouraging relatively small changes in physical activity and eating habits among people who are not yet overweight may be more promising [ 6 , 9 , 10 ]. Young adults are of particular interest for prevention of overweight. There is evidence that weight gain is most likely to occur among males and females between the ages of 25 and 34 [ 6 , 11 , 12 ]. It has also been suggested that the onset of weight gain may have a situational basis, with important events in the life cycle such as entering the workforce, marrying and/ or having children triggering energy imbalance [ 13 ]. Prevention of (further) weight gain is also important in young adults who are already overweight. To support relatively small behavioural changes to reverse the gradual increments in weight with age, it is important to identify correlates of people's motivation to prevent weight gain. Such correlates may be intermediate goals in weight gain prevention programs [ 14 ]. The Theory of Planned Behaviour [ 15 ] is one of the most widely-applied models to explain health behaviours [ 16 , 17 ]. According to this theory, behaviour in general is determined primarily by behavioural intention and postulates that this intention is determined by three conceptually independent constructs: attitude, subjective norms and perceived behavioural control. Attitudes are the overall evaluations of the behaviour by the individual; subjective norms consist of a person's beliefs about whether significant others think he or she should engage in the behaviour; perceived behavioural control is the individual's perception of the extent to which performance of the behaviour is under voluntary control. The present study aims to identify potential correlates of motivation to prevent weight gain in a population of young adults in preparation of the development of nation-wide mass media campaigns to prevent weight gain, from the Netherlands Nutrition Centre. Therefore, the present study is a first step to identify potential mediators that should be addressed in such campaigns [ 18 , 19 ]. Primarily, we investigated associations with the constructs from the Theory of Planned Behaviour (TPB) to identify potential correlates of intentions to prevent weight gain in a population of young adults. Additionally, we included constructs that have been identified as a possible extension of the TPB that may be relevant to explain weight maintenance behaviours: descriptive norms (modeling) and social support related to prevention of weight gain, overweight-related risk perceptions, and perceptions of personal weight status. A study by de Vries et al (1995) indicated that there are at least two other categories of social influences besides subjective norms that may determine health behaviour: descriptive norms, i.e. the observed behaviour of others in the direct social environment, and social support, i.e. the direct support people experience for preventive behaviour [ 20 ]. Risk perceptions were included as a separate potential determinant in the present study since important alternative health behaviour models such as the Health Belief Model [ 21 ] and the Protection Motivation Theory [ 22 ] emphasise risk perceptions as a determinant of prevention motivation. Further, weight perception, i.e. self-rated weight status, was included as an additional variable, since there is evidence that people underestimate their weight, which may reduce their perceived need to change and thus their prevention intentions [ 23 - 25 ]. Nowadays, behaviour change is generally conceptualised to occur in subsequent stages or phases [ 26 ]. Such stage models like the Transtheoretical model (TTM)[ 27 ] and the Precaution Adoption Process Model (PAPM) [ 28 , 29 ] suggest that people in different stages behave in qualitatively different ways and that the content of interventions to encourage people to move toward action should be stage-specific. Transition between stages can be viewed as barriers that must be overcome before action is taken. These stage-models generally distinguish between people who are unaware of or unengaged by a health issue (stage 1), 'engaged and deciding what to do' (stage 2), 'planning to act but not yet acting' (stage 3), 'acting' (stage 4) and 'maintaining' (stage 5). Further, PAPM explicitly distinguished a separate aware but not acting stage, i.e. a step out of the sequence toward action. There is now preliminary evidence that PAPM is applicable to investigate complex behaviour change, like diet and physical activity, and that people in different PAPM stages for these behaviours differ in psychosocial variables like attitudes and perceived behavioural control [ 30 ]. Therefore we also investigated how a young adult population was distributed over the different stages related to prevention of weight gain and if people in different PAPM-stages differ in demographic and psychosocial characteristics. Methods Participants and recruitment Data for the present study were derived from a cross-sectional survey conducted in November and December 2002. Data collection was performed using telephone questionnaires to obtain representative data of the non-obese Dutch population aged 25–35 years. Telephone numbers were selected at random by means of random digit dialling and stratified to different regions to ensure an equal distribution over the Netherlands. The participation rate among the population was 71.4% (6587 of the 23053 participants who were called refused to participate to the telephone interview). Those who did not meet the age and BMI inclusion criteria (n = 15449) were excluded. Only respondents aged between 25 and 35 years and with a BMI between 20 and 30 kg/m 2 could participate in the study. BMI was calculated from self-reported weight and height. Pregnant women (n = 38) were also excluded from the analysis, since weight gain can and should be expected during pregnancy. This resulted in a study population of 979 respondents of which 56.7% were female; 10.2 % was of non-Dutch origin; 57.5 % had intermediate or less than intermediate vocational training. The mean age was 30.0 years (SD = 3.0) and mean BMI was 23.7 (SD = 2.6), with a 30.1 % prevalence of overweight (BMI ≥ 25). Measures Administration of the telephone questionnaire took approximately 15 minutes. The questionnaire included items of demographics, psychosocial factors related to prevention of weight gain based on the TPB-model, descriptive norm, social support, risk perception and weight perception. Furthermore, the questionnaire obtained measures based on the Precaution Adoption Process Model. At the start of the interview, the interviewer introduced the topic by explaining that questions would be asked on prevention of weight gain. It was further explained that prevention of weight gain should be understood as 'watching one's weight' by implementing small changes in lifestyle, such as eating habits and physical activity (e.g. reducing the amount of calories of food in meals and snacks and activities to increase amount of physical activity), not only with the purpose to lose weight but particularly in order to avoid gaining weight. All items to assess TPB and other psychosocial constructs were measured on bipolar five-point scales. The interviewer read out the answering categories. For constructs that were assessed with multiple items, the mean item score was calculated after sufficient internal consistency was established. Cronbach's alphas and inter item correlations were calculated to analyse the internal consistence between the items related to behavioural determinants. Alpha > 0.5 were regarded as acceptable for the present exploratory study [ 31 ]. TPB constructs Attitude was assessed directly with three items asking how bad or good; unimportant or important; and unpleasant or pleasant they regarded prevention of weight gain (-2 = very bad; unimportant; unpleasant; 2 = very good; important; pleasant) (α = 0.53). Subjective norm was assessed with one item by asking if respondents thought that 'important others' (e.g. spouse, family, friends, colleagues) wanted them to prevent weight gain (-2 = no, certainly not; 2 = yes, certainly). Perceived behavioural control was assessed with two items asking how difficult or easy respondents thought it is to prevent weight gain is (-2 = very difficult; 2 = very easy) and how certain they were that they can prevent weight gain (-2 = no, certainly not; 2 = yes, certainly) (α = 0.53). Behavioural intention was measured with one item asking the respondent whether they intended to prevent weight gain (-2 = no, certainly not; 2 = yes, certainly). Other psychosocial constructs Descriptive norm (modeling) was measured by asking respondents to assess how many people they perceived in their direct social environment to actively try to prevent weight gain (-2 = very few people; 2 = almost all people). Social support was measured by asking respondents how much support they received from 'important others' to prevent weight gain (-2 = very little support; 2 = very much support). Risk perception of weight gain was calculated by multiplication of two items, one in which respondents were asked if they believed that they were at less or more risk for weight gain compared to others of same sex, age and height (1 = much less risk; 5 = much more risk) and one item asking how serious they perceived weight gain to be on a three point scale (1 = not serious; 2 = serious; 3 = very serious). This resulted in a risk perception scale of 1–15. Finally, weight perception was assessed by two items, one which asked whether respondents rated their body weight as low or high (-2 = far too light; 2 = far too high) and one in which respondents compared their weight to that of other people of the same sex and age (-2 = much lower weight; 2 = much higher weight) (α = 0.65). PAPM stages of weight gain prevention behaviour Following Weinstein's method [ 28 ], a staging algorithm was used to assign respondents into different PAPM stages (See Table 1 ). Table 1 PAPM staging-algorithm applied to weight gain prevention Respondents in stage: Agreed with statement: 1. Unengaged I have never thought about actively trying to prevent weight gain 2. Undecided to act I have thought about actively trying to prevent weight gain but I do not know (yet) whether I will do so 3. Decided not to act I have decided not to actively try to prevent weight gain 4. Decided to act I have decided to actively try to prevent weight gain but I am currently not doing so (yet) 5. Action and maintenance I do already actively try to prevent weight gain Analyses Methods Multiple logistic regression analysis was conducted in a 3-step process to assess the association of demographic characteristics (age, gender, education, ethnicity and BMI; step 1), TPB constructs (attitude, social norm and perceived behaviour control; step 2) and other psychosocial constructs (modeling, social support, risk perception and weight perception; step 3) with the intention to prevent weight gain. We used logistic regression analyses since we dichotomised the dependent variable 'intention to prevent weight gain' (negative or no intention = 0; positive intention = 1), because of a skewed distribution. Behavioural intention was dichotomised by categorising respondents who answered the intention-question with option 1 (yes, certainly) or 2 (yes, probably) as having a positive intention and those who answered with option -2 (certainly not), -1 (probably not) or 0 (not sure) as having a negative intention. One-way analysis of variance with Scheffe's multiple-comparison test (alpha = .05) and Chi Square (alpha = .05) was used to test for significant differences in demographic and psychosocial factors between the stages of change. All analyses were performed using the SPSS 11.0 statistical program for Windows. Results Eighty-five percent of respondents had a positive intention to prevent weight gain. On average, respondents had positive scores on attitude and perceived behavioural control, negative scores on subjective norm and social support, and neutral scores on descriptive norm, and weight perception (See Table 2 ). Table 2 Number of items on determinant constructs related to prevention of weight gain, the internal consistency between the items and the mean scores on the constructs, n = 979 Determinant (Range) Number of items Internal consistency (α) Mean score (SD) TPB construct: Intention (-2,2) 1 1.36 (1.18) Attitude (-2,2) 3 0.53 0.59 (0.65) Subjective norm (-2,2) 1 -0.67 (1.47) Perceive behavioural control (-2,2) 2 0.53 0.88 (0.92) Other psychosocial constructs: Descriptive norm (-2,2) 1 0.20 (1.19) Social support (-2,2) 1 -1.05 (1.03) Risk perception (1,15) 2 5.92 (3.69) Weight perception (-2,2) 2 0.65 0.12 (0.72) Predictors of intention to prevent weight gain With the first step, the logistic regression model explained 11.3% of the variance in intention to prevent weight gain (see Table 3 ). In this model the variables BMI, sex and age were significant predictors of the intention to prevent weight gain. With the second step, the model was extended with the TPB constructs and explained 45.5% of the variance. Additional to BMI, sex and age, were also attitude and subjective norms significant predictors of the intention to prevent weight gain. With the third step, the regression model was extended with the variables modelling, social support, risk perception and weight perception and explained 49.9% of the variance in intention to prevent weight gain. The results of the likelihood ratio test show that the inclusion of the variables at step 3 significantly increased the explained variance compared with the previous models. In the final model only age, attitude and risk perception were significant predictors of the intention to prevent weight gain (Table 3 ): respondents were more likely to have a positive intention to prevent weight gain when they were older, had a more positive attitude to prevent weight gain and had higher overweight-related risk perceptions. Table 3 Correlates of intention to avoid weight gain based on stepwise logistics regression analysis (n = 979): odds ratios (OR), 95% confidence intervals (95%CI) and explained variance (Nagelkerke R 2 ). Step 1 (R 2 = 0.113) Step 2 (R 2 = 0.455) Step 3 (R 2 = 0.494) Predictor: OR (95%CI) Predictor: OR (95%CI) Predictor: OR (95%CI) BMI (20–30 kg/m 2 ) 1.30 (1.20–1.42) BMI 1.22 (1.11–1.35) BMI 1.04 (0.91–1.19) Sex (0 = man; 1 = woman) 2.34 (1.61–3.39) Sex 1.81 (1.13–2.89) Sex 1.00 (0.58–1.71) Age (25–35y) 1.07 (1.01–1.13) Age 1.09 (1.01–1.17) Age 1.12 (1.04–1.20) Education (1 = low; 2 = high) 1.29 (0.92–1.81) Education 1.26 (0.89–1.78) Education 1.19 (0.84–1.70) Ethnicity (1 = Non Dutch origin; 2 = Dutch origin) 1.04 (0.58–1.89) Ethnicity 1.22 (0.60–2.50) Ethnicity 1.23 (0.58–2.59) Attitude 10.10 (6.85–14.89) Attitude 7.91 (5.33–11.74) Subjective norm 1.24 (1.04–1.47) Subjective norm 1.17 (0.97–1.40) Perceived behavioural control 1.00 (0.78–1.27) Perceived behavioural control 1.12 (0.88–1.44) Descriptive norm 1.19 (0.99–1.45) Social support 1.10 (0.82–1.47) Risk perception 1.24 (1.11–1.38) Weight perception 1.36 (0.81–2.30) Differences in factors across PAPM stages of change Table 4 shows that a majority of the respondents reported that they already act to prevent weight gain. Significant differences were detected between the PAPM stages in almost all socio-demographic and psychosocial variables with the exception of educational level and descriptive norms. Respondents who were in the decided-to-act and the action stage were on average older, more likely to be female and had a higher BMI compared with respondents in earlier stages. Furthermore, the respondents who were in the decided-to-act and action stage had a more positive attitude towards prevention of weight gain, reported a higher subjective norm, perceived more social support to prevent weight gain, had a higher overweight-related risk perception and evaluated their weight as significantly higher compared to those in the earlier stages. Finally, the results showed that the respondents who were in the decided-to-act stage reported a lower perceived behavioural control towards prevention of weight gain than respondents in most other stages. Table 4 Significant differences between PAPM stages on socio-demographic and psychosocial correlates (n = 979) UE N = 109 (11.1%) UD N = 64 (5.1%) DN N = 80 (8.2%) DA N = 85 (8.7%) A N = 641 (65.5%) Significant differences* Demographic variables: Age 30.00 30.28 30.35 31.28 30.98 A>UE Sex (% women) 30.3 29.7 37.5 54.1 64.1 DA, A>UE, UD A>DN Education (% higher education) 40.4 60.9 45.0 47.1 53.0 - BMI 22.33 23.12 23.02 24.79 23.97 DA> UE, UD, DN A> UE, DN TPB constructs: Attitude -0.095 0.35 -0.079 0.42 0.83 A> UE, UD, DN, DA UD, DA >DN, UE Subjective norm -1.06 -0.88 -1.14 -0.28 -0.57 A, DA > UE, DN Perceived behavioural control 0.82 0.87 0.74 0.38 0.96 UE, UD, A > DA Other psychosocial constructs: Descriptive norm -0.028 -0.078 -0.063 0.035 0.31 - Social support -1.49 -1.42 -1.54 -1.02 -0.88 A> UE, UD, DN DA> UE, DN Risk perception 2.96 3.97 3.89 6.51 6.79 DA, A > UE, UD, DN Weight perception -0.46 -0.094 -0.22 0.46 0.23 DA, A > UE, UD, DN UD>UE *Statistical significant at P < 0.05 Note: UE = unengaged; UD = undecided to act; DN = decided not to act; DA = decided to act; A = action Discussion The present study shows that a majority of Dutch non-obese adults aged between 25 and 35 years had a positive intention to prevent weight gain. Age, attitudes and risk perceptions related to weight-gain were the strongest predictors of this intention. Further, a majority of the respondents reported that they were already acting to prevent weight gain and respondents in the higher stages of change reported more positive psychosocial factors related to weight-gain prevention than respondents in earlier stages of change. However, respondents who had decided to act to prevent weight gain reported low perceived behavioural control, suggesting that low control was a barrier towards action. The study indicates that in order to motivate the minority with non-positive intentions to prevent weight gain, interventions should focus on attitude change and communication about the risks of weight gain and overweight. In order to help motivated people in the study population to act on their intentions, it may be important to increase perceived behavioural control. These results are in line with earlier studies in differences on psychosocial factors between stages of change for smoking cessation [ 32 ], fat reduction [ 33 ], and increasing fruit and vegetable consumption [ 34 ], that also concluded that attitudinal information and risk information should be provided in earlier stages of change to increase motivation, while self-efficacy or control information can assist people to move to the action stage. The importance of perceived behavioural control is also highlighted in studies on weight loss [ 35 , 36 ]. Our results further show that people who have decided to act have higher BMIs than people in earlier stages of change, which shows that people who have more reason to act may also be more ready for action. However, this result is not supportive to the 'prevention of overweight' goal, since it indicates that people become more inclined to prevention of weight gain when they already experience some overweight. The fact that women were more likely to be in later PAPM stages reflects results from earlier studies that show that women are more weight conscious [ 37 , 38 ]. The result from a secondary t -test analyses showed that women in the present study were significantly more weight concerned compared to men, which may also explain why the association of sex with the intention to prevent weight gain was non significant after adjustment for weight perception. The present study shows that a large majority of respondents regarded themselves as acting to prevent weight gain. However, this does not tell us if they have been successful in their self-perceived actions and if their actions would prevent weight gain in the future. Studies conducted in the Netherlands have shown that the prevalence of overweight is still increasing [ 1 ]. Taking this trend into account, the results of this study suggest that it is likely that respondents overestimated their actions to prevent weight gain or that they applied less effective strategies to balance their energy intake and expenditure. The present study further shows that about a quarter of respondents were still in the earlier stages of behaviour change. They may have perceived less need to prevent weight gain, since most of the respondents do not yet have a weight problem. This illustrates the challenge of prevention of weight gain: we need to encourage the non-overweight population to be attentive to and to act to prevent gradual changes in weight. For interpreting the results of this study, several limitations should be acknowledged. Firstly, the present study relied on self-reports, which may be subject to social desirability bias. No objective assessments of energy balance related behaviours were included in our measurements. Earlier studies show that errors in self-reporting increases directly with the magnitude of overweight and with age after the age of 45 years [ 39 , 40 ]. Since we studied a non-obese younger population group, self report-bias may have been limited but cannot be ruled out. Our measures of TPB and PAPM constructs were in accordance with those used in earlier studies [ 15 , 26 ], which were further pilot tested in a small sub-sample of the study population and submitted for review to an expert panel. For the present study, we framed all questions as related to 'watching your weight in order to prevent weight gain'. This formulation may not have been specific enough to embrace the complexity of energy balance related behaviour. Furthermore, some measures were based on few or single item assessments, which are more likely to have limited reliability. For future intervention development it will be more informative to investigate indirect measures of behavioural determinants, e.g. to identify what sort of specific beliefs may underlie people's weight gain prevention attitudes and control beliefs. For example, other studies indicate that people believe that low fat diets (an illustration of a specific weight control behaviour) are more expensive, more difficult to prepare and that eating less fat is difficult in general or in certain specific situations such as in the weekends and holidays [ 41 - 43 ]. In order to tailor education interventions, qualitative methods such as interviews or focus groups are needed to gain more knowledge about existing underlying salient beliefs of the target population [ 18 ]. This to determine which beliefs have to be changed by introducing new salient beliefs or by reinforcing existing beliefs [ 44 ]. Furthermore, we applied a cross-sectional research design and therefore were only able to study associations, and not causation [ 45 ]. Despite this limitation, results of meta-analyses revealed that TPB accounts for about 41% of the variance in intentions and 34% of the variance in behaviour across a variety of health-related behaviours [ 16 ]. These findings may establish to the credibility of behavioural intentions as the main predictor for weight maintenance-actions. Further, we also have to acknowledge limitations of the analyses since so many (85%) of the whole sample was found as having a positive intention. Because of this high percentage, we conducted additional analyses with more conservative data by classifying only those answering 'yes, certainly' as having a positive intention. The results of these analyses revealed that in the final step of the regression model, additional to the variables 'attitude' and 'risk perception' the variables 'sex', 'social support' and 'weight perception' became significant predictors of the intention to prevent weight gain. This means that respondents were also more likely to have a positive intention to prevent weight gain when they were female, be supported more by others and when they rated their weight as higher. For intervention development, these variables may be taken into consideration in order to motivate people who already have positive intentions to prevent weight gain, but who are still undecided on whether they will take action in the near future. Finally, the present study included only relatively few respondents in the earlier stages of behavioural change, and thus statistical power to detect differences between these stages was low. We therefore were not able to show with the present study that the PAPM was more useful than the TTM in order to investigate prevention of weight gain. Nevertheless, the PAPM might give additional information compared to TTM assessments when it is applied to more specific weight related behaviours. Our study population was a random sample of non-obese 'young' adults in the age group of 25–35 year. The mean BMI of our study population was indeed identical to that of the Dutch population aged 25–35 at large, although women were slightly over-represented and the respondents in this study were somewhat higher educated compared with the source population [ 46 , 47 ]. However, this is unlikely to have serious consequences for the generalisability of the findings, as results of the present study show that sex and educational level were not significant correlates of the motivation to prevent weight gain. Maybe the most striking result of this study is the high proportion of positive intentions to prevent weight gain and the high prevalence of respondents who reported to act in order to prevent weight gain. In this age group, weight gain is highly probable. The present study indicates that this occurs in spite of high motivation and self-perceived actions to avoid gaining weight. Andajani-Surjahjo and colleagues recently reported a lack of motivation related to physical activity and healthy eating for weight maintenance among women in the US [ 48 ]. This may illustrate the issue that people are motivated to avoid weight gain, but less motivated to engage in more specific actions that help to accomplish weight management. For a further understanding of the correlates of prevention of weight gain we need to investigate motivational factors (e.g. attitudes; subjective norms etc) related to specific weight-management actions such as 'avoiding calories', limiting the amount of food, and participation in regular physical activities [ 49 ]. Furthermore, we should not restrict our investigations to motivational factors. In the last decade, a number of reports have been published arguing that our present society fosters an 'obesogenic environment', i.e. a physical and social environment that promotes over-eating and discourages physical activity, and that such an environment may provide too high a barrier to avoid becoming overweight, despite high prevention motivation [ 50 ]. Further research is needed to investigate which energy balance related behaviours can help people deal better with the 'obesogenic environment' and may enable them to effectively act on their positive weight-gain prevention intentions. Competing Interests The author(s) declare that they have no competing interests. Authors' Contributions BW conceptualised the study, performed the statistical analyses, and drafted the manuscript. SK and BB both participated in the conceptualisation of the study, interpretation of the results and helped to draft the manuscript. JB conceived the study and assisted in the interpretation of the results and drafting the manuscript. All authors read and approved the final manuscript.

D:\keerthana\PMC001xxxxxx\PMC1079920.xml

1079921

Lumping or splitting: seeking the preferred areal unit for health geography studies

Background Findings are compared on geographic variation of incident and late-stage cancers across Connecticut using different areal units for analysis. Results Few differences in results were found for analyses across areal units. Global clustering of incident prostate and breast cancer cases was apparent regardless of the level of geography used. The test for local clustering found approximately the same locales, populations at risk and estimated effects. However, some discrepancies were uncovered. Conclusion In the absence of conditions calling for surveillance of small area cancer clusters ('hot spots'), the rationale for accepting the burdens of preparing data at levels of geography finer than the census tract may not be compelling.

Background The geographic study of cancer patterns can be an important tool in disease control and prevention [ 1 ], as well as a resource for generating hypotheses about pathogenesis [ 2 ]. Unfortunately, there is little practical guidance available as to whether or how to select an 'ideal' level of geography for surveillance of events with distinctive spatial autocorrelations [ 3 ]. Designating the geo-spatial locations of health events (i.e., 'geocoding') so as to be accurate (within acceptable error), precise (to a desired areal unit of analysis) and 'fit for use' (applicable to other available data) [ 4 ] can be vexing, even for those with great skill and experience [ 4 - 12 ]. On the one hand, small areal units containing few at-risk subjects will yield less reliable rates than larger units, whereas on the other hand, large areal units have potential to blur meaningful variation occurring within locales. Communicating and interpreting results that disentangle underlying risks from methodological artifact is important for public health workers and epidemiologists alike. Procedures for spatial analyses of suspected cancer 'hot spots' [ 13 ] may be unnecessary and even inappropriate [ 14 ] regarding studies of rate variation across large areas [ 15 ], as well as those intended to evaluate resource allocations [ 16 , 17 ]. At the same time, concerns to protect confidentiality of geographically referenced health data by those entrusted to collect and manage surveillance data may effectively eliminate some options for analysis. While the underpinnings of the 'modifiable areal unit problem (MAUP)' have been well described [ 18 , 19 ], there is neither guidance to effectively deal with the problem nor few real examples of whether or how differing aggregation units affect actual results. Armheim's treatment of simulated data suggests fewer disparities across findings with greater aggregation of data [ 19 ]. Krieger et al. , examining all-cause and selected cause-specific mortality and cancer incidence rates across Massachusetts and Rhode Island, found analyses by block group and census tract performed comparably [ 20 ], although tract-level analyses were found to offer greater linkage to area-based socio-economic indicators [ 21 ]. Sheehan et al . reported few differences for town, zip code or census tract-level analyses of breast cancer incidence across Massachusetts, but noted case counts fluctuated due to various geocoding problems [ 22 ]. Here, we address the problem of modifiable areal units while examining breast and prostate cancer incidence during a 5 year interval (1988–92) across Connecticut. Initially, we utilized geographically referenced data furnished us by the CT Tumor Registry to consider differences of incidence and late stage cases according to town and census tract. Evidence of either global or local clustering was evaluated using Oden's Ipop [ 23 ] and the spatial scan statistic [ 24 ]. Subsequently, we independently ascertaining census block group and exact latitude-longitude coordinates of recorded cases to consider whether greater precision of location modified/enhanced initial findings. Results Prostate cancer incidence Table 1 displays summary information for results of the Ipop global clustering test. Examining records aggregated by town, tract, or block group, the Ipop results indicated significant non-random clustering of cases throughout the state. Regardless of the analytic unit considered, approximately 40% of spatial clustering of prostate cancer incidence is attributed to the comparability of occurrences 'among' adjacent geographic locations, with any remaining clustering attributable to the incidence of cases within the given geographic units. Table 1 Ipop global clustering. Case count correlations for the geographic distribution of invasive and late-stage prostate or breast cancer incidence within or among selected areal units of analysis, Connecticut, 1988–92. Areal Unit Within % Among % p-value Prostate Cancer Incidence Block Group 60.0 40.0 0.0002 Tract 60.1 39.9 0.0002 Town 57.4 42.6 0.0002 Breast Cancer Incidence Block Group 72.1 27.9 0.0002 Tract 63.7 36.3 0.0002 Town 75.8 24.2 0.0002 Late Stage Prostate Cancer Incidence Block Group 99.7 0.3 0.0008 Tract 87.9 12.1 0.2190 Town 77.0 23.0 0.2740 Late Stage Breast Cancer Incidence Block Group 100.7 -0.7 <0.0001 Tract 100.2 -0.2 0.0002 Town 89.1 10.9 0.0846 Table 2 displays the latitude-longitude coordinates, approximate size, population at risk, numbers of cases and ratio of observed-to-expected cases for locations deemed likely clusters by the spatial scan statistic. Distances between the geographic coordinates of clusters identified for block group-level analyses (reference) and those by town, census tract or individual case coordinates are noted. The spatial scan statistic identified locales throughout the State, Depicted in Figure 1 , with potentially significant clustering of prostate cancers. Analysis by block group found four distinct locations with greater than expected incidence, findings for the tract level analysis identified two places and town level results indicated one significant site. The most likely locations for each level of analysis (i.e., primary clusters) depicted as shaded areas are common to North Central Connecticut (centroids of identified areas differed only by 11.1 km) with nearly identical ratios of observed-to-expected cases. The cluster identified at the town level appears more than 4-times the area of those based on census tracts or block groups, although it is much more comparable regarding the respective populations-at-risk (only 20% larger than others) and numbers of cases (11% difference). Additional locations where incidence was determined to be markedly greater than chance (i.e., secondary clusters depicted empty circles), were found in the southwest when analyzed by block group and southeast according to tract-level analysis. There were no significant secondary clusters based on the town-level analysis. Table 2 Spatial scan statistic clusters. Approximate locations with elevated invasive and late-stage prostate or breast cancer incidence according to selected areal units of analysis, Connecticut, 1988–92. Geocoded Records Coordinates (Lat.; Long.) Area (km 2 ) Population at-risk Cases in Cluster 0/E Distance (km) p-value Prostate Cancer Incidence Block Group 9,028 1 41.834; -72.727 1,504 254,092 2,651 1.28 Ref. <0.0001 2 41.311; -72.878 0 345 19 7.62 <0.0001 3 41.472; -73.225 0 148 12 6.97 0.0060 4 41.497; -73.218 0 97 13 5.32 0.0316 Tract 9,825 1 41.823; -72.735 1,297 238,007 2,673 1.26 1.4 <0.0001 2 41.463; -72.153 0 1,461 33 3.35 <0.0001 Town 10,054 1 41.995; -72.454 6,104 286,450 2,947 1.22 11.1 <0.0001 Breast Cancer Incidence Block Group 1 11,753 41.182; -73.510 573 85,084 952 1.22 Ref. 11,753 2 41.797; -72.775 115 27,066 391 1.31 0.0048 Tract 10,924 1 41.137; -73.391 854 147,066 1,554 1.21 11.2 <0.0001 2 41.787; -72.660 0 15 6 162.03 <0.0001 3 41.707; -72.647 87 32,358 401 1.26 0.0228 4 41.795; -72.756 64 20,737 284 1.31 0.0305 5 41.894; -72.368 0 2,137 34 2.34 0.0445 Town 12,518 1 41.960; -73.311 85 402 24 5.85 88.2 <0.0001 2 41.122; -73.346 584 87,795 1,009 1.14 0.0160 Late Stage Prostate Cancer Incidence Place of Residence 7,672 1 41.486; -73.065 2,057 1,651 549 1.19 2.0 0.0070 2 41.061; -73.458 65 72 41 2.04 0.0135 Block Group 7,672 1 41.501; -73.078 2,218 1,696 563 1.19 Ref. 0.0029 2 41.054; -73.478 44 61 35 2.05 0.0246 Tract 8,346 1 41.480; -73.075 1,895 1,596 541 1.22 2.4 <0.0001 Town 8,514 1 41.489; -73.052 1,959 1,932 644 1.19 2.5 <0.0001 Late Stage Breast Cancer Incidence Place of Residence 10,227 No significant clusters detected Block Group 10,227 41.666; -72.776 center16 105 center68 1.61 Ref. 0.0092 1 Tract 10,395 No significant clusters detected Town 11,854 No significant clusters detected Figure 1 Prostate cancer incidence. Geographic variation of prostate cancer incidence according to town, census tract and census block group units, Connecticut 1988–92. Primary clusters are indicated by solid circles and the statistically significant secondary clusters by hollow circles. Breast cancer incidence Significant global clustering was found at each level of analysis. According to Ipop test results, the percent of incident breast cancer cases clustering among geographic units was somewhat less than that for prostate cancer in results for town (24.2% vs. 42.6%) or block group (27.9 vs. 40.0%), but similar when examined according to census tract (36.3% vs. 39.9%). The spatial scan statistic applied to block group level data found two distinct locations, depicted in Figure 2 , with greater than expected breast cancer incidence, findings for the tract level analysis found five locations and town level results indicated two locations of possible clustering. There was good agreement regarding proximity and extent of risk between analyses at the block group and tract-level which identified Southwest Connecticut as the most likely location for clusters of incident breast cancers. Analysis by census tract identified a potential cluster with 49% greater area, 63% more cases and 73% larger population at risk than results for analysis by bock group. Those findings, by comparison, differed noticeably from the town-level analysis that identified the primary incidence cluster as single Northwestern Connecticut town (88 km from the center of the most likely cluster identified at the block group level) with a nearly 6-fold ratio of observed-to-expected cases. The town-level analysis yielded a secondary cluster with the locale of primary clusters found by the block group and tract analyses. The latter analyses, in turn, produced significant secondary clusters within North Central Connecticut. Figure 2 Breast cancer incidence. Geographic variation of breast cancer incidence according to town, census tract and census block group units, Connecticut 1988–92. Primary clusters are indicated by solid circles and the statistically significant secondary clusters by hollow circles. Proportion of late stage prostate cancer Results of the Ipop statistic for tract and town level analyses did not reveal global clustering of late-stage prostate cancers, but significant, albeit minimal clustering (i.e., only 0.3% of clustering was attributed to cases adjacent block groups) was indicated in analysis by block group. The spatial scan statistic using data for exact location of residence found two locations with proportions of late-stage cases significantly exceeding the statewide level; cases aggregated by block group revealed two locations while tract and town level analyses each produced one significant location. Results for primary clusters analyzed according to block group, tract, town, and exact place of residence, as illustrated in Figure 3 , yielded results with remarkable comparability regarding approximate location, affected areas, populations at risk, case counts and estimated effects. Figure 3 Late stage prostate cancer incidence. Geographic variation in proportion of late stage prostate cancer diagnoses according to town, census tract, census block group and exact place of residence units, Connecticut, 1988–92. Primary clusters are indicated by solid or hatch marked circles and the statistically significant secondary clusters by hollow circles. Proportion of late stage breast cancer Significant, but slight, global clustering of late stage breast cancer was found for analyses at the block group and tract level, but no clustering was found when analyzed according to town. According to Figure 4 , the spatial scan statistic was consistent in not locating statistically significant clusters with high proportions of late stage disease when cases were analyzed according to exact place, census tract or town of residence. However, analysis by block group found one area of Central Connecticut where late stage cases were 1.61 more likely among diagnosed cases than elsewhere around the State (p < 0.05). Figure 4 Late stage breast cancer incidence. Geographic variation in proportion of late stage breast cancer diagnoses according to town, census tract, census block group and exact place of residence units, Connecticut, 1988–92. Discussion Spatial analysis of health necessarily addresses issues about the accuracy of geocoded data, the requirements of time and training necessary to complete tasks, the threats to protecting confidentiality of sensitive health records and the interpretability of results for given areal units of analysis. Desire for greater precision challenges data safeguards as well as the technical capacity of available GIS systems. Surveillance by aggregating records into large areal units will yield greater proportions of accurate and protected records but possibly at the expense of capacity to identify discrete locales with elevated rates/proportions of health outcomes [ 16 ]. Our effort to contrast geographic analyses of prostate and breast cancers according to differing aggregation units across Connecticut yielded much, but not complete, consistency across analyses. Like others [ 20 , 22 ], we found in most instances that results obtained by block group level data mirrored those based on the census tract. As such, interpretations based on geocoded data available through the CTR were not appreciably enhanced by our further efforts to specify finer levels of geography. Global clustering of incident prostate and breast cancer cases was apparent for either level of geography and the test for local clustering found approximately the same locales, populations at risk and estimated effects. On the other hand, some discrepancies were uncovered. Secondary cluster locations varied by level of analysis. More importantly, analysis of breast cancer incidence by town yielded an approximate location of a significant primary cluster some distance from results based on block group or tract. It is possible that discrepancy is not a product of analytic scale but the consequence of differing ability to geocode records across all locales [ 25 ]. Test of this hunch requires analyses whereby cases excluded from one level of analysis would be excluded from all other analyses. As our intention was not a pure test of MAUP but a 'simulation' of the choice investigators might confront when selecting between a geographically referenced files in hand (CTR generated) or one independently created using original address data, we did not pursue this line of inquiry here. The local tests for late stage prostate cancer produced similar findings of significant clustering for analysis by exact coordinates, block group, tract or town, whereas results for the global clustering test were not significant for all but the block group analysis. Significant global clustering of late stage breast cancer was found using block group or tract, but not town or exact coordinates; significant local clustering was found only for the block group level analysis. Divergence across analyses could reflect distinctions among the levels of aggregation or merely subtly differences in the relative size of our data sets. It is noted that analysis of disaggregate (point) data raise issues separate from those specific to MAUP which we specifically address in this paper. It goes without saying that statistical procedures predicated on disaggregate (point) data would be unavailable if only aggregate files were available [ 26 ]. When analyzing geographic health data, concern regarding scale effects attributable to MAUP is unavoidable. Increased aggregation of data reduces power to detect very small clusters but stabilizes rate estimates. For now, the magnitude and direction of artifact generated by a given areal unit cannot be reliably predicted. Consequently, analysts will continue to be driven to select a preferred areal unit for analysis based on pragmatic rather than scientific consideration. In the absence of conditions calling for surveillance of small area cancer clusters ('hot spots'), the rationale for analysts to accept the technical, political and substantive burdens of preparing data at levels of geography finer than the census tract may not be compelling. The added protections to personal health data, the ease of interpretation and the applicability of similarly structured census and survey data organized argues for geographic studies to prioritize census tract level analyses. Methods The geographies of breast and prostate cancer incidence in Connecticut, 1988–1992, were evaluated in relation to the State's populations-at-risk within towns, census tracts and block groups for 1990 (1,160,886 males, and 1,282,917 females 20+ years of age according to seven age-categories: 20–29 years, 30–39, 40–49, 50–59, 60–69, 70–79, 80+) [ 27 ]. The at-risk populations are predominantly white (89.1%) and concentrated along Connecticut's southern shoreline and central river valley; eastern and northwestern sections of the State are considerably less densely populated. As shown in Table 3 , Connecticut's population is spatially organized within its 12,550 square mile area according to, 169 municipalities (towns), 834 census tracts and 2,905 block groups, as well as 50,569 census blocks, 330 zip codes, eight counties and two telephone area codes. Table 3 Spatial and population characteristics of selected areal units of Connecticut. Unit Places Area (sq. km) 1990 Population 20 & Over Persons 20 + years per sq. mile State 1 12,550 2,443,803 195 County 8 956 to 2,383 72,931 to 635,829 54 to 382 Town 169 13 to 160 443 to 100,552 6 to 2,426 Zip code 263 0.5 to 249 19 to 45,623 9 to 3,943 Census tract 834 <0.01 to 160 0 to 7,507 6 to 9,077 Block group 2,905 <0.01 to 86 0 to 5,415 0 to 21,333 Census block 50,569 Not available 0 to 2,796 Not available Between 1988 and 1992, the Connecticut Tumor Registry (CTR) recorded incidence and stage of diagnosis of 10,054 invasive cancers of the prostate (ICD-9-185) and 12,518 breast cancers (ICD-9-174) among State residents. The Institutional Review Boards of the University of Connecticut and Connecticut State Department of Public Health approved our access to, and analysis of information reported here. Geographic analyses of incidence by town and census tract were based on geographically referenced data files provided to us by the CTR. Every record identified an individual's town of residence and most assigned a census tract of residence to records (98% of prostate and 87% of breast cancer records). Total case counts are presented in Table 4 . Why some records were not assigned census tract identifiers by the CTR could not be determined here. Table 4 Geocoding of incident prostate and breast cancer cases, Connecticut, 1988–92. Prostate Cancers Breast Cancers Cases % Cases % Incident cases with town of residence recorded by the Connecticut Tumor Registry (CTR) 10,054 100 12,518 100 Census tract of residence recorded by CTR 9,825 98 10,924 87 Geocoded block group & street address of residence 9,207 92 11,864 95 Geocoded street address on 1 st try (stringent criteria) 4,546 5,926 Geocoded street address on 2 nd try (relaxed criteria) 4,661 5,938 Nursing home resident excluded for analysis by block group and exact coordinates 179 111 Record not geocoded 847 8 654 5 Post Office box listed 178 64 No street address listed 216 534 No house number listed 176 23 Listed address unable to geocode 277 33 To examine if geographic patterns of cancer incidence and late stage change at finer units of analysis, we subsequently used the full street address available within the CTR record to independently assigned latitude-longitude coordinates to census block group and place of residence for 9,207 prostate (92%) and 11,864 breast (95%) cancer records. Our purpose was neither to augment nor correct the CTR data, but to generate separate geographically-referenced files to study cancer patterns according to aggregation units otherwise unavailable to external researchers. This accounts for the seemingly incongruous observation that 11,753 records were geocoded (by us) to block group whereas only 10,924 records were geocoded (by CTR) to tract. The result of our effort, vis-à-vis data provided by the CTR, is summarized in Table 4 . As there is no 'gold standard' available to validate geocoded results, no effort was made to enumerate or resolve ambiguities that could be noted if files were directly compared. Approximately one-half of records geocoded in this manner were categorized using stringent coding criteria (i.e., an address conforms completely to a street location recognized by geocoding software); the remainder were completed using 'relaxed' procedures (i.e., an address bearing one or more incongruities was assigned to the 'most likely' street location by the geocoding software) [ 28 ]. We were unable to geocode 847 prostate and 654 breast cancer records because only a Post Office box was available, no street or house number was recorded or the recorded address could not be matched to a recognized street location. Records for individuals with addresses associated with nursing home were not included in this phase of analysis (179 prostate and 111 breast cancer records, respectively); leaving totals of 9,028 prostate cancers and 11,753 breast cancers for study. Numerous tests for spatial randomness (i.e., are geographical patterns due to random fluctuations/chance or true underlying variability?) are available [ 29 ]. For purposes of illustration, we selected one global clustering and one cluster detection test to evaluate geographic variations of disease rates. Oden's Ipop [ 23 ] indicates whether there is an overall pattern of spatial aggregation of cases throughout the study region, without regard to specific locations where aggregation might occur. Group data are used to generate a weighted correlation coefficient, adjusted for population size, that indicates the extent to which case counts within given locations are associated with values of neighboring locales (i.e., are places with high frequencies adjacent to places with similarly high frequencies?). The significance of the computed value is evaluated in relation to an expectation derived by a hypothetical null spatial distribution of data. Oden's Ipop was calculated using ClusterSeer v2.06 software [ 30 ]. The spatial scan statistic [ 24 ] looks for significant concentration of cases at specific locations within a study region without preconceptions about where concentrations might be found. The spatial scan statistic utilizes scanning circles of varying location and size so as to contain 0–25% of the State's population at risk to identify places where the number of observed cases exceeds expectation under a null hypothesis that incidence is proportional to population density. The spatial scan statistic was calculated using SaTScan 3.1 [ 31 ]. Among the available address matched records, 9,207 (92%) prostate cancer and 11,864 (95%) breast cancer records contained sufficient information for geographic analyses of 'late stage' disease across the State. Historical SEER summary stage classifications [ 32 ] were used where regional/distant prostate or breast cancers were noted among 2,198 (28%) and 4,119 (40%) records, respectively. Analyses of geographic distribution of disease stage (regional/ distant versus local) using Oden's Ipop and the spatial scan statistic were completed according to town, census tract and census block group of residence. The spatial scan also was applied using exact place of residence coordinates of cases; because necessary group boundaries for discrete residential locations are unavailable, Oden's Ipop could not be used with individual coordinates. Maptitude 4.5 software [ 28 ] was used to map cluster locations with markedly high incidence rates (Figures 1 and 2 ) or proportions of late-stage disease (Figures 3 and 4 ). Authors' contributions DG and MK conceived of the study; DG supervised all aspects of its implementation. LD and HS assisted with the study and completed the analyses. MK assisted with the study and analyses. DG, LD, and HS participated in drafting the manuscript. All authors helped review the manuscript and approved the final version.

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1079922

On geography and medical journalology: a study of the geographical distribution of articles published in a leading medical informatics journal between 1999 and 2004

Background Studying the contribution of individual countries to leading journals in a given discipline can highlight which countries have the most impact on that discipline, and also give some idea about the geographical outreach of those journals. This paper examines the number of countries that contributed articles to one leading medical informatics journal, Medical Informatics & the Internet in Medicine, and the amount of their contributions between 1999 and the first half of 2004. Methods The PubMed citations of all indexed articles from the chosen journal (n = 128) were retrieved online (up to Volume 29, Number 2/June 2004 issue, the latest indexed issue as at 28 January 2005). The country of corresponding author's affiliation for each retrieved citation was recorded. The five-year-and-half corpus of abstracts retrieved from PubMed was further explored using MetaCarta Geographic Text Search . Results The examined journal has an international outreach, with authors from 24 countries, spanning four continents, contributing to the journal during the studied period. The journal is dominated by a very large number of articles from Europe (81.25% of all articles counted in this study), and in particular from the UK (15.63%) and Greece (15.63%). There were no contributions from Africa or South America. Conclusion A detailed discussion and interpretation of these results and ideas for future analyses are provided. MetaCarta can prove very useful as a bibliometric research tool.

Background The study of the geographical distribution of journal publications as an indicator of the research output of individual countries has become a field of interest [ 1 , 2 ]. Studying the contribution of individual countries to leading journals in a given discipline can highlight which countries have the most impact on that discipline, and also give some idea about the geographical outreach of those journals. Medical Informatics & the Internet in Medicine (MEDLINE Abbreviation: Med Inform Internet Med) is one of the most prestigious journals in the field of medical and health informatics, with a 2001 Impact Factor of 1.909 (ISI 2001 Journal Citation Reports-Science Edition – . In this paper, we examine the number of countries that contributed to Med Inform Internet Med and the amount of their contributions between 1999 and the first half of 2004. We also demonstrate the use of MetaCarta Geographic Text Search appliance (GTS – to sift through the corpus of Med Inform Internet Med abstracts from the same period through a geographical 'lens'. Founded by a team of MIT researchers in 1999 with funding from the US CIA, MetaCarta bridges the gap between Geographical Information Systems (GIS) and text search, allowing users to link information to geography and to discover geographical themes within their documents. MetaCarta GTS scans documents and extracts geographical references, intelligently handling any geographical name ambiguities or inconsistencies it encounters. Users can search a geo-parsed document collection for the occurrence of any keywords (geographical or otherwise). GTS presents search results as icons on a map of the region of interest. The results are linked to the full documents. Users can further narrow searches by zooming in to specific geographical locations (the map acting as a filter), and by modifying their query string. Methods The US National Library of Medicine PubMed service was used to retrieve the PubMed citations of all indexed articles from Med Inform Internet Med as at 28 January 2005 (query string: 'Med Inform Internet Med [jour]'). The country of corresponding author's affiliation for each retrieved citation was recorded. If the affiliation of an entry could not be obtained from PubMed, it was looked up in the online version of the journal. A simple 'pen and paper' and Microsoft Excel analysis of the country of provenance of all retrieved citations was performed. A MetaCarta GTS online demo account was created . The abstracts of all retrieved citations from PubMed were saved as individual HTML pages, one for each abstract. These were then compressed into a single zip archive and uploaded as a document collection to the MetaCarta demo account. Results From 1999 (Volume 24, Number 1/March 1999 issue) to 2004 (Volume 29, Number 2/June 2004 issue) 128 articles from 24 countries were published in Med Inform Internet Med (Tables 1 and 2 ). Table 1 Country contributions to Med Inform Internet Med Countries that contributed to Med Inform Internet Med and the amount of their contributions between 1999 and the first half of 2004. Country Number of papers (1999 – June 2004) Australia 1 Cuba 1 Denmark 1 Hong Kong 1 India 1 New Zealand 1 Poland 1 Singapore 1 Slovenia 1 The Czech Republic 1 Taiwan 2 Israel 3 Finland 4 Austria 5 Spain 5 Sweden 5 USA 7 Japan 6 The Netherlands 6 France 7 Italy 13 Germany 15 Greece 20 UK 20 Total: 24 countries 128 papers Table 2 Breakdown per year of Med Inform Internet Med papers and country contributions Breakdown per year of Med Inform Internet Med papers, contributing countries, and top countries and their individual contributions. Year Number of papers Number of Countries Top countries (number of papers) 1999 24 12 UK (6), Greece (4) 2000 20 9 Greece (5), UK (3) 2001 23 10 Germany (7), Greece (3), USA (3) 2002 24 14 UK (4), Greece (3), Italy (3) 2003 22 10 Greece (5), UK (4) 2004 (first 2 issues) 15 12 Austria (2), Italy (2), USA (2) Authors from the UK contributed 15.63% and their colleagues from Greece another 15.63% of all articles published in Med Inform Internet Med during the period between 1999 and June 2004. Authors from Germany and from Italy were responsible for 11.72% and 10.16%, respectively, of all articles during the same period (Figure 1 ). Figure 1 Top contributing countries to Med Inform Internet Med and their share in per cent . The leading countries in Med Inform Internet Med and their share in per cent during the period between 1999 and June 2004. The five-year-and-half corpus of Med Inform Internet Med abstracts retrieved from PubMed was further explored in MetaCarta by searching for the occurrence of different keywords in it and observing the results plotted on interactive geographical maps at various zoom levels. The search results are linked to the respective abstract pages, and the latter are usually linked to the full text of the articles on the publisher's Web site. Keywords can be: • a geographical name, e.g., 'Amsterdam' (Med Inform Internet Med 2003 Sep, 28(3):209-19) • an author's surname, e.g., 'Darmoni' (Med Inform Internet Med 2001 Jul-Sep, 26(3):165-78 and 2001 Oct-Dec, 26(4):325-30) • the name of a project, e.g., 'Hip-Op' (Med Inform Internet Med 2002 Jun, 27(2):71–83 and 2003 Mar, 28(1):59–71) • any other text string, e.g., 'electronic patient records' or 'NHS' (Figure 2 ), or Figure 2 Searching the corpus of Med Inform Internet Med abstracts (1999–June 2004) in MetaCarta . Searching the corpus of Med Inform Internet Med abstracts (1999–June 2004) in MetaCarta for the keyword 'NHS' yielded two abstracts, one mapped to Oxford, UK, with two occurrences of the word 'NHS' in it (Med Inform Internet Med 2003 Jun, 28(2):129-34) and the other mapped to York, UK, with only one occurrence of the same word (Med Inform Internet Med 1999 Jul-Sep, 24(3):223-9). • a combination of any of the above to narrow searches. Discussion Med Inform Internet Med has an international outreach, with authors from 24 countries, spanning four continents, contributing to the journal during the period between 1999 and June 2004, and contributions from at least nine countries to each yearly volume of the journal in the same period of time. However, the journal is dominated by a very large number of articles from Europe (104 or 81.25% of all articles counted in this study), and particularly from the UK and Greece (which together account for 31.26% of all articles), with a small number of articles by comparison from the USA (5.47%), Japan (4.69%), Australia and the rest of the world (and none from Canada). These findings might be partially explained by the fact that Med Inform Internet Med is a UK-based journal and also a European journal by geography (the UK being part of Europe). Previous results from other disciplines have also shown that a large proportion of papers originating from the UK appeared in British journals [ 1 ]. The observed geographical variation could be due to differences in the numbers of manuscript submissions from different countries and/or differences in rejection and acceptance rates for submitted manuscripts from different countries, with the possibility of some selection bias coming into play, e.g., reviewers (and perceived readership) preferring and evaluating more favourably papers from their own countries or some other country [ 1 , 3 , 4 ]. However, no data are available from Med Inform Internet Med to support or investigate such possibilities. There were no contributions from any African or South American country during the same period. For Africa, this might be partially explained by the developing nature of the economies and infrastructures of the continent and the dampening effects this has on the research funding and productivity of African countries, and on their access to the Internet and the latest scientific periodicals. The 'brain drain' phenomenon might also be a contributory factor, with many African scientists and researchers being attracted to the West and conducting and publishing their research under the umbrella of Western institutions. In South America similar factors operate [ 5 , 6 ], besides the fact that Spanish and Portuguese, rather than English, dominate the continent as the main spoken languages. The theory of a bias against publications from the developing world might also partially explain the lack of contributions from Africa and South America [ 7 ]. It is noteworthy that Africa and South America were found to have the lowest number of biomedical publications per million population per year in a study by Rahman and Fukui published in 2003 [ 8 ]. MetaCarta as a bibliometric research tool Information resources and large textual datasets can be organised and navigated based on their geographical attributes. These geographical aspects of textual information are sometimes very useful as an index to information, providing an intuitive way of accessing, mining, and understanding it [ 9 ]. In these respects, MetaCarta can prove very useful as a bibliometric research tool. For example, by searching the relevant corpus of literature, MetaCarta can help identifying potential collaborators in one's research area of interest from one's same geographical region or across the world (geographically-aware information retrieval). Metacarta geo-parses all geographical references in searched documents. For example, if an abstract by an author affiliated to a US institution is describing research that has been conducted in Germany and referring to related research conducted in Austria, then the abstract will be mapped to the three countries (USA, Germany and Austria). Although this could sometimes cause problems (if what one is looking for is to map or link each document to only one single location in some particular context), it could also prove a very powerful feature in other contexts. Ideas for future research Other ideas for future and better analyses of this kind include: • It is possible for some articles resulting from international cooperation to have authors from more than one country, but this study only considered the country of corresponding author's affiliation. It would be useful to consider this possibility in future studies. PubMed and MEDLINE only include the corresponding author's institutional affiliation and address, so the actual journal articles will have to be checked for the details of other authors. • Comparing the geographical distribution of the readership of Med Inform Internet Med (using data from subscriptions to the printed edition and the access logs of the online journal articles) and that of the articles it publishes might help interpreting some of the geographical variation in the latter, since authors usually select journals they can access and read or are at least aware of to publish their papers in. • Smaller affluent nations like Belgium, Finland, Israel and Sweden often have higher publication activity rates than larger countries like Germany, the UK and the USA, but this is not readily apparent when looking at the gross or absolute publication productivity numbers of the different countries. A good way around this is to normalise publication productivity data to population size or GDP of respective countries (publications per million inhabitants/year or publications per billion US dollars GDP/year) [ 10 - 13 ]. • It would be interesting to repeat the same study after five years and compare the results. Reference [ 14 ] provides a related example. • It would also be useful to see how Med Inform Internet Med compares to other leading and newer journals in the field of medical and health informatics regarding the geographical distribution of their published articles, and to see if there are any common trends across journals. • Also, breaking down the geographical distribution of articles published in a given journal or collection of journals by article/study type and by research topic/theme over the studied period of time could help revealing how medical and health informatics thinking and applications are evolving worldwide, as well as any regional themes. Reference [ 15 ] provides a related example. • Tracking multiple articles by the same authors and institutions covering same, similar or different research topics or projects could also prove useful in identifying evolving research trends, centres of expertise, existing collaboration nuclei (and potential collaborators) in different research areas from around the world [ 16 ]. • Studying the geographical distribution of editorial board members of leading medical and health informatics journals and relating the findings to the geographical distribution of articles published in the same journals could yield additional useful insight. References [ 17 ] and [ 18 ] provide two related examples. Conclusion This paper has provided a detailed discussion and interpretation of the results of a study of the geographical distribution of articles published in a leading medical informatics journal between 1999 and 2004. Studying the contribution of individual countries to leading journals in a given discipline can highlight which countries have the most impact on that discipline, and also give some idea about the geographical outreach of those journals. Ideas for future analyses were also presented. MetaCarta can prove very useful as a bibliometric research tool.

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1079923

Scale and shape issues in focused cluster power for count data

Background Interest in the development of statistical methods for disease cluster detection has experienced rapid growth in recent years. Evaluations of statistical power provide important information for the selection of an appropriate statistical method in environmentally-related disease cluster investigations. Published power evaluations have not yet addressed the use of models for focused cluster detection and have not fully investigated the issues of disease cluster scale and shape. As meteorological and other factors can impact the dispersion of environmental toxicants, it follows that environmental exposures and associated diseases can be dispersed in a variety of spatial patterns. This study simulates disease clusters in a variety of shapes and scales around a centrally located single pollution source. We evaluate the power of a range of focused cluster tests and generalized linear models to detect these various cluster shapes and scales for count data. Results In general, the power of hypothesis tests and models to detect focused clusters improved when the test or model included parameters specific to the shape of cluster being examined (i.e. inclusion of a function for direction improved power of models to detect clustering with an angular effect). However, power to detect clusters where the risk peaked and then declined was limited. Conclusion Findings from this investigation show sizeable changes in power according to the scale and shape of the cluster and the test or model applied. These findings demonstrate the importance of selecting a test or model with functions appropriate to detect the spatial pattern of the disease cluster.

Background Over the past several years, there has been an increased interest in the detection of focused clusters of disease, or disease clusters associated with a known pollution source [ 1 ]. Along with this interest, the development and use of various cluster detection methods have grown. Wartenberg and Greenberg [ 2 ] emphasized the importance of statistical power as a criterion in the selection of appropriate method for cluster investigations. A limited number of power evaluations in the literature have examined the issues of scale or shape with regard to focused disease cluster detection. As part of a more extensive evaluation, Sun [ 3 ] examined the power of Stone's [ 4 ] and Tango's Tests [ 5 ] to detect clusters of varying size. Waller [ 6 ] described two shapes of clusters, hot spot and clinal [ 2 ], and evaluated the power of Stone's and Besag and Newell's Tests to detect these types of clusters at varying levels of aggregation. Additional focused cluster shapes also have been proposed, such as a cluster of angular shape which could result from the effect of a dominant wind dispersing environmental contaminants [ 7 ]. However, this cluster shape has not been included in previous power evaluations. Additionally, information is lacking regarding the power of generalized linear models to detect focused clusters of varying scale and shape. This investigation evaluates the power of a number of focused cluster tests and generalized linear models to detect clusters of varying scale and shape. Count data are simulated for three total numbers of events (N = 200, 500 and 1000) to represent clustering around a single, centrally located pollution source in spatial patterns consistent with pollution dispersion principles. Results Focused cluster test results Data simulated with distance decline (DD) in relative risk The LRS Test for Distance Decline, Tango's Focused Test, Stone's Test and the Radial Score Test demonstrated the best power curves for the range of total simulated events ( N = 200, 500, 1000). Stone's Test showed slightly less power than the other best-performing tests; and for N = 500 and N = 1000 events the Radial Score Test performed with slightly lower power than Tango's Focused Test and the Radial Score Test. Power of 100% was reached only by Tango's Test ( τ = 1 and 5) and the LRS Test for Distance Decline in the N = 1000 simulation at α 1 = 2 (Figure 1 ). For all total event scenarios, power curves for the four best-performing tests were similar, with low power at lower values of α 1 (< 2), peaking at α 1 = 2 and decreasing slightly at α 1 = 10. For N = 500 and N = 1000, Cuzick and Edwards' Test reached modest power ranging from 30% to 50% at the highest values for α 1 . As opposed to the focused cluster tests that showed the best power curves, the power for Cuzick and Edwards' Test continued to increase up to the highest value of α 1 ( α 1 = 10) rather than declining slightly. All other focused cluster tests that were examined showed less power than those previously described. Figure 1 Power curves for DD with N = 1000 Data simulated with peaked distance decline (PDD) in relative risk The LRS Test for Distance Decline, Tango's Test, the Radial Score Test, Besag and Newell's Test, Stone's Test, and Cuzick and Edwards' Test showed the best power curves for PDD clusters. However only modest power was achieved (50%) for N = 200 by the focused cluster tests with the best power curves: LRS Test for Distance Decline, Tango's Test, and the Radial Score Test. Power increased with increasing events. Eighty percent power was achieved for N = 500, and 100% power was achieved for N = 1000 (Figure 2 ). As expected, power curves were similar to those for DD at the lowest values of α 2 ( α 2 < 0.5), with power increasing as α 1 increased until a slight drop at the highest α 1 value ( α 1 = 10). However as α 2 increased, the power curves changed. For N = 200, power curves for α 2 = 0.5 and α 2 = 1 were similar and showed the Besag and Newell's Test reached the highest power of any focused cluster test at lower values of α 1 . Power for this test declined as α 1 increased; whereas the power increased for Tango's Test, Stone's Test, the LRS Test for Distance Decline, the Radial Score Test, and Cuzick and Edwards' Test k = 7. For N = 500 and N = 1000 at α 2 = 0.5 and 1, the Radial Score Test demonstrated the highest power among all focused cluster tests at lower values of α 1 . At these same α 1 values, power for the Radial Score Test increased as α 2 increased to 1. The Besag and Newell's Test at k = 4 and 7 for N = 500 and at k = 7 and 10 for N = 1000 showed a trend similar to the Radial Score Test but with consistently lower power. Tango's Test and the LRS Test for Distance Decline showed the highest power, though still poor, at higher α 1 values. The power for these tests declined as α 2 increased to 1. None of the other focused cluster tests we examined performed as well. Figure 2 Power curves for PDD, α 2 = 1, with N = 1000 Data simulated with a directional effect (DIR) in relative risk The LRS Test for Direction demonstrated the highest power among the focused cluster tests we examined for detecting DIR clusters (Figure 3 ). The Directional Score Test showed similar power curves, reaching 100% power for each total event scenario. Besag and Newell's Test achieved modest power 30%, 40% and 40% respectively for N = 200 with k = 4, N = 500 with k = 10 and N = 1000 with k = 2. However other tests had less power. Generally, the tests showed less power at wider angles ( α 3 and α 4 < 0.2) and increasing power as the angle narrowed. Figure 3 Power curves for DIR with N = 1000 Data simulated with a distance decline and directional effect (DDIR) in relative risk Figure 4 demonstrates the power curves for N = 1000 at α 3 and α 4 = 0.5. Power curves at the lowest α 3 and α 4 values were generally similar to the DD power curves, and power curves at the highest α 3 and α 4 values resembled the DIR power curves. Overall, the LRS Test for Direction was the best-performing test with respect to power at α 1 < 1, and the Directional Score Test had similar power curves as α 3 and α 4 increased. Power for the LRS Test for Direction and for the Directional Score Test improved with increasing values of α 3 and α 4 . Both tests attained 100% power for lower α 1 values at α 3 and α 4 = 1 for 200 events and at α 3 and α 4 = 0.5 for N = 500 and N = 1000. For α 1 ≥ 1, Tango's Test and the LRS Test for Distance Decline, followed by the Radial Score Test and Stone's Test, showed the most power at lower α 3 and α 4 values. As α 3 and α 4 increased, these tests gradually increased in power, with all of these tests showing 100% power at the highest values for α 1 , α 3 and α 4 . For these higher α 1 values, power also improved for the Directional Score Test and the LRS Test for Direction as α 3 and α 4 increased. In most cases of higher α 1 values, the power for both of these directional tests matched or surpassed the power for Tango's Test, the LRS Test for Distance Decline, the Radial Score Test and Stone's Test at the highest values of α 3 and α 4 . Cuzick and Edwards' Test k = 7 and 10 also reached 100% power at the highest values for α 1 , α 3 and α 4 for N = 500 and N = 1000. Figure 4 Power curves for DDIR, α 3 and α 4 = 0.5, with N = 1000 Data simulated with a peaked distance decline and directional effect (PDDIR) in relative risk We begin by presenting the findings for lower values of α 2 ( α 2 = 0.05 and 0.1). Results for these two α 2 levels were similar to one another and are represented by Figure 5 . For N = 200, N = 500 and N = 1000, the best-performing tests for lower values of α 1 ( α 1 < 1) were the LRS Test for Direction followed by the Directional Score Test. Though at lower α 3 and α 4 values, the highest power achieved was poor. As α 3 and α 4 increased, the LRS Test for Direction and the Directional Score Test improved in power. Tango's Test and the LRS Test for Distance Decline, followed by the Radial Score Test and Stone's Test, demonstrated the most power at higher levels of α 1 ( α 1 > 0.1), increasing as α 3 and α 4 increased. However, at α 3 and α 4 = 0.5 and above, the LRS Test for Direction and the Directional Score Test generally demonstrated the highest power for all values of α 1 except α 1 = 10. At this α 1 value, Tango's Test, the Radial Score Test, Stone's Test and the LRS Test for Distance Decline were more powerful until α 3 and α 4 reached 2; at which point, other tests matched their power. For N = 500, Cuzick and Edwards' Test at k = 7 and k = 10 also reached 100% power at α 3 and α 4 = 2 and α 1 = 10. Power was low for all other values of these coefficients for this focused cluster test. Figure 5 Power curves for PDDIR α 2 = 0.05, α 3 and α 4 = 0.5, with N = 1000 Figure 6 represents the power curves for higher values of α 2 ( α 2 = 0.5 and 1), though results differed somewhat among the three total events scenarios. For N = 200, the LRS Test for Direction showed the best power overall. All focused cluster tests examined showed lower power for α 3 and α 4 = 0.05. But several tests improved as α 3 and α 4 increased to α 1 = 2. Besag and Newell's Test at k = 7 and 10 showed similar power to the LRS Test for Direction at lower values of α 1 , α 3 and α 4 . The Directional Score Test showed the second best power curve for values of α 3 and α 4 > 0.2. For each value of α 3 and α 4 , the Directional Score Test, the LRS Test for Direction, and Besag and Newell's Test declined in power as values of α 1 decreased. The Radial Score Test demonstrated an unusual U-shaped curve, most pronounced at α 3 and α 4 = 2. For N = 500 and N = 1000, the Radial Score Test showed the best power curve at α 3 and α 4 < 0.5, with power decreasing as α 1 values declined. As α 3 and α 4 increased from 0.5, the LRS Test for Direction demonstrated the highest power, followed by the directional score and Radial Score Tests. For each of these tests, power decreased as α 1 increased. Tango's Test and the LRS Test for Distance Decline showed an unusual trend of no power at values of α 1 under 10, then a sudden increase in power at α 1 = 10. This trend became more pronounced as α 3 and α 4 increased. Figure 6 Power curves for PDDIR, α 2 = 1, α 3 and α 4 = 0.5, with N = 1000 Focused cluster model results Figures 7 through 11 demonstrate that better power curves were achieved for larger numbers of events. Figure 7 shows the power curve resulting from comparing the alternative hypothesis model that included a function for a distance decline in relative risk to the null hypothesis model with null relative risk. Power was low when the α coefficients were small, peaked when α 1 = 2 and decreased slightly as α 1 increased to 10. For 200 events, low to moderate power was achieved; however, better power was evident for 500 and 1000 events. Use of the null model versus the model with a function of directional elevated relative risk showed power increasing with increasing values of α 3 and α 4 , or as the angle of effect narrowed (Figure 8 ). One hundred percent power was achieved in each of the three total event situations (N = 200, 500 and 1000). Figure 9 shows that power was inversely related to the α 1 coefficient value when α 2 was larger, or the peak was further from the source ( α 2 = 1). Power increased directly with α 1 values when α 2 was smaller, or the peak was closer to the source ( α 2 = 0.1). Only the 1000 event situation achieved moderate to high power. Overall, better power curves were evident for the comparisons between null models and the models that included a function for distance decline and directional relative risk (Figure 10 ). For all variations of total events, 100% power was achieved across all α 1 values for higher values of α 3 and α 4 ( α 3 and α 4 = 2). At α 3 and α 4 = 0.5, 100% power was shown for the 500 and 1000 event situations at lower α 1 values ( α 1 < 10), however power decreased at the highest value of α 1 ( α 1 = 10) for 500 and 1000 events. For 200 events, lower overall power was achieved, and power began decreasing immediately with increasing α 1 values. At the lowest α 3 and α 4 values ( α 3 and α 4 < 0.5), power increased directly with values of α 1 but decreased at α 1 = 10 for 500 and 1000 events. Results from the final null model comparison, the null model versus a model including a spatial function for peaked distance decline and direction, is shown in Figure 5 . For 1000 events and α 3 and α 4 = 2, the power of the test from the alternative model was more powerful; and the 500 event situation was similar, with power decreasing slightly at α 1 = 10. The overall trend for all three total event counts at α 3 and α 4 = 0.5 showed very good power at lower levels of α 1 ( α 1 < 10 for 500 and 1000 events, α 1 < 1 for 200 events), but power decreased with increasing values of α 1 . At α 3 and α 4 = 0.1a different overall trend was evident, with power generally increasing directly with values of α 1 . Power trends for α 2 = 1, for all α 3 and α 4 values and all total event counts, demonstrated an inverse relationship between power and α 1 value. Additionally, power increased with increasing values of α 3 and α 4 . Figure 7 Null model vs. DD model Figure 8 Null model vs. DIR model Figure 9 Null model vs. PDD model Figure 10 Null model vs. DDIR model Figure 11 Null model vs. PDDIR model, α 2 = 0.1 Comparison of the distance decline relative risk model to a model including a peaked distance decline function showed that no power was gained from adding the parameter for a peak. Power was less than 10% for all total event scenarios and variations of the α coefficients. However, better power curves resulted from other model comparisons. At values of α 3 and α 4 = 0.5 and 2, adding direction as a parameter to models with a function for distance decline vastly improved power for detecting clusters with distance decline and directional distributions. The increase in power was low for the addition of the directional parameter when α 3 and α 4 = 0.1. Similar trends were evident for the addition of parameters for a peak and for direction when compared to a distance decline only relative risk model. In general, power was high at all but the highest α 1 value ( α 1 = 10) when α 3 and α 4 = 0.5 and 2. Yet, improvements in power were not very large for lower levels of α 3 and α 4 ( α 3 and α 4 < 0.2). Under certain conditions, power greatly increased when parameters were added to a directional only model. When comparing a directional only model to a model with parameters for distance decline and direction, power tended to increase with increasing α 1 , α 3 and α 4 values. However a slight decrease in power was evident at α 1 = 10 for most model comparisons. The power curves were very similar to those for the comparison between a directional-only model to models with parameters for peaked distance decline and direction at lower values of α 2 ( α 2 < 0.5). At higher levels of α 2 ( α 2 > 0.1), power trends differed greatly, with the highest power at lowest levels of α 1 and greater decreases as α 1 increased to 10. Power curves from models with parameters for peaked distance decline were also compared to models with parameters for peaked distance decline and direction. Generally, power trends were similar for the two α 2 levels with power inversely related to α 1 value and directly related to α 3 and α 4 values. The improvement in power was very low for the addition of the directional component at the lowest α 3 and α 4 values ( α 3 and α 4 < 0.2). As with previous model comparisons, the addition of a parameter for peak generally resulted in low power when parameters for distance decline and direction were already in the model. The highest power achieved was modest (about 40%) and was evident only for 1000 events at the highest values for α 2 , α 3 and α 4 values ( α 2 = 1, α 3 and α 4 = 2) at α 1 = 1. Conclusion Focused cluster tests Based on the results presented, we found that Tango's Test, the LRS Test for Distance Decline, Stone's Test and the Radial Score Test showed the most power for detecting a significant difference in relative risk simulated with DD from a centrally-located pollution source at a fixed location. As expected, power increased directly with number of total cases of disease involved in the cluster. For these tests, power also tended to increase as the slope of the DD became steeper or, in other words, as the relative risk changed more rapidly with increasing distance from the pollution source. The results of our power evaluations are not surprising as the best-performing cluster tests for detecting radial clusters with DD were generally developed for detecting these cluster shapes. Power for detecting PDD clusters with N = 200 was generally low, with reasonable power demonstrated at N = 500 and N = 1000 for some combinations of peak distances from the pollution source and slopes of decline in risk. Evaluating the power of focused cluster tests to detect PDD clustering with peaks closest to the source revealed that the LRS Test for Distance Decline, Tango's Test, Stone's Test and the Radial Score Test showed the highest power among the tests evaluated. Power generally increased for these tests as the slope of the cluster became steeper. This trend reversed as peaks increased in distance from the pollution source so that power generally decreased as the slope became steeper. The Radial Score Test demonstrated the most power for detecting peaks furthest from the pollution source for N = 500 and N = 1000, while Besag and Newell's Test performed best for N = 200. Supporting our hypothesis that focused cluster tests containing functions appropriate to the spatial pattern of pollution dispersion would be more powerful, the LRS test for direction and the Directional Score Tests revealed the most power for detecting focused clusters with DIR. Power increased for these tests with increased number of total events and as the angle of effect became more pronounced. For data simulated with DDIR, the LRS Test for Direction performed best with wider angles of effect and the flattest declines in slope. But as the angle of effect became stronger, the LRS Test for Direction was powerful at all slopes. Overall, power to detect clusters with a DDIR distribution generally increased with narrower angles of effect and as the total number of events simulated increased. As the slope of the decline became steeper, Tango's Test, LRS Test for Distance Decline, Stone's Test and the Radial Score Test showed comparable power. Power for the Directional Score Test greatly improved with stronger angles of effect, becoming one of the best-performing tests for all slope levels. To summarize the power evaluation results for detecting DDIR clusters, directional tests demonstrated the most power with flatter declines in cluster slopes; whereas the radial distance decline tests were more powerful with steeper declines in slope. Both types of test improved with narrowing angles of effect. With those cluster patterns, directional tests became more powerful at all slope levels and distance decline tests remained powerful with steeper slopes. In order to simulate data with clusters of PDDIR, only one component was added to the simulation equation for clusters of DDIR: α 2 * log( d ). Therefore, as one would expect, at very low values of α 2 (e.g. 0.005), the results of power evaluations for detecting PDDIR clusters were very similar to those for detecting DDIR clusters. However, as α 2 values increased, or as the peak of the cluster increased in distance from the pollution source, findings differed. With the widest angles, overall power for detecting PDDIR with N = 200 was low. At these same angles, the Radial Score Test proved best for detecting clustering with N = 500 and N = 1000. This test decreased in power with increasing steepness of slope. As the angle of the directional effect narrowed, the LRS Test for Direction and the Directional Score Test also showed comparable power, particularly with a greater number of events. The power for these tests decreased as the cluster slope flattened. Interestingly, the Radial Score Test also followed this trend and demonstrated power second only to the directional tests. These results show that although the Radial Score Test could detect DIR effects combined with PDD effects, it was generally less powerful than directional tests at the narrowest angles of effect. Focused cluster models Overall, the addition of model parameters for peaks did not appear to contribute to improved power, particularly when a distance decline parameter was already included in the model. Only at α 1 < 1, or the flattest slope, did the null versus peaked distance decline model comparison show higher power than the null versus distance decline model comparison. Also, very low power resulted when comparing the distance decline only model to the peaked distance decline model and when adding a parameter for peak to models already containing distance decline and directional parameters. Lastly, the power curves in which only a directional component was added are very similar to those resulting from the addition of peaked and directional components. Low power to detect a peaked distance decline cluster of elevated risk may be related to the variation in relative risk. For example, Figure 3 shows higher power for lower values of α 1 when α 2 = 1. For α 2 = 1, the range of relative risk is much greater at lower values of α 1 and decreased directly with α 1 . The addition of a directional parameter improved the power of tests from models, particularly for detecting disease clusters distributed over narrower angles of effect. Evidence of this outcome was provided by the power curves resulting from the comparison between the model of null relative risk and the model with a directional component. When a directional parameter was added to distance decline models and to peaked distance decline models, power was very high for clustering with gentler declines ( α 1 < 2) and narrower angles ( α 3 and α 4 > 0.1). However, power tended to decrease as α 1 increased from 2 to 10 and as the angle of effect widened, or as α 3 and α 4 increased. Similarly, distance decline also appears to be an important parameter to include in focused cluster models, with respect to power. In opposition to the directional component, parameters for distance decline appeared most beneficial with clusters of steeper declines (larger α 1 values). However, this observation may be due to the main effect extending outside the window of the simulated region for clusters with gentler declines. Comparison of the null model to the distance decline model showed power generally increasing as the steepness of the cluster slope increased. Also, the addition of a parameter for distance decline to a directional model showed similar power curves. One interesting effect in many of the model comparisons involving a distance decline parameter was the decrease in power from α 1 = 2 to α 1 = 10. This may be caused by the steepness of the slope resulting in fewer data points demarcating the decline in slope. As the number of observations decreases, power will also decrease. Overall conclusions Though a variety of spatial scales and shapes of clusters were examined, further power evaluations are needed in order to explore fully the range of disease clustering that could result from various pollution dispersion spatial patterns. Given the overall finding that more complex spatial cluster patterns can be more difficult to detect, the development and use of additional sampling schemes for power evaluations of these clusters would be beneficial. We chose to simulate three total numbers of events because count data are typically most accessible in cluster investigations. Work is currently underway to examine power for case-event data, however further investigations are needed to examine a greater range of total numbers of events for count data. Smaller numbers of total events are of particular importance in cluster investigations of rare diseases or sparsely populated areas. A number of variations were examined for the four α coefficients, yet a more comprehensive range of coefficients, representing additional changes in shape and scale, would provide important information. Other spatial components also should be examined, such as azimuth, which could be of primary concern for air pollution sources located in valleys. Additionally, these power evaluations were performed simulating dispersion from a single, centrally-located pollution source. Further power evaluations are needed to address cluster detection in situations where pollutants are dispersed from multiple sources. Williams and Ogston [ 8 ] compared observed and simulated spatial distributions of environmental exposures. Additional comparisons between measured levels of environmental pollutant spatial dispersion with those simulated here would also be useful in evaluating the accuracy of spatial functions in a variety of situations. Similar power evaluations of focused cluster tests and models using individual-level data are also needed to improve cluster investigation techniques, and work in this area is proceeding. It should also be noted that the results from this investigation are subject to Monte Carlo error; though this error has a maximum value of 0.05 with a sample size of 100 simulated data sets. In order to estimate the uncertainty, we determined the Monte Carlo standard errors for the power estimates. Table 2 provides examples of these error estimates for the focused cluster tests, and Table 3 demonstrates the errors for the tests from the fitted models. If an approximate normal distribution is assumed for the power estimates, confidence intervals could be computed via standard procedures. However, the overall conclusions of this investigation were based on many results and should not be overly influenced by the level of error. Table 2 Sample Monte Carlo standard errors for focused cluster tests with N = 1000 Radial Score Test DIR Score Test Besag and Newell's Test ( k = 7) Cuzick and Edwards' Test ( k = 7) Tango's Test ( τ = 5) LRS DIR Test LRS DD Test Stone's Test DD α 1 = 0.005 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.02 α 1 = 0.05 0.02 0.02 0.01 0.02 0.03 0.02 0.03 0.02 α 1 = 0.1 0.02 0.02 0.01 0.02 0.03 0.02 0.03 0.03 α 1 = 1 0.05 0.02 0.01 0.03 0.04 0.02 0.04 0.05 α 1 = 2 0.03 0.02 0.01 0.04 0.02 0.03 0.01 0.03 α 1 = 10 0.04 0.02 0.01 0.05 0.03 0.02 0.03 0.03 DIR α 3 and α 4 = 0.005 0.03 0.04 0.01 0.02 0.03 0.04 0.03 0.02 α 3 and α 4 = 0.1 0.03 0.04 0.02 0.02 0.02 0.05 0.02 0.02 α 3 and α 4 = 0.2 0.03 0.03 0.01 0.01 0.03 0.01 0.03 0.02 α 3 and α 4 = 0.5 0.03 0.00 0.02 0.01 0.02 0.00 0.02 0.02 α 3 and α 4 = 1 0.03 0.00 0.03 0.00 0.03 0.00 0.03 0.02 α 3 and α 4 = 2 0.03 0.00 0.04 0.00 0.04 0.00 0.04 0.03 PKDD α 1 = 0.005 α 2 = 0.05 0.03 0.02 0.02 0.02 0.01 0.02 0.02 0.01 α 2 = 0.1 0.04 0.02 0.02 0.01 0.01 0.02 0.01 0.00 α 2 = 0.5 0.03 0.02 0.04 0.01 0.00 0.02 0.00 0.00 α 2 = 1 0.01 0.02 0.04 0.01 0.00 0.02 0.00 0.00 Table 3 Sample Monte Carlo standard errors for fitted models with N = 1000 Null vs. DD α 1 = 0.005 α 1 = 0.05 α 1 = 0.1 α 1 = 1 α 1 = 2 α 1 = 10 Standard Error 0.02 0.02 0.03 0.05 0.02 0.04 Null vs. DIR α 3 and α 4 = 0.005 α 3 and α 4 = 0.1 α 3 and α 4 = 0.2 α 3 and α 4 = 0.5 α 3 and α 4 = 1 α 3 and α 4 = 2 Standard Error 0.04 0.05 0.03 0.00 0.00 0.00 Null vs. PKDD α 1 = 0.005 α 2 = 0.05 α 2 = 0.1 α 2 = 0.5 α 2 = 1 Standard Error 0.03 0.03 0.04 0.00 This study examined the power of a number of focused cluster tests and generalized linear models to detect a wide range of simulated focused cluster shapes and scales. The results of this study provide information that can improve the choice of statistical method in focused cluster investigations. To summarize the overall findings from this investigation: 1) Focused cluster tests and tests from models containing functions appropriate to the spatial pattern of pollution dispersion are more powerful. DIR tests were more powerful detecting clusters with narrower angles of effect and DD tests were more powerful detecting clusters with steeper declines in slope. 2) Power increased with stronger DD (steeper slopes) and DIR (narrower angles) effects. 3) Power for detecting clusters with peaked effect patterns was generally low. Methods Data simulation As count data are generally more widely available in disease mapping studies than individual-level data, count data were simulated for this study. A basic Poisson model was assumed for the counts: y i ~ Poisson( e i θ i ), where an expected count e i is modified by a relative risk θ i and y i , i = 1,..., M , is the count of disease in the i th region. Clusters of three sizes ( N = 200, 500 and 1000 events) were simulated from the multinomial distribution, where the probability of a case in the i th region is . Five shapes of clusters were simulated, including: 1) distance decline, where risk declines with increasing radial distance from the pollution source (DD, Model 1, Additional File 1 : Simulated models of the five focused cluster shapes); 2) peaked distance decline, where risk peaks and then declines with increasing radial distance from the source (PDD, Model 2, Additional File 1 ); 3) direction, increasing disease risk in a particular angular direction from the pollution source (DIR, Model 3, Additional File 1 ); 4) distance decline combined with a directional effect (DDIR, Model 4, Additional File 1 ); and 5) peaked distance decline combined with a directional effect (PDDIR, Model 5, Additional File 1 ). These shapes correspond to frequently encountered air pollution dispersion patterns from point sources. For instance, shape 1 typifies dispersion from a ground level source with a relatively uniform distribution of wind directions, shape 3 represents ground level dispersion with a dominant wind direction and shape 5 represents dispersion from an elevated source with a dominant wind direction. The count data simulated for these five cluster shapes under the alternative hypothesis as well as data simulated under the null hypothesis of randomly distributed counts of disease were assigned to regional centroids of a 16*16 unit square grid. The grid of 256 regions of uniform size and shape, unitless in geographic terms, also contained a centrally located pollution source. Expected disease rates were considered to be uniform throughout the regions composing the simulated study area. The model for the relative risk at location x , θ ( x , β ) represents the relationship between the pollution source and spatial distribution of associated disease, for some choice of parameters β . As shown in Additional File 1 : Simulated models of the five focused cluster shapes, coefficients in the model equations were varied in order to represent further variations of scale for the five main cluster shapes (DD, PDD, DIR, DDIR, and PDDIR). Power evaluation methods for focused cluster tests In this study, we evaluated the power to detect the five general cluster shapes for eight widely known focused cluster tests, including: Stone's Maximum Likelihood Test [ 4 ], the focused adaptation of Besag and Newell's Test [ 1 ], Cuzick and Edwards' Test [ 9 ], Tango's Focused Test [ 5 ], variations of the Lawson-Waller Score Test [ 7 , 10 ], and variations of Bithell's Linear Risk Score (LRS) Test [ 11 ]. Power was evaluated through the use of Monte Carlo significance testing with 100 datasets simulated under the null hypothesis and 100 datasets simulated under each alternative hypothesis (i.e., each variation of cluster shape, size and scale). The formulations used for each test are briefly described. For Stone's Maximum Likelihood Test [ 4 ] (hereafter referred to as Stone's Test), we selected a number of distances ( d 1 ,..., d k ) as bins and placed regions falling between these distances in the appropriate bin. Stone's Test was then defined as follows: where is the vector of maximum likelihood estimates under the alternative hypothesis of decreasing risk with increasing distance from the cluster center, and θ 0 is the relative risk under the null hypothesis of constant risk. For the purposes of this investigation, the focused cluster adaptation of Besag and Newell's Test [ 1 ], Waller and Lawson reference} was defined as: M = min( i : D i ≥ k ), where D i is the number of cases accumulated among i regions and k is defined as the number of cases specified to define a cluster. Four variations of k (2, 4, 7 and 10) were evaluated. In the one sample approach of Cuzick and Edwards' Test [ 9 ], data are ordered by distance to cluster center; and the test statistic is defined as: where n 0 is the number of regions required until we have k events ( k = the number of cases designating a cluster). We examined four values of k : 2, 4, 7 and 10. The application of this test involves the construction of increasingly larger circles around the point source of interest until the number of cases in the regions contained by the circle equals k cases. We applied the following formulation of Tango's Focused Test [ 5 ]: C F = A ( r - p ), where, A is a vector with i th element given by a i = exp(- d i / τ ), d i = distance of the i th region centroid from the pollution source, and r and p are vectors with i th element y i / N and e i / N respectively. For the purposes of this study, τ was defined as 1 and 5. Bithell [ 11 ] indicates that LRS Tests can incorporate various functions of distance and rank to describe exposure. We applied functions of distance decline and direction, as described by Lawson [ 7 ], to represent exposure to environmental contaminants from a centrally located pollution source. Bithell's LRS Test statistic formulation [ 11 ] is described as: where θ 1 i is the area-specific relative risk based on the alternative hypothesis. We therefore defined: H 1 : distance decline in risk and: for H 1 : directional effect of risk where μ = the mean angle between the regional centroid and the pollution source. Two formulations of the Lawson-Waller Score Test [ 7 , 10 ] also were evaluated. We applied the Radial Distance Decline Score Test as defined by Lawson [ 7 ]: Lawson's [ 7 ] formulation of the Directional Score test also was used: where μ is the mean angle estimated under the null hypothesis during model fitting; however, during data simulation, was selected. Power evaluation methods for focused cluster tests from models Power evaluations of tests from generalized linear Poisson regression models to detect focused clustering were conducted with Monte Carlo significance testing of differences in model residual deviances. These regression models take the form: θ ( x i , β ) = exp( x i ' β ) where x i is the region centroid, x i ' is the i th row of a covariate design matrix (that can include x i ), and β is a parameter vector describing the relationship between the spatial distribution of disease and the pollution source. Detailed equations of the fitted models are presented in Additional File 2 : Fitted models for the five focused cluster shapes. For Monte Carlo testing, models with fewer parameters describing the spatial cluster shape were compared to models containing more parameters, or a more comprehensive function describing the spatial cluster pattern. Essentially, the resulting power curves represent the capability of detecting disease clusters when more complete information regarding the cluster pattern shape and scale is included in the model. A list of the model comparisons is presented in Table 1 . For each of these power evaluations, the model with fewer spatial parameters was fit, and any α coefficients were estimated. The comparison model, containing additional spatial parameters to describe the spatial cluster pattern, was then fitted, including fixed α estimates obtained from fitting the model with fewer spatial terms. The residual deviance difference between the two models was then determined. In order to obtain the critical value for the Monte Carlo testing, the previously described procedure was followed using 100 datasets simulated with no disease clustering to represent the expected counts and 100 datasets simulated under the model with fewer spatial parameters to represent the observed counts. The difference of the residual deviances obtained from comparing the two models was determined as the critical value. This procedure was repeated using the 100 datasets simulated with no disease clustering to represent the expected counts and the 100 datasets simulated under the alternative hypothesis of the more explicit spatial distribution to represent the observed counts. The 100 residual deviance differences were then compared to the critical value obtained from the null hypothesis testing. The number of test statistics under the alternative hypothesis that were greater than the critical value provided the power estimate. Table 1 Model comparisons Base Model Spatial Distribution Function of Alternative Model Base Model Observed Dataset More Explicit Model Observed Dataset Null Distance Decline Relative Risk = 1 Distance Decline Relative Risk Null Direction Relative Risk = 1 Directional Relative Risk Null Peaked Distance Decline Relative Risk = 1 Peaked Distance Decline Relative Risk Null Distance Decline and Direction Relative Risk = 1 Distance Decline and Direction Relative Risk Null Peaked Distance Decline and Direction Relative Risk = 1 Peaked Distance Decline and Direction Relative Risk Distance Decline Distance Decline and Direction Distance Decline Relative Risk Distance Decline and Direction Relative Risk Distance Decline Peaked Distance Decline Distance Decline Relative Risk Distance Decline and Direction Relative Risk Distance Decline Peaked Distance Decline and Direction Distance Decline Relative Risk Peaked Distance Decline and Direction Relative Risk Direction Distance Decline and Direction Directional Relative Risk Distance Decline and Direction Relative Risk Direction Peaked Distance Decline and Direction Directional Relative Risk Peaked Distance Decline and Direction Relative Risk Peaked Distance Decline Peaked Distance Decline and Direction Peaked Distance Decline Relative Risk Peaked Distance Decline and Direction Relative Risk Distance Decline and Direction Peaked Distance Decline and Direction Distance Decline and Direction Relative Risk Peaked Distance Decline and Direction Relative Risk Authors' contributions RCP carried out the simulations, performed the statistical analysis and drafted the manuscript. ABL conceived of the study and acquired funding. ABC assisted with programming the simulations and the statistical analyses. DEP, TEA, CEF, ABC, ABL and JRH had scientific input from the project's inception, through early development of the protocol, and in drafting the manuscript. All authors read and approved the version of the manuscript submitted here. Supplementary Material Additional File 1 Simulationmodels.pdf contains simulation models of the five focused clusters shapes Click here for file Additional File 2 Fittedmodels.pdf contains fitted models for the five focused cluster shapes Click here for file

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1079924

Gallbladder carcinoma: a retrospective analysis of twenty-two years experience of a single teaching hospital

Background The purpose of this study was to retrospectively evaluate our experience with gallbladder cancer since the establishment of a tumour registry in our institute. Methods Between 1975 and 1998, 23 consecutive patients with gallbladder cancer were identified using the tumour registry database. There were 18 females (78%) and 5 (22%) males. The mean age at diagnosis was 70.6 (range 42–85) years. The diagnosis was achieved either intra-operatively or following the histological analysis of the gallbladder (n = 17), following gallbladder or liver biopsy (n = 4) or at autopsy (n = 2). Presenting symptoms included upper abdominal pain, weight loss, nausea, vomiting, fever, painless jaundice, hepatomegaly, upper abdominal mass, upper abdominal tenderness, and gastrointestinal haemorrhage. Results Histological examination revealed 20 adenocarcinomas (87%), 2 squamous cell carcinomas (9%) and one spindle cell sarcoma (4%). At presentation, 14 (61%) gallbladder cancers were stage IV, 5 (22%) were stage III and 4 (17%) were stage II. Kaplan Meier analysis revealed a mean survival of 3.2, 7.8 and 8.2 months for stage IV, III, and II disease respectively. Out of 14 patients with stage IV disease, 8 patients received adjuvant chemotherapy and survived for 4.6 months whereas six patients who did not receive adjuvant chemotherapy survived for 1.3 months. This difference was statistically significant (p = 0.04). Conclusion The majority of patients with gallbladder cancer presented with advanced stage disease (stage IV) which carries a dismal prognosis. Patients who received chemotherapy with stage IV disease, however, did better than those who did not, but this is probably a reflection of patient selection.

Background Carcinoma of the gallbladder is a rare malignancy accounting for approximately 7,100 new cases and 3,500 deaths per annum in the US. It is the most common biliary tract malignancy and the fifth most frequent gastrointestinal malignancy [ 1 ]. Its clinical presentation is non-specific and the majority of patients have advanced disease at presentation. The aim of this article is to review our experience of gallbladder carcinoma since the establishment of a tumour registry in our institute. Methods Between 1975 and 1998, 23 consecutive patients with histological proven gallbladder cancer treated at St. Joseph's Hospital, Omaha, Nebraska were identified using the tumour registry database. There were 18 females (78%) and 5 (22%) males. All but one patient were Caucasian. The mean age at diagnosis was 70.6 (range 42–85) years. In 17 (74%) patients the cancer was diagnosed either intra-operatively or following the histological analysis of the gallbladder. In 4 patients, due to the extensive nature of the disease, the diagnosis was confirmed following gallbladder or liver biopsy. In the remaining two patients it was discovered at autopsy. Family history of other types of cancers was positive in 5 patients (22%), negative in 11 patients (48%) and unavailable in 7 (30%) patients. Presenting symptoms included upper abdominal pain, weight loss, nausea, vomiting, fever, painless jaundice, hepatomegaly, upper abdominal mass, upper abdominal tenderness, and gastrointestinal haemorrhage. Surgical procedures and other therapies were reviewed and their impact on survival noted. The survival of the patients discharged from the hospital was determined using Kaplan Meier analysis. P < 0.05 was considered significant. The software used was PRISM, GraphPad Software San Diego, California. Results Histological examination revealed 20 adenocarcinomas (87%), 2 squamous cell carcinomas (9%) and one spindle cell sarcoma (4%). At presentation, 14 (61%) gallbladder cancers were stage IV, 5 (22%) were stage III and 4 (17%) were stage II (Table 1 , Table 2 ). Kaplan Meier analysis revealed a mean survival of 3.2, 7.8 and 8.2 months for stage IV, III, and II disease respectively (Figure 1 ). Only one patient was alive (16.6 months with stage II disease) at the time of analysis of this data. Out of 14 patients with stage IV disease, six patients did not receive adjuvant chemotherapy and survived for a mean period of 1.3 months. On the other hand, 8 patients who received adjuvant multi-agent chemotherapeutic treatment survived for a mean period of 4.6 months. This difference was statistically significant (p = 0.04). Table 1 American Joint Commission on Cancer (AJCC) Staging TNM Definition Tumour Location Tis Carcinoma in situ T1a Gallbladder wall: mucosa T1b Gallbladder wall: muscle T2 Perimuscular connective tissue T3 Serosa or one organ, liver <2 cm T4 Two or more organs, liver >2 cm N1 Hepatoduodenal ligament nodes N2 Other regional lymph nodes M0 No distant metastases M1 Distant metastases Figure 1 Survival of patients according to the AJCC staging classification. Discussion DeStoll described carcinoma of the gallbladder on the bases of two autopsies in 1777 [ 2 ]. Since that time, primary carcinoma of the gallbladder has remained a uniformly fatal neoplasm. The reasons being (a) its late presentation; (b) early spread by lymphatic; haematogenous and direct route; (c) high propensity to seed the peritoneal surfaces after tumor spillage and (d) lack of effective adjuvant therapy. The majority of reports suggest that the gallbladder carcinoma is two to six times more prevalent in women and the incidence peaks in the seventh decade of life. In our series the female to male ratio was approximately 4:1 and the mean age at the diagnosis was 70.6 (range 42–85) years. Despite advances in hepatobiliary imaging techniques [ 3 , 4 ], the preoperative diagnosis of gallbladder carcinoma remains a daunting task. This in part is related to the disease's non-specific presentation and its similarity to benign biliary tract disorders. In our series the majority of patients underwent ultrasound scan (USS) of the upper abdomen (on the basis of their symptoms) which revealed either the presence of gallstones with our without thick walled gallbladder or liver metastases in advanced cases. In those cases where the tumour was suspected, the findings of contrast enhanced computerised tomography (CE-CT) were not helpful either. The CE-CT findings ranged from a mass in the right lobe of the liver, dilated gallbladder, thick walled gallbladder, dilatation of intrahepatic bile ducts, abscess cavity in the right lobe of the liver and liver metastases. Other studies have similarly confirmed low sensitivity and specificity of USS and CE-CT in achieving preoperative diagnosis of carcinoma of the gallbladder [ 5 - 12 ]. The most common finding described for gallbladder carcinoma on both USS and CE-CT is diffuse thickening of the gallbladder. However this is also commonly reported in the inflammatory conditions of the gallbladder and therefore does not aid in the diagnosis. In our series the majority of gallbladder carcinomas were diagnosed either intra-operatively or subsequent to histological analysis following a cholecystectomy. In the era of an laparoscopic cholecystectomy this situation poses a major dilemma as laparoscopic technique has been associated with an early and rapid dissemination of the disease both intraperitoneally and at the port site in patients with proven gallbladder carcinomas and therefore precludes potentially curative resection [ 13 - 16 ]. It has therefore been suggested that laparoscopic surgery should not be undertaken if radiological or clinical diagnosis of gallbladder carcinoma is suggested. Moreover, if such a diagnosis is suspected during initial laparoscopy then the procedure should be abandoned to maximize the chance of curative resection. As our experience ranged over the twenty years period, only a handful of cholecystectomies were performed laparoscopically and in all these patients the diagnosis was revealed only after the histological analysis of the specimen. The overall outcome of this disease is dismal with the 5-year survival rate being less than 5% with a median survival of 5 to 8 months. Piehler and Circhlow [ 17 ] reviewed 5836 patients with carcinoma of the gallbladder from 1960 to 1978 and found an overall 5-year survival of 4.1% and one year survival of 11.8%. Only 25% were resected for cure and of these only 16.5% survived 5 years. However this figure dropped to 2.9% if the surgeon identified a tumour at the time of exploration. The best survival was achieved in patients whose cholecystectomy specimens were found to have incidental tumour. Even then only 14.9% survived 5-year. Similarly Cubertafond et al [ 18 ] reported a median survival of 3 months, a 5-year survival of 5% and a 1-year survival of 14% amongst 724 carcinomas of the gallbladder. They observed no differences among the different surgical procedures adopted and concluded that no progress had been made in the treatment of gallbladder carcinoma. This clearly demonstrates the minimal impact that surgical treatment has had on this disease. However two recent reviews from Japan have contradicted all these previous reports. Ogura et al [ 19 ] have reported an impressive 50.7% 5 year survival for 984 patients undergoing radical resection compared to only 6.2% for 702 patients undergoing conservative management. Similar Todoroki et al [ 20 ] have also shown that radical resection for gallbladder carcinoma improves the prognosis even for stage IV disease, provided that complete gross tumour resection is combined with radiotherapy. These results suggest the possible role of surgery ± adjuvant therapy in changing the natural history of gallbladder carcinoma. In the current series none of the patients underwent any extensive surgical resection either because of incidental findings of gallbladder carcinoma following a cholecystectomy or the tumour was too extensive at the time of laparotomy. The longest survival recorded was 18.7 months with a median survival of 5.1 months. Subset analysis revealed that patients with well differentiated adenocarcinoma had the best outcome although none of them survived even two years. The stage at which the disease presents certainly has a direct impact on the survival of the patient (Table 1 , 2 ). A number of authors have reported between 71% to 100% 5 year actuarial survival for stage I carcinoma both following a simple and extended cholecystectomy [ 21 , 22 ], between 22% to 100% for stage II carcinoma only following an extended cholecystectomy [ 23 , 24 ], between 8% to 63% for stage III carcinoma [ 25 , 26 ] and between 8% to 25% for stage IV carcinoma following an extended resection ± adjuvant therapy [ 19 , 26 ]. We have not been able to demonstrate such impressive survival figures in our patients. The survival of our patients certainly was better in the lower stage disease compared to the advanced stage yet no patient even made it to 2 years (Figure 1 ). This may be due to the fact that an extended cholecystectomy was not performed in any of our patients. This validates the point raised by Ogura et al [ 19 ] in their review that extended therapy improves long term survival. Table 2 Staging of gallbladder carcinoma Stage TNM Modified Nevin 0 Tis N0 M0 In situ carcinoma I T1 N0 M0 In situ carcinoma II T2 N0 M0 Mucosal or muscular invasion III T1 N1 M0 T2 N1 M0 T3 N0 M0 T3 N1 M0 Transmural direct liver invasion IV A T4 N0 M0 T4 N1 M0 Lymph node metastasis IV B Any T N2 M0 Any T Any N M1 Lymph node metastasis ± distant metastasis Adjuvant chemotherapy and/or radiotherapy for patients with gallbladder cancer has not altered the dismal prognosis, but may marginally improve survival. Chao et al [ 27 ] reported no survival benefit between the two groups of patients, one receiving and other denied adjuvant chemotherapy and/or radiotherapy. On the other hand Oswalt and Cruz [ 28 ] and Morrow et al [ 29 ] have shown an improved median survival amongst the cohort of patients receiving chemotherapy and/or radiotherapy following their surgery. Similarly Makela and Kairaluoma [ 30 ] have demonstrated that superselective intra-arterial chemotherapy with mitomycin for gallbladder cancer had a 48% response rate and the responder had a significantly better survival (34 months) compared to the non-responders (8 months). However this type of therapy was only effective in those patients whose tumours were confined to gallbladder wall. Similarly in our study, patients who received adjuvant multi-agent chemotherapeutic treatment for stage IV disease had a significantly longer mean survival period compared to the ones who did not receive such treatment. This may simply reflect patient selection bias and therefore it is impossible to credit minimal improvements in survival in ours and other series to chemotherapy alone without a large randomised trial. An important point to make is that none of the patients who received chemotherapy in our series survived beyond two years – indeed a disappointing outcome. Conclusion Gallbladder carcinoma not only presents a diagnostic dilemma but also poses a difficult treatment option in the era of laparoscopic cholecystectomy. The majority of patients with gallbladder cancer present with advanced stage disease (stage IV) which carries a dismal prognosis. Chemotherapy seems to have some survival benefit in stage IV disease, but no randomized controlled trials exit to define its role in the adjuvant setting. The prognosis of this disease is dismal and even a 2 year survival seems to be the exception rather than the rule. Authors' contributions MAM was responsible for acquisition, analysis and interpretation of data; MAM, SA, MHS and BM have been involved in drafting the manuscript and revising it critically for important intellectual content and have given final approval of the version to be published. All authors have participated sufficiently in the work to take public responsibility for its content.

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1079925

Interpretation of the post-surgical Somatostatin Receptor Scintigram of a Primary Neuroendocrine Tumor of the Thymus: a case report and literature review

A case of a thymic neuroendocrine tumor and the interpretation problems in a post-surgical Somatostatin Receptor Scintigraphy are presented. In a 53-year-old man with superior vena cava obstruction syndrome an atypical carcinoid of the thymus ( neuroendocrine carcinoma of intermediate grade 2 ), was found at surgery. During his first year of follow-up a Somatostatin Receptor Scintigraphy was recommended. An area of abnormal concentration of the radiopharmaceutical was revealed in the mediastinum at this time. A thorough understanding of the mechanisms of the radiopharmaceutical uptake and of the various clinical settings in which uptake can occur are essential for a proper evaluation of the scintigraphic findings and result in the optimal use of this valuable modality. The literature review provides an overview of this rare type of tumor and insight into the clinical significance of Somatostatin Receptor Scintigraphy.

Introduction Primary neuroendocrine tumors of the thymus, previously known as thymic carcinoids, are unusual tumors that account for less than 5% of all anterior mediastinal neoplasms. They affect patients over a wide age range, with the median age being 43 years. Men are more frequently affected than women, with a male to female ratio of 3:1 [ 1 ]. Clinically, these tumors manifest in one of four ways: 1) as an incidental finding on routine chest radiography, 2) with symptoms of thoracic structure displacement or compression, 3) with symptoms related to an associated endocrinopathy or 4) with symptoms and signs relating to a distant metastasis, most commonly in the liver, lung, pancreas, pleura or bone. At least 20% of affected patients have metastatic disease at presentation, with the frequency of extra-thoracic metastasis being 20% – 30 % [ 2 , 3 ]. Approximately one-half of thymic neuroendocrine tumors (TNET) are functionally active and manifest with clinical hormone-excess syndromes, such as Cushing syndrome and are part of the autosomal dominant syndrome of multiple endocrine neoplasia (MEN) [ 4 ]. Type I (MEN) syndrome is characterized by hyperparathyroidism, islet cell tumors of the pancreas, and pituitary adenomas. Carcinoids, adrenal adenomas or carcinomas, lipomas and follicular thyroid adenomas are less frequently associated neoplasms. The majority of patients with TNET and type I MEN syndrome are male. Additional associated conditions found in patients with TNET include type 2 MEN syndrome, inappropriate secretion of antidiuretic hormone, polymyositis, finger clubbing, polyarthropathy, and myocarditis. Thoracic CT and MRI and nuclear medicine imaging, including Somatostatin Receptor Scintigraphy (SRS), meta-iodobenzylguanidine (MIBG) scintigraphy with I-123 or I-131 and bone scan, are useful studies in the evaluation of these patients [ 5 ]. Radio labeled somatostatin receptor analogues can detect biochemical lesions. This constitutes an advanced option of "functional" imaging. The success of imaging a certain lesion depends on the somatostatin receptor subtypes (SSRT) that are expressed by the tumor. In-111-DTPA-D-Phe 1-pentetreotide (pentetreotide) has shown highest affinity for SSRT2, SSRT3 and SSRT5. Its total sensitivity in detecting neuroendocrine tumors is reported to be 71% – 100% depending on the histological type of the tumor. MIBG scan sensitivity has been reported to be 16% – 96% in these tumors [ 6 ]. Few reports of cross-sectional imaging features of TNET have been published; these lesions have been described as anterior mediastinal masses indistinguishable from thymomas at CT. Invasion of focal structures and calcification within the tumor have both been reported. However, pentetreotide has also been shown to concentrate in primary and metastatic thymic tumors, including thymoma, thymic carcinoma, and TNET [ 7 - 9 ] where subtype SSRT2 is present in high density. SRS also may have a role in the follow-up of these patients [ 9 ]. Another potential application of the pre-surgical SRS is its use as a guide for octreotide therapy [ 10 ] and for radionuclide therapy with radio labeled analogues under specific conditions and criteria. Systemic injection of 111 In-Pentetreotide has yielded tumor growth inhibition in animal models and in patients with somatostatin receptor positive tumors, this therefore could be a clinical application in this kind of tumor [ 11 - 13 ]. Case presentation A 53-year-old man noticed some swelling of his face. A chest x-ray showed a large mass projecting over the right lung field (fig 1 ). A CT scan of the chest showed an enormous anterior mediastinal mass contiguous with the posterior aspect of the sternum and the right ribs, occluding the superior vena cava and compressing the heart (fig 2 ). Blood tests were normal, tumor markers and Acetyl Choline Receptor Antibodies were negative. The patient underwent bronchoscopy and the biopsy showed the features of a carcinoid tumor. Figure 1 The chest x-ray, showing a large mass projecting over the right lung field. Figure 2 A pre-surgical CT scan of the chest showing the tumour. He underwent excision of the tumor, which required resection of the superior vena cava and insertion of a graft, resection of the right phrenic nerve and plication of the diaphragm. The finding at surgery was an enormous spherical tumor with an extremely vascular capsule due to the venous collaterals. The tumor was 20 cm from pole to pole and 15 cm in diameter. On the left it bulged into the left pleural space, displacing the phrenic nerve laterally. Superiorly it obliterated the left innominate vein and extend into the strap muscles. There was an enlarged lymph node. On the right it had obliterated the confluence of the innominate veins and the first centimeter or so of the superior vena cava. It enclosed the phrenic nerve over its complete length and was adherent to the medial aspects of the upper and middle lobes of the right lung. There was some thickening of the visceral pleura in this area and there appeared to be some superficial infiltration. Posteriorly it was adherent to the pericardium and there was some thickening of the ascending aorta, consistent with longstanding pressure effects. There was a small bloody pericardial effusion and a rather large right pleural effusion. There was no evidence of any pleural deposits or intrapericardial deposits. The histology of the resection specimen showed a tumor weighing 2.1 kg. The tumor appeared encapsulated with an irregular surface. The tumor invaded the surrounding fat and pleura. There was infiltration into the underlying lung tissue lymphatics. There was some residual thymic tissue within the surrounding fat. Metastatic deposits were found in the right hilar lymph node and the node removed from the left cervical horn of the thymus. The tumor was classified as an atypical carcinoid of the thymus ( neuroendocrine carcinoma of intermediate grade 2 ). One month later he underwent a CT scan of the chest. There were findings consistent with a recent sternotomy and a graft of the superior vena cava. There was a fluid filled cyst at the base of the right lung and areas of atelectasis and pleural thickening were noted in both lungs. An I-123 MIBG scan was performed with negative results for detection of any residual or recurrent pathology. He was treated with postoperative radiotherapy and chemotherapy. One year later, a follow-up CT scan of the chest was obtained with no findings of recurrence or abnormal lymph nodes in the mediastinum. Linear atelectasis was detected in the lower lobes of both lungs and elevation of the right hemi diaphragm was noted. In addition a bone scan was performed with a positive finding at the posterior arch of the 8 th right rib. The patient was advised to have a SRS, for a more accurate evaluation of his status, as it was considered that "functional" imaging performed by Nuclear Medicine examinations, might detect pathological processes earlier than "structural" images, such as CT. Spot images of head-neck, thorax, abdomen and lumbar region were obtained 3 hr after injection of 5 mCi In-111-DTPA-D-Phe 1-pentetreotide. The scan was carried out on a Sopha single headed tomographic gamma camera coupled to a dedicated NXT computer system. Spot images of the areas of interest were obtained with a high-energy all-purpose collimator using 20% windows centered at 171 and 245 keV. The SRS was reported as showing an area of abnormally increased concentration of the radiopharmaceutical at the anatomical area of the mediastinum due to either a mass remnant or a recurrence of the disease or an inflammatory process (fig. 3 ). The bone scan finding did not take up the radiopharmaceutical. A repeat bone scan after a further 9 months was negative. Figure 3 SRS with In-111-DTPA-D-Phe 1-pentetreotide showed an area of abnormally increased concentration of the radiopharmaceutical at the anatomical area of the mediastinum. Thus, the main questions that we had to answer were: is there a mass remnant, or a recurrence of the disease? and, can we exclude a metastatic lesion at the site of the finding on the bone scan? Discussion Although there was no pre-operative SRS, requesting a post-operative SRS for this patient was justified because of the histological type of his tumor, the extensive lesions and the extended and complicated surgical management. Additionally, it is possible that during surgical maneuvers some cells were transported and implanted in other organs, so that post-surgical metastasis had occurred. Another serious reason for referring such a patient for a SRS is the finding on the bone scan [ 14 ]. Regional lymph node and distant metastases, including osteoblastic bone metastases, have been reported in up to 73% of cases and can occur late. An accidental injury could also be detected by the bone scan and for a period of 1 year. In the SRS, no lesion was found compatible with the finding in the bone scan. This, together with the fact that the bone scan lesion was solitary, reduces the probability of a metastatic bone lesion, though the possibility of variation of expression of SSRTs between metastases and primary tumor remains. This explains, in part, why the MRI scan is reported to be a more sensitive method than SRS [ 5 ]. The patient underwent a post-surgical 123 I MIBG scan that was negative. The 123 I MIBG scan was also performed in order to detect neuroendocrine tissues, but its sensitivity is unknown in this tumor. We note that SRS detects primary and metastatic thymic tumors, including TNET [ 7 , 8 , 14 ]. As to the interpretation we gave to the SRS, this could not be more precise in answering whether there is a mass remnant or a recurrence of the disease, for the following reasons: 1. There wasn't a pre-surgical SRS for comparison. So it was not known whether or not the primary mass could be imaged with pentetreotide. A negative scan may result when the SSRTs of the tumor are different from those detected by the radiopharmaceutical. 2. There have been extended and complicated surgical maneuvers that may have cause anatomical changes. 3. An inflammatory process may exist in the same area which could also cause accumulation of the radiopharmaceutical. This inflammatory component may be due to post radiotherapy fibrosis or to co-existing inflammatory reaction in the atelectasis of the lungs, because of the alveolar wall thickening and fibrosis. The atelectasis, may have been caused by the surgical maneuvers but it is known that linear or disc-like atelectasis is common in the inferior lung lobes secondary to pleural, pericardial or mediastinal lesions, as was present in our patient. It has also been recorded that if an atelectatic lesion has not resolved after three months, it will not return to normal tissue and constitutes a favourable site for mild infections (bronchectasis and pulmonary fibrosis). In these cases total blood count and ESR remain normal. Pentetreotide is accumulated in surgical trauma because of the healing process in which macrophages are recruited. This fact makes a SRS unable to distinguish the disease from the healing process. This applies for two months after the surgery. In the case of our patient, the SRS was performed one year after the surgery, so this possibility should be excluded, although the radiotherapy, which our patient had undergone, may have prolonged the healing period. Two years after the SRS, the patient is free of disease according to his clinical condition, biochemical data and imaging modalities, including a MRI scan. A second SRS that could clarify our finding was not performed since the MRI scan was negative, and a further study of our finding was not possible. Thus, the SRS finding has to be interpreted, based on the above follow up data, as a radiopharmaceutical accumulation at the site of an inflammatory process and not as a mass remnant or a recurrence of the disease. To our best knowledge, 28 cases of TNET in which SRS was performed have been reported to date in the literature (table 1 ) [ 5 , 7 - 10 , 15 - 18 ], and only one study in abstract form has reported a patient with TNET with MEN 1 having a "false" negative SRS [ 17 ]. Tiffet et al. [ 19 ] in a study of 12 neuroendocrine tumors arising in the thymus, found that none of the 12 tumors stained positively for somatostatin receptors, while Boix et al. in a case of a 33 year-old-woman with MEN 1 described positive somatostatin receptor staining by immunohistochemistry [ 20 ]. From the first study, it would seem that SRS is not advisable for post-operative follow up, while from the second, a post-operative SRS could be performed even if pre-operative SRS was not done. Table 1 Series of Primary Neuroendocrine Tumors (PNET) of the Thymus, studied by Somatostatin Receptors Scintigraphy (SRS) with 111 In-DTPA-D-Phe 1-pentetreotide. Author Year No of cases Results Zahner et al [15] 1994 1 Positive Cadigan et al [7] 1995 1 Positive Teh et al [16] 1998 3 Positive Lastoria et al [8] 1998 3 Positive Satta et al [9] 1999 2 Positive Grimfjard et al [17] 2002 3 2 positive/1 Negative Gibril et al [5]* 2003 8 Positive Plachcinska et al [18]** 2004 1 Positive Loehrer et al [10] 2004 6 Positive * 5 cases with initial and 3 with recurrent thymic carcinoid ** SRS with 99 m Tc-EDDA/HYNIC-TOC Conclusion Since CT and MRI are more accurate than SRS in the detection of TNET, the main role of pre-operative SRS is in the planning of follow up and possible therapeutic approaches for these patients. The post-operative SRS should be performed at the right time, so that the healing process is over and a possible remnant or recurrence can be detected. All the radiopharmaceutical accumulation mechanisms have to be taken into account in order to proceed to a valid differential diagnosis and give precise information to the clinician. Competing interests The author(s) declare that they have no competing interests.

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1079926

Time for sex: nycthemeral distribution of human sexual behavior

Background Nycthemeral (daily) oscillation has been documented in a variety of physiological and behavioral processes. The present study was carried out to evaluate the existence of a nycthemeral rhythm of human sexual behavior and to identify environmental factors responsible for the rhythmic pattern. Methods Non-traditional university students (ages 18 to 51 years) recorded the times of day when they went to sleep, when they woke up, and when they had sex for 3 consecutive weeks. They also answered a questionnaire designed to identify the causes of their selection of time for sex. Results The majority of sexual encounters took place at bedtime (11 pm to 1 am). The most common explanations for this temporal pattern were the rigidity of the professional work schedule and family obligations and the availability of the partner, which reduced the opportunity for sexual encounters at other times of the day. Conclusion Most sexual encounters take place around bedtime. Although the presence of an endogenous component responsible for this temporal pattern cannot be excluded, the evidence indicates strong environmental forcing, particularly from the work/family schedule of the individuals and from partner availability.

Background Practically all physiological parameters in the animal and human body exhibit nycthemeral (daily) or circadian oscillation [ 1 , 2 ]. Epidemiological studies have documented nycthemeral oscillation in a variety of aggregate variables, such as heart attacks [ 3 - 5 ], births [ 6 - 8 ], and suicides [ 9 - 11 ]. One study conducted in 1982 provided evidence of the existence of a nycthemeral rhythm of sexual activity in young married couples [ 12 ]. The present study sought to verify this nycthemeral rhythmicity in a sample of human adults with a wider age range and to identify environmental factors responsible for the rhythmic pattern. Methods Part 1 The first part of the study, conducted during the winter of 2003, involved 15 non-traditional university students in South Carolina (6 males, 9 females, ages 18 to 51 years). The subjects participated in the study as partial fulfillment of course requirements. They were given a data sheet and were asked to record, each day for three weeks, the times of day when they went to sleep, when they woke up, and when they had sex. Subjects were allowed to use their own definition of sex, which did not necessarily involve vaginal intercourse. Complete forms were received from 11 subjects, anonymously. All subjects were married or were involved in a steady relationship with a partner. The accumulated nycthemeral distribution of sexual encounters was analyzed in three ways. A Kolmogorov-Smirnov test [ 13 ] was used to determine whether the experimental distribution differed significantly from a flat distribution. A Rayleigh test [ 14 ] was used to determine whether the experimental distribution conformed to a sinusoidal pattern. A cosinor test [ 15 ] was used to determine the acrophase (time of peak) of the nycthemeral oscillation. Part 2 The second part of the study, conducted during the winter of 2005, involved a separate group of 38 university students (14 males, 24 females, ages 18 to 43 years). The subjects were given a brief survey containing two main questions: "What time of day do you usually have sex?" and "Why do you have sex at these times (as opposed to other times of day)?". Answers could be written in for both questions, but potential answers were provided for the second question, as follows: __ I feel more sexual at these times. __ These are the only times when my partner is available. __ My work/family schedule does not allow me to have sex at other times. __ I'm already in bed, so why not have sex? __ Other. Specify: ________________________________ Ten subjects (4 males, 6 females, aged between 18 and 20 years) had never had sex and, therefore, were excluded from the study. The percentages of the answers to the second question were computed and compared by the Kolmogorov-Smirnov test. Results and Discussion Part 1 Although there were not enough data for the analysis of the distribution of sexual encounters according to day of the week, the mean number of sexual encounters per week (2.3) was comparable to that found in the broader adult population of the United States (twice a week) [ 16 ]. The nycthemeral distribution of sexual encounters is shown in Figure 1 . Of 71 recorded encounters, 17 took place at midnight, which was the average to-bed time for the subjects. A smaller peak occurred at 06:00, which was the average wake-up time for the subjects. The Kolmogorov-Smirnov test indicated that the distribution was significantly different from a flat distribution ( D = 0.201, p < 0.01), and the Rayleigh test indicated that the distribution conformed to a sinusoidal pattern ( nR 2 = 15.706, χ 2 (2) = 31.412, p < 0.0001). The cosinor test indicated an acrophase at 01:00. Figure 1 Nycthemeral distribution of sexual encounters. Of 71 recorded encounters, 17 took place at midnight, which was the average to-bed time for the subjects. The mean bedtime of 00:00 and mean waketime of 06:00 are within the range of bedtimes and waketimes observed in various societies around the world [ 17 - 23 ]. The observed bedtime peak of sexual encounters is in agreement with a previous study of young married couples [ 12 ]. The results are also in agreement with those obtained in a survey about the time of day of the first intercourse of young women, in which it was found that over 85% of the subjects had lost their virginity either in the evening or at night [ 24 ]. Part 2 Because the subjects in Part2 were asked only to recall their usual time of sex, the precision of the results was lower than in Part 1. The answers were categorized as "morning" (20%), "afternoon" (5%), "evening" (10%), and "night" (65%). This distribution is significantly different from a flat distribution ( D = 0.400, p < 0.001) and is consistent with the results from Part 1. The distribution of responses to the question "Why do you have sex at these times?" is shown in Figure 2 . Although the subjects were allowed to give multiple answers, most of them gave only one answer, and the few who chose "Other" actually wrote in one the four offered options. Consequently, the percentages of answers conveniently added up to 100%. Only 28% of the respondents gave an answer that would characterize an endogenous rhythm of sexual appetite ("I feel more sexual at these times"). In contrast, 33% of the respondents attributed the time selection to restrictions imposed by work/family schedule, and 23% attributed it to partner availability. The distribution of percentages does not differ significantly from a flat distribution ( D = 0.090, p > 0.05), which indicates that none of the four responses was consistently chosen more frequently than the others. Thus, the response based on an endogenous rhythm of sexual appetite was chosen by only a quarter of the respondents. Even this relatively small fraction may be an overestimate, as the subjects may have failed to identify environmental factors other than those offered as potential answers. Figure 2 Frequency distribution of answers to the question "Why do you have sex at these times?" Most subjects indicated that the selection of time for sex is determined by external factors, such as work schedule and partner availability. In addressing the issue of exogenous causation, it must be emphasized that this study was conducted under normal living conditions, where environmental factors can "mask" the expression of an endogenous rhythm of sexual appetite. In men, it is known that there is daily rhythmicity in plasma concentration of testosterone [ 25 , 26 ], which likely has an endogenous source and could be responsible for rhythmicity in sexual appetite. However, the results of this study clearly indicate that, if there is an endogenous rhythm of sexual appetite, it is not the main determinant of the selection of time for sex in individuals living under normal societal conditions. Instead, the time for sex seems to be determined predominantly by environmental forcing. Conclusion Although human adults seem to find opportunities for sex at practically any time of the day, most sexual encounters occur around bedtime (11 pm to 1 am). A smaller peak in sexual activity occurs around wake time. Because the study was conducted in the presence of external time cues, the issue of the endogenous source of this nycthemeral variation cannot be directly addressed. However, self-reports indicate the presence of strong environmental forcing, particularly from the work/family schedule of the individuals and from partner availability. Competing Interests The author(s) declare that they have no competing interests.

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1079927

Development of daily rhythmicity in heart rate and locomotor activity in the human fetus

Background Very little is known about the perinatal genesis of circadian rhythmicity in the human fetus. Some researchers have found evidence of rhythmicity early on in fetal development, whereas others have observed a slow development of rhythmicity during several years after birth. Method Rhythms of fetal heartbeat and locomotor activity were studied in women with physiological course of pregnancy at 16 to 40 gestational weeks. Observations were conducted continuously for 24 h using the method of external electrocardiography, which provided simultaneous detection of the changes in maternal and fetal heartbeat as well as assessment of daily locomotor activity of the fetus. During the night-time, electroencephalogram, myogram, oculogram and respiration of the mother were registered in parallel with fetal external electrocardiography. Results Although we found no significant daily rhythmicity in heart rate per se in the human fetus, we developed a new method for the assessment of 24-h fetal cardiotachogram that allowed us to identify daily rhythmicity in the short-term pattern of heart beating. We found that daily rhythmicity of fetal electrocardiogram resembles that of the mother; however, the phase of the rhythm is opposite to that of the mother. "Active" (from 9 a.m. to 2 p.m. and from 7 p.m. to 4 a.m.) and "quiet" (from 4 a.m. to 9 a.m. and from 2 p.m. to 7 p.m.) periods of activity were identified. Conclusion A healthy fetus at gestational age of 16 to 20 weeks reveals pronounced rhythms of activity and locomotion. Absence of distinct rhythmicity within the term of 20 to 24 weeks points to developmental retardation. The "Z"-type fetal reaction, recorded during the "quiet" hours, does not indicate unsatisfactory state, but rather is suggestive of definite reduction of functional levels of the fetal physiological systems necessary for vital activity.

Background Animal homeostasis is based on rhythmic activity of the physiological systems of an organism, whereas these rhythmic processes bring the physiological functions into line with environmental rhythmicity. Studies that highlight the regulating role of the suprachiasmatic nucleus in the course of circadian rhythmic activity [ 15 , 21 , 27 ], that confirm the significance of melatonin in the development of sleep-wakefulness rhythms [ 12 , 20 , 24 , 26 ], and that underscore the importance of cryptochromal proteins in the generation of daily rhythmic activity [ 11 , 25 , 28 ] have provided profound insights into the rhythmic mechanisms of human physiological functions. However, much remains to be learned. Among the challenging questions is that concerning the perinatal genesis of circadian rhythmicity of the human fetus. Few investigations have been conducted in this area. Some researchers have found evidence of rhythmicity early on in fetal development, whereas others have observed a slow development of rhythmicity during several years after birth [ 19 , 22 , 23 ]. The Research Institute of Perinatal Medicine, Obstetrics and Gynecology has been conducting investigations on fetal rhythmicity since 1978 with three major goals: 1. Determination of rhythmicity of the heartbeat and locomotor activity of the human fetus, including the interdependence between maternal and fetal biological rhythms (Research works 1978–1979); 2. Differentiation of the characteristics of the human fetus response to exertion (workload) tests with regard to natural changes in the functional state of the fetus related to the run of its biological clock (Research works 1980–1986); and 3. Determination of the time of onset and formation of fetal biological rhythms (Research works 1989–1993). Method Stages 1 and 3 Subjects The subjects in Stage 1 were 18 volunteer pregnant women, aged 19 to 26 years, at the gestational period of 36 to 40 weeks. They were informed of the format and extent of the trials beforehand. The subjects in Stage 3 were 28 pregnant women examined at the gestational period of 16 to 28 weeks (16 to 17 weeks: n = 1; 17 to 18 weeks: n = 3; 18 to 19 weeks: n = 2; 19 to 20 weeks: n= 2; 20 to 21 weeks: n = 3; 21 to 22 weeks: n = 2; 22 to 23 weeks: n = 4; 23 to 24 weeks: n = 3; 24 to 28 weeks: n = 8). Catamnestic data for the 46 pregnant women observed showed that in all cases newborns were healthy, being evaluated by Apgar Scale as 8–9 points (19 cases) and 9–10 points (27 cases). Procedure Recording sessions were conducted in electrically isolated rooms following 2 or 3 days of preliminary adaptation. Routine living conditions were maintained as much as possible. The participants were thoroughly screened and selected for physiological pregnancy. The observation was performed uninterruptedly during 24 hours, using the method of external electrocardiography (ECG) to identify maternal and fetal heart beat as well as fetal locomotor activity. External ECGs of the fetus and EEGs of the mother were recorded by means of 16 channel electroencephalograph " MEDICOR ", Hungary. Paper-tape velocity was 30 mm/sec. The recordings obtained were processed by the calculation of maternal and fetal heart rate (FHR) in 5-minute intervals and transferred onto the cardiotachogram representing a graph of intra-minute fluctuations of HR. Based on 24-hour uninterrupted recording of fetal external ECG, a 24-hour cardiotachogram was plotted for every hour (from 1 to 2, 2 to 3, 3 to 4 etc.). The analysis of hourly cardiotachogram was done according to the following parameters: hourly fluctuations in maternal and fetal HR, intra-minute fluctuation of HR, and baseline rhythm. The baseline was defined as the level obtained at 7 a.m., under the conditions of basic metabolic rate. The values of the parameters under study at 12 p.m., 5 p.m., 10 p.m., and 2 a.m. were compared to the levels at 7 a.m. (39, 40). Fetal movements were fixed at oscillation of isoelectrical line of the external ECG of the fetus, calculated by hour and a graph of hourly fetal movements was plotted. At night-time, external ECGs of the fetus and EEGs of the mother were recorded simultaneously with the above procedures. The EEGs were subjected to visual analysis, which consisted of the evaluation of wave duration, sleep amplitude, shape and general structure, sleep cycles, and phasic composition and duration. Stage 2 A total of 2,500 fetuses from carefully selected pregnant women with physiological course of pregnancy at the gestational term of 32 to 40 weeks were studied in Stage 2. Single-time recording of external ECG or external cardiotachogram (CTG) was conducted during 20–25 min. To determine the intrauterine condition of the fetus, functional tests were carried out 10–12 min following the commencement of the background recording. Breath-hold at expiration, physical exertion, sound stimulation, and non-stress test (NST) were used as functional loads. Breath-hold at expiration was carried out during 20–25 sec. For "sound load", a telephone handset was placed anteriorly on the abdominal wall in the fetal head projection area and a permanent sound was delivered at the frequency of 500–1000 Hz for 60 sec at an intensity of 105 decibel. The mothers wore ear-plugs during the stimulation. Physical exertion was given in the form of bending and straightening of the trunk 10 times for 45–50 sec. Functional state of the fetus was evaluated based on the analysis of a one-time CTG (focus being placed on the HR fluctuations, baseline rhythm, intra-minute oscillation, fetal reactivity to functional loads, and number of variable accelerations and decelerations). The trials were dynamically conducted during both "active" (9 a.m. to 2 p.m.) and "quiet" (2 p.m. to 7 p.m.) periods. The electrographic findings of the intrauterine fetus were correlated with evaluations by Apgar scale as well as with the data regarding the course of postnatal period. The time of the recordings was strictly fixed for each observation of the fetus. Differences between the parameters under study were considered reliable at p < 0.05. Results and Discussion Stage 1 The methodical approach utilized in our study of daily rhythms of external ECG allowed us to perform a digital analysis of hourly values of maternal and fetal heart rate during 24-hours of uninterrupted recording. We were able to evaluate changes in the functional levels of two processes in the fetus: heart action and locomotor activity. The uninterrupted recordings of fetal ECGs allowed us to use a new self-designed technique for analysis of daily cardiotachograms (CTG) of the fetus and the pregnant woman. The analysis of heart rate changes in healthy pregnant women showed that function enhances from 7 a.m. (66.5 bpm) to 12 p.m. (79.5 bpm), slightly reduces, and then continues to increase until 5 p.m. (76.5 bpm). Minimal heart rate is observed at 2 a.m. (61 bpm). Thus, in healthy pregnant women, the daily rhythms of HR fluctuations seem to be preserved (Table 1 ). Table 1 Hourly layout for 24-hour period frequencies of maternal and fetal heart rate rhythms. (36 – 40 weeks gestation) Time of day (h) Mother (bpm) Fetus (bpm) 7 – 8 66.5 ± 1.3 * 133 ± 1.9 ** 8 – 9 68 ± 1.2 * 133 ± 2.1 ** 9 – 10 68.5 ± 1.3 ** 132 ± 2.0 ** 10 – 11 72 ± 1.4 ** 136 ± 2.1 ** 11 – 12 76 ± 1.4 ** 135 ± 2.3 ** 12 – 13 79 ± 1.3 * 136 ± 2.3 ** 13 – 14 79 ± 1.2 * 134 ± 1.9 * 14 – 15 75 ± 1.2 * 136 ± 2.1 ** 15 – 16 76 ± 1.1 * 133 ± 2.3 * 16 – 17 76 ± 1.1 * 132 ± 1.8 ** 17 – 18 76.5 ± 1.2 * 133 ± 2.2 ** 18 – 19 79 ± 1.0 * 135 ± 1.8 ** 19 – 20 71 ± 1.1 * 131 ± 1.8 * 20 – 21 72 ± 1.1 ** 133 ± 2.0 * 21 – 22 69 ± 1.2 * 135 ± 1.9 ** 22 – 23 70 ± 1.1 * 134 ± 1.8 ** 23 – 24 65 ± 1.0 * 136.5 ± 2.0 ** 24 – 1 64 ± 1.2 * 138.5 ± 1.9 * 1 – 2 65 ± 1.1 * 138.5 ± 2.2 * 2 – 3 61 ± 1.2 * 133.5 ± 1.9 ** 3 – 4 64 ± 1.1 * 136 ± 1.8 ** 4 – 5 62 ± 1.1 * 132 ± 1.9 ** 5 – 6 66.5 ± 1.1 * 133 ± 2.1 ** 6 – 7 68 ± 1.0 * 134 ± 2.2 ** * p < 0.001 (compared to heart rate at 7 am). ** p > 0.05. The study of 24-hour FHR fluctuations demonstrated that the HR magnitude at 7 a.m. was 133.0 ± 1.9 bpm; at 10 p.m. it was 136.5 ± 2.0 bpm, and at 2 a.m. the value approximated that at 7 a.m. (133.5 ± 1.6 bpm). Statistical processing of fetal HR magnitude during the 24-hour period did not show any significant difference between day and night measurements (Fig. 1 , Fig. 2 ). Consequently, analysis of daily FHR fluctuations using the method of frequency changes computing did not permit to judge on the presence of daily periodicity of the fetal heart rhythm. Figure 1 Hourly layout for 24-hour period frequencies of maternal and fetal heart rate rhythms (36 – 40 weeks gestation) Figure 2 Hourly layout for 24-hour period frequencies of maternal and fetal heart rate rhythms (36 – 40 weeks gestation) Karr et al [ 16 ] and Hellbrugge [ 13 ] investigated 24-hour heart rate rhythms of the mother and the fetus using single auscultations of maternal heart rate and pulse with 2-hour intervals throughout 24 hours. N.N. Konstantinova [ 10 ] and Hoppenbrowers [ 14 ] directed their investigations mainly toward the study of fetal heart rate changes during the mother's sleep. Our findings agree with Hellbrugge's [ 13 ] results indicating the occurrence of typical 24-hour HR rhythms in the pregnant woman. At the same time, FHR is more or less identical during the day time and at night, averaging 133.0 ± 5.0 bpm. Hoppenbrouwers [ 14 ] found that fetal HR did not undergo substantial changes during the sleep and waking periods of the mother. N.N. Konstantinova [ 10 ] reported a dependence of fetal HR on maternal sleep-wakefulness cycles. We have undertaken the task of investigating the principles of conformity between maternal and fetal rhythms with the result of taking notice of heterogeneity in fetal cardiotachograms, which allowed us to classify four types of oscillations occurring on an hourly cardiotachogram in different combinations, and sequences, and continuously replacing one another. Depending on the differences in form, amplitude and duration, each of the oscilation types was designated by a proper term: "peak-like", "rounded", "flat" and "mixed" (Fig. 3 ). Type I ("peak-like") is characterized by rapid fluctuations of FHR during 5–10 sec with the intra-minute fluctuations of ± 12–30 bpm. Type II ("rounded") is characterized by a gradual intensification of fetal HR by ± 18–34 bpm and further gradual return to the initial values. Type III ("flat") is characterized by low intra-minute fluctuation of ± 1–4 bpm. Type IV ("mixed") is characterized by the baseline rhythm of 120–150 bpm and intra-minute fluctuation of ± 7–15 bpm. Figure 3 Types of cardiotachograms Having separated the 24-hour cardiotachograms into hourly parts, we carried out a careful visual and digital analysis of the findings and discovered certain irregularities in the distribution of one or another type of oscillations across a daily rhythmogram. Further, having computed the amount of time necessary for each type of cardiotachogram to repeat within one hour of the observation, we paid attention to the findings that the period when the background cardiotachogram is presented by "flat (III) type" oscillation (51% and 55 % of the recording time) was observed to occur twice during 24 hours, predominating over the other three types (Fig. 4 , Fig. 5 ). The periods from 4 a.m. to 9 a.m. and from 2 p.m. to 7 p.m. were termed "quiet" hours. During the rest of the day, i.e. from 9 a.m. to 2 p.m., and from 7 p.m. to 4 a.m., the background cardiotachogram was represented by "mixed" oscillations with the prevalence of types I, II and IV (87% and 89% of the recording time). These periods were termed "active" hours (Table 2 , Table 3 , Table 4 , Fig. 6 , Fig. 7 ). Table 2 ECG and locomotor activity of the fetus in "quiet" hours (4 a.m. to 9 a.m., 2 p.m. to 7 p.m.) (36 – 40 weeks gestation) Time (h) Type Duration Time (h) Duration Oscillation type Fetal movements Recording Oscillation type Fetal movements Recording Min. Sec. Min. Sec. Min. Min. Sec. Min. Sec. Min. 4–5 I - 20 ± 0.2 - - 54 ± 0.3 14–15 - 10 ± 0.2 - - 40 ± 0.4 II 16 ± 0.3 - - 50 ± 0 3 ± 0.4 - - 50 ± 0.1 III 20 ± 0.2 - - - 30 ± 0.4 - - - IV 18 ± 0.2 - - - 7 ± 0.1 - - - 5–6 I - - 1 ± 0.1 - 46 ± 0.2 15–16 1 ± 0.2 - - - 54 ± 0.3 II 7 ± 0.4 - - - 7 ± 0.2 - 2 ± 0.1 - III 30 ± 0.3 - - - 24 ± 0.1 - - - IV 9 ± 0.3 - - - 21 ± 0.2 - - - 6–7 I - 30 ± 0.2 - - 59 ± 0.2 16–17 - 20 ± 0.3 - - 45 ± 0.1 II 18 ± 0.1 - 1 30 ± 0.3 12 ± 0.1 - 1 ± 0.3 - III 26 ± 0.1 - - - 21 ± 0.1 - - - IV 15 ± 0.2 - - 11 ± 0.1 - - - 7–8 I - 50 ± 0.1 - - 50 ± 0.5 17–18 - 20 ± 0.1 - - 45 ± 0.3 II 12 ± 0.4 - - - 8 ± 0.2 - 1 50 ± 0.1 III 22 ± 0.4 - 1 5 ± 0.1 20 ± 0.1 - - - IV 15 ± 0.3 - - - 16 ± 0.1 - - - 8–9 I - 20 ± 0.1 - - 40 ± 0.2 18–19 - 10 ± 0.1 - - 42 ± 0.1 II - - - 50 ± 0.1 4 ± 0.3 - - 40 ± 0.2 III 28 ± 0.3 - - - 25 ± 0.2 - - - IV 12 ± 0.3 - - - 12 ± 0.3 - - - Table 3 ECG and locomotor activity of the fetus in "active" hours (9 a.m. to 2 p.m.) (36 – 40 weeks gestation) Time (h) Type Duration Time (h) Duration Oscillation type Fetal movements Recording Oscillation type Fetal movements Recording Min Sec Min Sec Min Min Sec Min Sec Min 9–10 I - 45 ± 0.2 7 ± 0.2 - 48 ± 0.2 12–13 - 10 ± 0.2 8 ± 0.1 - 30 ± 0.3 II 6 ± 0.2 - - - 10 ± 0.1 - - 50 ± 0.1 III 7 ± 0.3 - - - 2 ± 0.1 - - - IV 35 ± 0.1 - - - 17 ± 0 - - - 10–11 I - 50 ± 0.4 3 ± 0.1 - 24 ± 0.3 13–14 2 ± 0.3 - 4 ± 0.3 - 48 ± 0.1 II 8 ± 0.1 - - - 8 ± 0.1 - - - III 3 ± 0.1 - - - 8 ± 0.2 - - - IV 12 ± 0.01 50 ± 0.1 - - 30 ± 0.1 - - - 11–12 I - 30 ± 0.2 4 50 ± 0.1 38 ± 0.2 Total from 9–14 6–42 ± 0.16 45 ± 0.26 26 50 ± 0.16 188 ± 0.22 II 10 ± 0.3 - - - 25 ± 0.24 III 5 ± 0.3 - - - 116 IV 22 ± 0.2 - - 50 ± 0.12 Table 4 EEG and locomotor activity of the fetus in "active" hours (from 7 p.m. to 4 a.m.) (36 – 40 weeks gestation) Time (h) Type Duration Time (h) Duration Oscillation type Fetal movements Recording Oscillation type Fetal movements Recording Min Sec. Min Sec. Min Min Sec. Min Sec. Min 19–20 I 3 ± 0.1 - 4 50 ± 0 54 ± 0.1 24–1 2 ± 0.2 - 4 ± 0.1 - 53 ± 0.2 II 15 ± 0.2 - - - 14 ± 0.1 - - - III 4 ± 0.1 - - - 4 ± 0.4 - - - IV 32 ± 0.2 - - - 33 ± 0.4 - - - 20–21 I - 10 ± 0.2 4 ± 0.2 - 30 ± 0.2 1 – 2 1 ± 0.1 - 6 ± 0.3 - 34 ± 0.4 II 10 ± 0.1 - - - 11 ± 0.2 - - - III 5 ± 0.1 - - - 7 ± 0.4 - - - IV 15 ± 0.2 - - - 15 ± 0.2 - - - 21–22 I - - 3 ± 0.1 - 39 ± 0.3 2 – 3 1 ± 0.2 - 4 ± 0.2 - 53 ± 0.1 II 12 ± 0.4 - - - 18 ± 0.1 - - - III 22 ± 0.4 - - - 7 ± 0.1 - - - IV 15 ± 0.3 - - - 27 ± 0.2 - - - 22–23 I I 40 ± 0.2 16 ± 0.2 - 26 ± 0.3 3 – 4 - 30 ± 0.3 3 30 ± 0.2 31 ± 0.2 II 7 ± 0.1 - - - 9 ± 0.1 - - - III - - - - 7 ± 0.1 - - - IV 18 ± 0.3 - - - 15 ± 0.3 - - - 23–24 I 1 ± 0.3 - 3 ± 0.2 - 58 ± 0.1 Total from 19-4 12 20 ± 0.21 43 20 ± 0.16 388 ± 0.21 II 23 ± 0.3 - - - 123 ± 0.15 - III 5 ± 0.2 - - - 44 ± 0.17 - IV 29 ± 0.4 - - - 200 ± 0.26 - Figure 4 ECG of the fetus in "quiet" hours (4 a.m. to 9 a.m.) 36–40 weeks gestation Figure 5 ECG of the fetus in "quiet" hours (2 p.m. to 7 p.m.) 36 – 40 weeks gestation Figure 6 ECG activity of the fetus in "active" hours (9 a.m. to 2 p.m.) 36 – 40 weeks gestation. Figure 7 ECG activity of the fetus in "active" hours (from 7 p.m. to 4 a.m.) 36–40 weeks gestation Our investigation demonstrated that concentration of type I oscillations on a cardiotachogram during "quiet" hours was four times lower than in "active" hours; concentration of type II and IV oscillations was twice as low in "quiet" hours as compared to "active" periods. The "flat" (III) type oscillations were four times as prevalent during "quiet" hours as during "active" hours. Statistical processing of the findings regarding the duration of each type of the oscillations and "active" and "quiet" hours of the fetus yielded a reliable result (p < 0.001) (Fig. 8 , Fig. 9 ). Figure 8 Fetal cardiotachogram for "active" period Figure 9 Fetal cardiotachogram for "quiet" period The analysis of locomotor activity during the defined periods of rest and activation of the fetus showed that in "quiet" hours the recording of fetal movements lasted for 11 min and 5 sec, which corresponded to 2,3% of the recording time. In "active" hours, fetal locomotor activity augmented by 7–8 times and was equal to 91 min and 10 sec, which corresponded to 16% of the recording time (Fig. 10 ). Figure 10 Fetal locomotor activity in "quiet" and "active" hours (36 – 40 weeks gestation) Apart from the analyses mentioned, we thought it reasonable to calculate the number of fetal heart contractions with the values lower than 120 bpm and higher than 150 bpm, which were encountered in hourly portions of daily cardiotachograms. The data obtained pointed to a significant prevalence of the frequencies lower than 120 bpm in "quiet" hours, whereas frequencies higher than 150 bpm were either solitary or were not registered at all. ECGs recorded from the pregnant women were also analyzed. It was found that during nocturnal sleep the duration of "flat" type cardiotachograms exceeded significantly that of types I, II, and IV. In the day-time, during mother's waking period, "flat" type cardiotachograms were either absent or occasionally appeared on hourly recordings within a short space of time (2–5 min). Our investigation has demonstrated that "active" periods of the fetus are characterized by the elevation of the levels of physiological functions, which is expressed by the predominance of "peak-like", "rounded" and "mixed" oscillations with high levels of intra-minute fluctuation and variability of HR, as well as by the prevalence of frequency values higher than 150 bpm and enhanced locomotor activity of the fetus. "Quiet" hours show typical reduction of HR variability, predominance of "flat" (type III) oscillations, significant prevalence of frequencies lower than 120 bpm, and sharply decreased locomotor activity [ 2 , 4 , 8 , 9 , 18 , 29 ]. This evidence suggests that the fetus develops a sleep-like state from 4 a.m. to 9 a.m. and from 2 p.m. to 7 p.m. The duration of night-time sleep in the healthy pregnant women was 8 h 10 min. The EEG analysis showed that common structural characteristics of sleep together with each of its phases are typical of those of healthy individuals being in the relative resting state [ 6 ]. The analysis of fetal locomotor activity during night sleep of the pregnant women revealed 1051 ± 2.6 fetal movements, observed from 10 p.m. to 4.30 a.m. (sleep cycle I – 207 ± 1.4 movements, II – 315 ± 2.3, III – 11 ± 1.4, IV – 508 ± 2.1). At the same time, from 4.30 a.m. to 8 a.m. there were only 136 ± 1.2 movements (sleep cycle V – 196 ± 1.1, VI – 40 ± 1.6). During "active" hours (7 p.m-4 a.m.), irrespective of the mother's sleep phase, the fetus was observed to be active, as inferred from the increased number of its movements. On the other hand, locomotor activity of the fetus was 7–8 fold decreased in "quiet" hours (from 4 a.m. to 9 a.m.). Thus, locomotor activity of the fetus did not affect the course of nocturnal sleep and its cyclicity. Consequently, as in adults, acceleration and deceleration of physiological activity take place in a healthy full-term fetus. The curve depicting changes in the levels of fetal physiological functions bears a biphasic character; the levels are reduced in the morning (4 a.m. to 9 a.m.) and in the afternoon (2 p.m. to 7 p.m.) and increased during the day-time (9 a.m. to 2 p.m.) and evening-night (7 p.m. to 4 a.m.) periods (Fig. 11 ). Figure 11 Correlation of quiet and active periods for mother According to many authors, changes in the majority of physiological processes in humans (body temperature, activity of the cardiovascular system, respiration rate, etc.) manifest themselves in constant elevations of the levels from 8 a.m. to 1 p.m., with a slight decrease between 1 p.m. and 2 p.m., and continued elevation reaching maximal values by 4 p.m. to 6 p.m. The second minimal value of the parameters is observed at 2 to 3 a.m. The daily rhythm oscillation of blood adrenalin and adrenohypophyseal system activity fluctuate within the range of an opposite phase, reaching peak values at 6 a.m. to 9 a.m. These data suggest that the periods from 4 a.m. to 9 a.m. and from 7 p.m. to 1 a.m. are transitional stages during which the mother's physiology shifts from one functional level to another, which is a natural functional load for both the mother and the fetus. It is possible to suggest that the process involved in the intrauterine development of the fetus requires the availability of a relatively persistent homeostasis; the fetus employing its intrinsic adaptive capacities of responding to the rhythmic changes in the levels of maternal physiological functions becomes active when these levels are reduced and decreases its own physiological activity when they are elevated. The data obtained has led us to the conviction that the levels of functioning of the fetal physiological systems do comply with the state of maternal organism but run with a reverse phase. Stage 3 Issues regarding the onset and formation of the fetal intrauterine rhythm were studied on 28 pregnant women at the gestational term of 16–28 weeks (at 16–20 weeks – 8 pregnant participants, at 21–24 weeks – 12, at 26–28 weeks – 8). The self-designed methodological approach previously employed in our investigations was entirely preserved. Analyses of daily rhythmograms were performed using the original method described above [ 7 , 17 , 19 ]. The results showed regular rhythms of daily fluctuations of HR in all 28 pregnant women. Sleep duration in this group was 8 hrs 32 min. The general structural characteristics and each phase of sleep were typical of those of a healthy individual. The analysis of 24-hour CTGs of the fetus demonstrated a clear-cut daily rhythm of heart rate and locomotor activity in 21 fetuses (16–20 weeks gestation – 6 cases; 21 – 24 weeks gestation – 8 cases; 26–28 weeks gestation – 7 cases). Just as in a full-term fetus at 16–28 weeks gestation, the fetus' background cardiotachogram for "active" hours, from 9 a.m. to 2 p.m. and from 7 p.m. to 4 a.m., was represented by mixed (type IV) oscillations with markedly expressed "peak-like" and "rounded" types. During "quiet" periods (4 a.m. to 9 a.m. and 2 p.m to 7 p.m.), the background rhythmogram was represented by "flat" (type III) oscillations. The locomotor activity in "active" hours was 11 (in a full-term fetus 6–7) times as intensive as compared to that in "quiet" periods of the day. Consequently, as early as at 16–20 weeks pregnancy the fetus clearly expressed 24-hour rhythms of the heartbeat and locomotor activity. On the other hand, in 7 cases (out of 28) the analysis of daily cardiotachograms showed mostly "mixed" oscillations during both "active" and "quiet" periods with "peak-like, "rounded" and "flat" types (types I, II, III) being observed with different intensity against the background cardiotachograms. In these 7 cases (at 16–20 weeks' gestation – 2 cases; 21–24 weeks' gestation – 4 cases; 26–28 weeks' gestation – 1 case), the concentration of "flat" (type) oscillations in "active" hours was equal to 17 ± 0.6 min, i.e. 3.03% of the total recording time from 9 a.m. to 2 p.m. and from 7 p.m. to 4 a.m. In "quiet" hours the concentration of flat oscillations was 14 ± 1.03 min, corresponding to 28% of the total recording time from 4 a.m. to 9 a.m. and from 2 p.m. to 7 p.m. The locomotor activity of these 7 fetuses was 3.5 fold higher in "active" hours than that in "quiet" periods. Comparative analysis of these findings for 21 fetuses at 16–28 weeks' gestation with expressed fetal rhythms showed that the concentration of "flat" (type III) oscillations in "active" hours made up 49 ± 0.6 min, i.e. 8.7% of the total recording time. During "quiet" hours, the concentration of "flat" oscillations was 56.2% of the total recording time. Fetal movements in "active" periods were observed to be 11 times more than in "quiet" hours. The study allows assuming that, at the gestational age of 16–20 weeks, the hypothalamo-hypophyseal system of a healthy fetus reaches the degree of maturation which is sufficient to provide well-developed capacities for adaptation to the environment. A healthy fetus at the gestational age of 16–20 weeks has pronounced daily rhythms of the heartbeat and locomotor activity. The fetuses in which we failed identify any distinctly expressed rhythms of heart beat and locomotor activity were included in the "risk" group. They were given a repeated observation following 3–4 weeks. Absence of clear-cut rhythmicity at 20–24 weeks gestation indicates developmental retardation. The periods that we classified as "active" are characterized by the predominance of "peak-like" and "rounded" types of cardiotachograms. These oscillations appear against the background of "mixed" types, which occupy the major portion of the 24-hour period. "Quiet" hours are characterized by the prevalence of "flat" cardiotachograms. We have determined "active" (9 a.m. to 2 p.m. and 7 p.m. to 4 a.m.) and "quiet" (4 a.m. to 9 a.m. and 2 p.m. to 7 p.m.) periods for the fetus. Stage 2 According to literature data [ 31 - 38 ], there are three types of fetal response to functional testing: "acceleration", "deceleration" and "zero-type" reaction. The best response to functional load is "acceleration". "Deceleration" points to a decrease in compensatory mechanisms, while "zero-type" reaction is indicative of an unsatisfactory condition of the fetus. The analysis of 24-hour fetal CTGs showed that in "active" hours the background cardiotachograms were represented by mixed-type (IV) oscillations within the range of 126.0 ± 3.4 bpm to 148.6 ± 3.8 bpm; the intra-minute fluctuations equaled 7.5 ± 2.0 bpm. At functional loading, an "acceleration" type response was observed. The baseline rhythm following the loading was 136.8 ± 1,3 bpm. Response time to loading made up 32.0 ± 1.1 sec [ 1 , 3 , 5 , 8 , 30 ]. Of the 2,500 fetuses investigated, FHR accelerations were seen in 2,004 (80.2%) of cases at fetal movements. The remaining 496 fetuses showed deceleration and "zero-type" oscillations induced by locomotor activity. During "quiet" hours (2 p.m. to 7 p.m.), the fetal background CTG's were represented by "flat" (type III) oscillations. FHR fluctuations were observed within the range of 131.6 ± 3.1 bpm with the average of 136.2 ± 2.9 bpm. The baseline rhythm was 138.2 ± 2.6 bpm. The intra-minute fluctuations were 1.9 ± 0.8 bpm to 4.5 ± 0.7 bpm with the average of 3.2 ± 0.7 bpm. Out of the 2,500 recordings of fetal external ECGs in "quiet" hours, fetal movements were recorded in 37 cases. No changes in FHR were observed in these cases. At functional loading in "quiet" hours the fetuses revealed "zero-type" response, i.e. there was no reaction at all. The baseline rhythm level following the loading was 137.6 ± 1.7 bpm. Evaluation of the 2,500 fetuses by Apgar Scale identified 2,257 cases (90.7%) with 8 to 10 points and 133 cases (9.3%) with 7 to 8 points. In summary, our investigation has clearly showed that in "active" hours a fetus with efficient compensatory-adaptive mechanisms responds to functional loads by HR acceleration (Fig. 12 ). No reaction is observed in "quiet" periods. However, the "zero"-type fetal reaction recorded by us within the period from 2 p.m. to 9 p.m. does not indicate unsatisfactory condition of the fetus but rather is suggestive of a definite reduction of functional levels of the fetal physiological systems, which is necessary for vital activity. Although conventionally recognized as an indicator of poor state of the fetus, this type only calls for precise attention when recorded in fetal "active" hours (Fig. 13 ). Figure 12 Response of the fetus to functional load in "active" hours ("acceleration" type) Figure 13 Response of the fetus to functional load in "quiet" hours ("zero" type) Conclusion Although we found no significant daily rhythmicity in heart rate per se in the human fetus, we developed a new method for the assessment of 24-hur fetal cardiotachogram that allowed us to identify daily rhythmicity in the short-term pattern of heart beating. The analysis of four types of oscillation – designated as "peak-like", "rounded", "flat," and "mixed" – revealed that "acceleration" and "deceleration" in the physiological functions of the fetus occurs in a way similar to that of an adult. "Active" hours (9 a.m. to 2 p.m. and 7 p.m. to 4 a.m.) and "quiet" hours (4 a.m. to 9 a.m. and 2 p.m. to 7 p.m.) were determined for the fetus. Fetal locomotor activity did not influence the course and cyclicity of the mother's nocturnal sleep. It can be assumed that the fetus, during the intrauterine development requires the availability of a relatively persistent homeostasis; the fetus employing its intrinsic adaptive capacities of responding to the rhythmical changes in the levels of maternal physiological functions becomes active when these levels are reduced and decreases its own physiological activity when they are elevated. A healthy fetus at the gestational age of 16–20 weeks has pronounced daily rhythms of the heartbeat and locomotor activity. Absence of clear-cut rhythms at 20–24 weeks gestation indicates developmental retardation. We showed that a fetus with efficient compensatory-adaptive mechanisms responds to functional loading by the heart beat acceleration in "active" hours. No reaction is observed in "quite" periods. However the "zero"-type fetal reaction recorded does not point to unsatisfactory condition of the fetus, but rather is suggestive of a definite reduction of functional levels of the fetal physiological systems which is necessary for vital activity. The "zero" type, conventionally recognized as an indicator of poor state of the fetus, should be taken into consideration merely in "active" fetal hours. The results of this study provide empirical bases for the assessment of the intrauterine state of the fetus, thus advancing the prognosis for both pregnancy and labor. The investigations have laid down the foundation for fetal chronobiology and chronotherapy, having established the principle of interdependence and conformity between maternal and fetal biological clock. Competing interests The author(s) declare that they have no competing interests. Authors' contributions Kintraia P – Supervisor of the study. Performed data analysis. Zarnadze M – Chief experimenter. Collected material for stages 1, 2, and 3. Conducted data analysis. Wrote the article. Kintraia N – Collected material for stages 2 and 3. Conducted data analysis. Kashakashvili I – Collected material for stage 3. List of abbreviations bpm – beats per minute CTG – cardio tachogram ECG – electrocardiogram EEG – electroencephalogram FHR – fetal heart rate h – hour(s) HR – heart rate NST – non-stress test sec – second(s)

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1079928

Molecular mechanisms in uterine epithelium during trophoblast binding: the role of small GTPase RhoA in human uterine Ishikawa cells

Background Embryo implantation requires that uterine epithelium develops competence to bind trophoblast to its apical (free) poles. This essential element of uterine receptivity seems to depend on a destabilisation of the apico-basal polarity of endometrial epithelium. Accordingly, a reorganisation of the actin cytoskeleton regulated by the small GTPase RhoA plays an important role in human uterine epithelial RL95-2 cells for binding of human trophoblastoid JAR cells. We now obtained new insight into trophoblast binding using human uterine epithelial Ishikawa cells. Methods Polarity of Ishikawa cells was investigated by electron microscopy, apical adhesiveness was tested by adhesion assay. Analyses of subcellular distribution of filamentous actin (F-actin) and RhoA in apical and basal cell poles were performed by confocal laser scanning microscopy (CLSM) with and without binding of JAR spheroids as well as with and without inhibition of small Rho GTPases by Clostridium difficile toxin A (toxin A). In the latter case, subcellular distribution of RhoA was additionally investigated by Western blotting. Results Ishikawa cells express apical adhesiveness for JAR spheroids and moderate apico-basal polarity. Without contact to JAR spheroids, significantly higher signalling intensities of F-actin and RhoA were found at the basal as compared to the apical poles in Ishikawa cells. RhoA was equally distributed between the membrane fraction and the cytosol fraction. Levels of F-actin and RhoA signals became equalised in the apical and basal regions upon contact to JAR spheroids. After inhibition of Rho GTPases, Ishikawa cells remained adhesive for JAR spheroids, the gradient of fluorescence signals of F-actin and RhoA was maintained while the amount of RhoA was reduced in the cytosolic fraction with a comparable increase in the membrane fraction. Conclusion Ishikawa cells respond to JAR contact as well as to treatment with toxin A with rearrangement of F-actin and small GTPase RhoA but seem to be able to modify signalling pathways in a way not elucidated so far in endometrial cells. This ability may be linked to the degree of polar organisation observed in Ishikawa cells indicating an essential role of cell phenotype modification in apical adhesiveness of uterine epithelium for trophoblast in vivo.

Background Human embryo implantation is a complex sequence of events which starts with adhesion of the blastocyst to the endometrial lining of the receptive uterus. In order to permit adhesion, uterine epithelial cells develop competence to bind trophoblast to their apical (free) poles [reviewed by [ 1 - 4 ]]. This receptive state of uterine cells is marked by numerous cellular modifications including a reduced thickness of the glycocalyx and a changed composition of proteins and glycoproteins in their apical plasma membrane [ 5 - 8 ]. Also the lateral and basal compartments of these cells are involved in those transformations showing e.g. increased depth and geometrical complexity of tight junctions laterally and a reduced contact to the substratum at the basal cell poles [reviewed by [ 2 ]]. Additionally, organisation of the cell architecture and signalling systems seem to be changed [ 3 , 9 - 16 ]. These modifications point to a reduction or destabilisation of apico-basal polarity of uterine epithelial cells in preparation for trophoblast adhesion during early embryo implantation [ 2 , 17 - 19 ]. In order to get more detailed insight into molecular processes in uterine epithelial cells when in contact with trophoblast, we have established an in vitro model simulating this cell-to-cell contact [ 20 - 22 ]. We use multicellular spheroids of human trophoblastoid JAR cells as a model for blastocyst trophoblast. These spheroids are delivered onto monolayers of human uterine epithelial RL95-2 cells serving as a model for the receptive uterine epithelium. We previously demonstrated that formation of stable cell-to-cell bonds depends in this model on RL95-2 cells' actin cytoskeleton (F-actin) and small GTPases of the Rho family, most likely RhoA. Data suggested that activation of Rho GTPases and co-ordinated rearrangement of F-actin within the apical and the basal poles of uterine epithelial cells in response to trophoblast binding are part of a generalised structural and functional reorganisation of the cellular architecture of uterine epithelial cells [ 23 - 25 ]. Although RL95-2 cells have proven a useful tool for these investigations, e.g. concerning the interaction between signal transduction events and cytoskeleton [ 14 , 23 ], this model can be criticised since these cells stably and rigidly express a non-polar epithelial phenotype [ 21 , 22 ]. Thus, they do not mimic exactly the in vivo uterine epithelium which destabilises and partially down-regulates (but nevertheless maintains a degree of) apico-basal polarity at receptivity. Therefore, an optimised model should consist of a human uterine epithelial cell line that has maintained polar organisation to a higher degree than RL95-2 cells but permits trophoblast attachment in order to simulate the in vivo situation more closely. As shown in the present communication, Ishikawa cells [ 26 ] meet these criteria at least to a certain extent. In addition, this cell line represents a possible alternative model for processes involved in human embryo implantation [ 27 - 30 ]. Data show that Ishikawa cells respond to JAR contact as well as to treatment with toxin A with rearrangement of F-actin and a redistribution of the small GTPase RhoA. Nevertheless, this reorganisation differs in various aspects from the characteristics of reaction known from other endometrial model cells investigated so far, e.g. RL95-2 cells [ 24 , 25 ]. This is probably due to the expression of a specific polar phenotype by Ishikawa cells indicating an essential role of cell phenotype modification in apical adhesiveness of uterine epithelium for trophoblast in vivo. Methods Antibodies and fluorescent dyes Mouse monoclonal antibody against RhoA (26CH:sc-418) was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Mouse monoclonal antibody against E-Cadherin (C20820) was obtained from Transduction Laboratories (Heidelberg, Germany). Mouse monoclonal antibody against α v integrin subunit (0770) was obtained from Dianova (Hamburg, Germany). 5-Chloromethylfluorescein diacetate (CMFDA, Cell Tracker Green; C-2925), the secondary antibodies AlexaFluor 488 goat anti-mouse IgG (A-11001), AlexaFluor 488 goat anti-rat IgG (A-11006) and AlexaFluor 633 goat anti-mouse IgG (A-21052) were obtained from Molecular Probes/MoBiTec (Goettingen, Germany). Tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin was obtained from Sigma (Taufkirchen, Germany). Cell culture The human endometrial cancer cell line Ishikawa was derived from an endometrial adenocarcinoma [ 26 ] and was cultured as previously described by the group of Schulz [ 31 , 32 ] from which the cells were obtained. The cells were maintained in MEM Dulbecco medium (Gibco, Eggenstein, Germany) supplemented with 15% FCS, 5 ml L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Boehringer, Mannheim, Germany). For electron microscopical experiments, cells were grown on poly-D-lysine-coated thermanox coverslips. For immunohistochemical stainings poly-D-lysine-coated glass coverslips were used as described previously [ 20 , 21 ]. As an invasive trophoblast model, multicellular spheroids of human choriocarcinoma JAR cells (ATCC: HTB 144) [ 33 ] were placed onto the free surface of endometrial cell monolayers. JAR spheroids were prepared as described previously [ 20 ], i.e. a suspension of 450,000 JAR cells per 6 ml RPMI 1640 medium (Gibco, Eggenstein, Germany) supplemented with 10% fetal calf serum (FCS) was agitated at 37°C on a gyratory shaker (Certomat R; Braun, Melsungen, Germany) at 110 rpm in order to form multicellular spheroids 72 h after initiation of culture. Attachment assay The JAR cell attachment assay was performed as described previously [ 20 ] with the following modifications [ 24 ]: In brief, JAR spheroids were stained with the fluorescent vital dye 5-chloromethylfluorescein diacetate (CMFDA) for 45 min at 37°C on a gyratory shaker. After incubation in JAR growth medium without CMFDA, spheroids were gently delivered onto confluent monolayers of endometrial Ishikawa cells. Poly-D-lysine-coated glass coverslips were taken as controls. After 60 min of co-culture in JAR medium at 37°C and 5% CO 2 , spheroid adhesion to the monolayers was quantified by centrifugation of the coverslips at 12 g for 5 min with spheroid surface facing down. As a measure for adhesiveness of monolayers, attached spheroids were counted and expressed as a percentage of the number of spheroids seeded initially. To inactivate small GTPases of the Rho family, confluent monolayers of Ishikawa cells were incubated with Clostridium difficile toxin A [ 34 ] for 24 h at a concentration of 100 ng/ml in growth medium. Then, the attachment assay was performed as described above. Controls were cultured for 24 h without toxin A. Preparation of subcellular fractions and Western blotting Ishikawa cells were suspended in ice-cold buffer A (20 mM Tris/HCl, pH 7.4, 2 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulphonylfluoride, 50 μg/ml soybean trypsin inhibitor, 10 μM pepstatin, 10 μM leupeptin and 2 μg/ml aprotinin) and thereafter disrupted by three cycles of freeze-thawing, using liquid nitrogen and a 37°C water bath. The resulting lysates were centrifuged at 17,000 × g for 5 min, thereby separated into the cytosolic fraction and the pellet. The latter was resuspended in ice-cold buffer A, supplemented with 1 % Triton X-100, sonicated 5 times for 10 s each and centrifuged as above, to produce in the cytoskeletal fraction [ 24 ]. The samples were boiled for 5 min in Laemmli buffer and proteins were separated by SDS-PAGE on 12.5 % acrylamide gels (per lane: lysate 10 μg, membrane and cytosolic fraction as well as cytosol 100 μg each), transferred to nitrocellulose membranes, and stained with an specific anti-RhoA antibody (dilution 1:1000; 1 h incubation). The membranes were then incubated with secondary antibody, and immunoreactivity was visualised by enhanced chemiluminescence (Amersham Pharmacia Biotech, Freiburg, Germany) as described before [ 24 ]. Immunofluorescence and F-actin staining Immunostaining was performed as described before [ 24 , 25 ]. Samples were fixed and permeabilised by a 1 + 1 mixture of ethanol-acetone for 10 min at room temperature. After rinsing, non-specific binding sites were blocked by incubation with 0.5% bovine serum albumine (BSA) in phosphate buffered saline (PBS) for 15 min. Then, samples were incubated for 90 min at 37°C with the primary antibody (see above), which was omitted in control stainings. Thereafter, cells were rinsed in PBS/0.5% BSA, incubated with the corresponding fluorescence-conjugated secondary antibody (see above) for 90 min at 37°C, again rinsed and mounted in PBS supplemented with 90% glycerol and 1% p-phenylendiamine. For staining of F-actin, samples were fixed with 3% paraformaldehyde for 15 min at room temperature, permeabilised by incubation with 0.05% Triton X-100 for 2 min and then incubated for 15 min with TRITC-phalloidin which was omitted in controls. Afterwards, samples were mounted in PBS/glycerol/phenylendiamine. In order to combine immunofluorescence and F-actin staining, samples were fixed with 3% paraformaldehyde for 15 min at room temperature and permeabilised in Triton X-100 according to the F-actin staining protocol. Then, primary antibody and TRITC-phalloidin were applied simultaneously for 90 min at 37°C. Both were omitted in controls. Thereafter, the immunostaining reaction was performed as described above. All samples were examined by confocal laser scanning microscopy. Confocal laser scanning microscopy, image analysis and processing Confocal microscopy was performed using a Zeiss Axiovert 100 M microscope attached to a confocal laser scanning microscopy system (CLSM) (model LSM 510; Carl Zeiss, Jena, Germany) as described previously [ 24 ]. As excitation sources, an argon laser with output at 488 nm and two helium-neon lasers with output at 543 and 633 nm, respectively, were available. Fluorescence emission of CMFDA was encoded as 'blue' after passing a 505–530 nm bandpass filter, emission of TRITC as 'red' after passing a 560–565 nm bandpass filter, and emission of Alexa Fluor 633 and Alexa Fluor 488 as 'green' after passing a 650 nm longpass filter and a 505–530 nm bandpass filter, respectively. Optical tomography was performed at 0.5 μm intervals using a 40-fold oil immersion objective with a numerical aperture of 1.3 NA and a pinhole size corresponding to a value of 1.0 of the airy disk. To improve the signal to noise ratio, each slice was scanned 8 times followed by averaging. All measurements were performed on several single Ishikawa cells. In monolayers without contact to JAR cells, examined Ishikawa cells were selected randomly from the entire photographic field. In monolayers with contact to JAR spheroids, only Ishikawa cells with definite membrane contact to JAR cells were included. Mismatching with JAR cells was avoided by pre-labelling those with CMFDA. To obtain semi-quantitative data, intensities of fluorescence signals of each channel were measured and expressed as average grey scale values (gsv) using the CLSM software (version 2.8 SR 1; Carl Zeiss). The apical-most and the basal-most slices of each stack were selected for analysis. Image Pro Plus software was used for image enhancement as well as spatial measurements of RhoA-positive granules (version 4.5; Media Cybernatics, Crofton, Md., USA). It was additionally equipped with a Gaussian filter module [ 35 ] and a homomorphic filter plugin [ 36 ]. Adobe Photoshop software (version 7.0; Adobe Systems, San Jose, Calif., USA) was used for the arrangement of RGB-colour images out of single grey scale images each representing the signal of one colour channel. Transmission electron microscopy Cells were grown as monolayers on thermanox coverslips as described above, rinsed twice in PBS and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 30 min at room temperature. After several washings in cacodylate buffer, samples were postfixed with 1% OsO 4 in cacodylate buffer, dehydrated with graded ethanol and propylene oxide, and embedded in epoxy resin mixture [ 37 ]. The embedded monolayers were separated from the thermanox coverslip by short-term heating on a hot plate. Ultrathin sections were mounted on 200-mesh copper grids, double-stained with uranyl acetate and lead citrate and examined with a Zeiss 902 A at 80 kV (Carl Zeiss, Jena, Germany). Statistical analysis In Fig. 4 , data are presented as medians (first – third quartile) with n denoting the number of experiments. Statistical analysis was performed by using both the Kruskal-Wallis test for global differences between groups and the Wilcoxon Signed Rank Sum test for their pairwise comparison. A value of p < 0.05 was considered significant. In Fig. 6 , data are presented as means ± SEM with n denoting the number of experiments. Paired and unpaired t-tests were applied as appropriate. A value of p < 0.05 was considered significant. Results I. Morphology of Ishikawa monolayers Ultrastructural features and localisation of adhesion molecules Ishikawa cells typically grow as monolayers as demonstrated by electron microscopy (Fig. 1 ). Individual cells showed a uniform size and a cylindrical cell shape. Nuclei were located predominantly in the basal compartment of the cells. Endoplasmatic reticulum, Golgi apparatus and mitochondria were arranged predominantly in the supranuclear region of the cells (Fig. 1A ). At their apical poles, membrane protrusions were found which varied in length and shape. Laterally (Fig. 1B ), tight junctions were located in the subapical regions and numerous interdigitations of adjacent membranes were seen. Additionally, adherens junctions and desmosomes were scattered along the lateral membranes whereas regular junctional complexes consisting of tight junction, adherens junction and desmosomes in apico-basal sequence were rarely found. As shown by immunohistochemistry, E-cadherin (Fig. 2A–D ) and integrin subunits α v (Fig. 2E–H ) as well as β 1 (data not shown) were detected in all regions of the plasma membrane of Ishikawa cells, including their apical membrane (Fig. 2A,E ). Figure 1 Ultrathin sections of Ishikawa cells. A : Ishikawa cells before treatment with toxin A. Cells grow as monolayers and show apico-basal polarity with nuclei located at the base of the cells and organelles found predominantly in the supranuclear region of the cells. Insert (light microscopy, cross section) shows overview of Ishikawa cells growing as monolayers. B : Lateral cell membranes show tight junctions, adherens junctions and desmosomes in varying combinations, whereas regular junctional complexes consisting of tight junction, adherens junction and desmosomes in apico-basal sequence were rarely seen. C : Ishikawa monolayers after treatment with toxin A. Standard electron microscopy showed no substantial differences between untreated (A) and toxin A-treated cells (C). Me: cell culture medium; N: nucleus; oo: coverslip; *: desmosome; arrow: adherens junction. Figure 2 Localisation of E-cadherin and integrin subunit α v . A-D : E-cadherin; E-H : integrin subunit α v , both detected by confocal laser scanning microscopy. Note that both adhesion molecules are located in all plasma membrane domains, including the apical one. Typical patterns are presented for apical (apical) and basal (basal) cell poles as well as for the middle part (mid) of cells. D,H: negative controls. F-actin and RhoA Staining of F-actin revealed a small band of strong fluorescence signal in the periphery of the cells (Fig. 3A,D ). Small F-actin-positive aggregates were observed apically corresponding in size and shape to membrane protrusions seen in electron microscopy (see above). Numerous stress fibres and focal contacts were detected in the basal cell poles (Fig. 3D ). In addition, the cytoplasm stained weakly. The intensity of F-actin staining was 1.6x higher in the basal compared to the apical cell poles of Ishikawa cells (31 (27–38) gsv apically vs. 50 (46–60) gsv basally; Fig. 4A ). Figure 3 Localisation of F-actin and RhoA in Ishikawa monolayers before binding of JAR spheroids. A, D : F-actin (red); B, E : RhoA (green); C, F : Merger of F-actin and RhoA signal. xy-sections representing second slices from the apical (apical) and basal (basal) cell poles, respectively. Typical patterns are presented. G, H : negative controls. Figure 4 Quantification of F-actin and RhoA. A: Quantification of F-actin in the apical and basal regions of monolayer cultured Ishikawa cells before (mono) and after (contact) binding of JAR spheroids as well as after treatment with toxin A (mono + toxin A). For semi-quantitative evaluation of fluorescence, grey scale values (gsv) of each colour channel were determined within double-labelled cells. Stacks of 6 xy -sections at 0.5 μm intervals were collected with the first marked slice at the apical cell surface and basal cell pole, respectively. Numbers of Ishikawa cells tested: n = 14 (mono), n = 12 (contact), n = 15 (mono + toxin A). Values differ significantly (p < 0.05) between these experimental groups but not between apical and basal cell poles within Ishikawa cells being in contact with a JAR spheroid (n.s.). Data are presented as medians (first – third quartile). B: Quantification of RhoA in the apical and basal regions of monolayer cultured Ishikawa cells before (mono) and after (contact) binding of JAR spheroids as well as after treatment with toxin A (mono + toxin A). Semi-quantitative evaluation of fluorescence was done as described under A. Values differ significantly (p < 0.05) between these experimental groups but not between apical and basal cell poles within Ishikawa cells being in contact with a JAR spheroid (n.s.). Data are presented as medians (first – third quartile). C: Diameter of RhoA-positive granules in Ishikawa cells before (mono) and after contact (contact) with JAR spheroids, number of cells tested: n = 14 (mono), n = 12 (contact). Values differ significantly (p < 0.05) between apical and basal cell poles in contact situation as well as between basal cell poles in contact vs. non-contact situation (*). n.d.: not detectable. Data are presented as medians (first – third quartile). In Ishikawa cells, measurement of RhoA fluorescence intensity revealed a 1.2x higher signal in the basal compared to the apical cell poles (26 (23–32) gsv apically vs. 30 (30–40) gsv basally; Fig. 4B ). A granular staining pattern was seen with single bigger granules within dominating fine-grained signals (Fig. 3B,E ). These granules showed a diameter of apically 550 (490–600) nm and of basally 520 (480–560) nm (Fig. 4C ) and were arranged preferentially in the periphery of the cells and along the F-actin aggregates (Fig. 3C,F ). Western blotting showed a relatively homogeneous distribution between membrane fraction and cytosol whereas the cytoskeletal fraction was nearly free of RhoA (Fig. 5 ). Figure 5 Immunoblot analysis of endogenously expressed RhoA. Ishikawa monolayers remained untreated (- toxin A) or were treated with 100 ng/ml toxin A for 24 h (+ toxin A). Preparation of cell lysates (lys), membranes (mem), cytoskeleton fraction (TX) and cytosol (cyt) of cells was performed as described. Proteins (lysates: 10 μg/lane; membranes, cytoskeleton fraction, cytosol: each 100 μg/lane) were separated by SDS-PAGE and subsequently immunoblotted. Data shown are typical for three independent experiments. Figure 6 Apical adhesiveness of Ishikawa cells. Apical adhesiveness for JAR cell spheroids of monolayer-cultured Ishikawa cells was determined in the centrifugal force-based adhesion assay before (mono; n = 349) and after (mono + toxin A; n = 358) treatment with Clostridium difficile toxin A. For comparison, adhesiveness of poly-D-lysine-coated glass coverslips (glass) for spheroids is shown ( n = 335). Values differ significantly (p < 0.05) between experimental groups except for Ishikawa cells treated with and without toxin A (n.s.). Data are presented as means ± SEM. II. Molecular mechanisms of apical adhesiveness of Ishikawa monolayers for JAR spheroids Adhesiveness for JAR spheroids In order to determine adhesiveness of the apical cell poles of Ishikawa cells for trophoblastoid cells, multicellular spheroids of JAR cells were placed onto confluent monolayers of Ishikawa cells. As shown in Fig. 6 , the majority of seeded JAR spheroids adhered to the Ishikawa monolayers (80 ± 14 %). 37 ± 8 % of JAR spheroids adhered to poly-D-lysin-coated glass coverslips serving as controls. Therefore, significantly more JAR spheroids adhered to Ishikawa monolayers as compared to controls, i.e. Ishikawa cell monolayers permitted JAR adhesion. Morphology of the contact site Morphology and cytoskeletal details of the contact site was examined by fluorescence confocal microscopy after tracking JAR spheroids with CMFDA and staining of F-actin in Ishikawa cells and JAR cells with phalloidin-TRITC (Fig. 7A ). CLSM revealed that a large membrane contact area was formed between the JAR spheroid and the Ishikawa monolayer. All cells of the lower part of the spheroid that were exposed to the monolayer and all adjacent cells of the latter participated in the adhesion interaction. Figure 7 Localisation of F-actin and RhoA in Ishikawa monolayers after binding of JAR spheroids. A : xz-section through contact site of Ishikawa cells and JAR spheroid after tracking of JAR cells with the vital dye CMFDA (blue). red: F-actin cytoskeleton. B -I: Localisation of F-actin (red) (B, E) and RhoA (green) (C, F) in the apical (apical) and basal (basal) regions of Ishikawa monolayers after binding of JAR spheroids tracked with CMFDA (blue). xy-sections represent second slice from the apical and basal cell poles, respectively. D, G: Merger of F-actin and RhoA signal. Typical patterns are presented. H, I: negative controls. F-actin and RhoA at the contact site After confrontation culture of Ishikawa monolayers and JAR spheroids (Fig. 7A ) the distribution of F-actin and of RhoA was examined by CLSM in detail (Fig. 7B–I ). The asymmetric distribution of F-actin along the apico-basal axis found in Ishikawa monolayers without contact to JAR spheroids disappeared in the contact area. The distinct F-actin aggregates observed in the apical poles of non-contact Ishikawa cells were not detectable any longer but an increased amount of stress fibres basally as well as of homogeneous staining was seen (Fig. 7B,E ). Thus, a significant increase in fluorescence intensity was detected in the contact zone with regard to F-actin, i.e. 103 (95–112) gsv apically and 106 (103–109) gsv basally when compared to values before contact (Fig. 4A ). The apico-basal asymmetry seen in the pre-contact situation was lost after contact also with respect to RhoA (Fig. 7C,F ). The overall signal of RhoA increased significantly in the apical and basal regions of the cells after attachment of JAR spheroids, i.e. 66 (63–68) gsv apically vs. 65 (62–68) gsv basally (Fig. 4B ). Also the diameter of RhoA-positive granules increased significantly at the basal cell poles from 520 (480–560) nm to 650 (590–690) nm in the contact area (Fig. 4C ). Apically, the mean diameter (540 (510–610) nm) did not differ significantly from the non-contact situation (550 (490–600) nm; Fig. 4C ). Ishikawa monolayers after treatment with Clostridium difficile toxin A In order to determine the role of RhoA for adhesiveness of Ishikawa cells, Rho GTPases were inhibited by treatment with Clostridium difficile toxin A (toxin A). Standard electron microscopy showed no substantial difference between untreated (Fig. 1A ) and toxin A-treated cells (Fig. 1C ). Upon treatment, F-actin fluorescence decreased significantly to 24 (22–25) gsv in the apical and 40 (39–41) gsv in the basal cell poles. (Fig. 4A ). In contrast, RhoA intensities increased significantly in the apical as well as in the basal cell poles (after treatment: 48 (43–50) gsv apically vs. 82 (80–85) gsv basally; Fig. 4B ). A changed subcellular distribution of RhoA was found by Western blotting (Fig. 5 ). The amount of RhoA was reduced in cytosolic fraction with a comparable increase in the membrane fraction while the cytoskeleton fraction remained free of detectable RhoA. After incubation of Ishikawa cells for 24 h with 100 ng/ml toxin A and subsequent attachment assay, 86 ± 15 % of seeded JAR spheroids adhered to the monolayers (Fig. 6 ). This was no significant difference to untreated monolayers where 80 ± 14 % of JAR spheroids adhered. Discussion The formation of stable cell-to-cell bonds to trophoblast cells requires a rearrangement of the cytoskeleton (F-actin) and redistribution of small GTPases of the Rho family in endometrial cells [ 23 - 25 ]. Consistent with this, F-actin and RhoA increase significantly in Ishikawa cells being in contact with JAR spheroids. This may be part of a generalised structural and functional reorganisation of the cellular architecture as the co-ordinated rearrangement is observed in the apical as well as in the basal cell poles. Interestingly, Ishikawa cells equalise F-actin and RhoA signals in the apical and basal cell poles in response to JAR contact. This is in contrast to another endometrial cell line, e.g. RL95-2, which inverts the gradient between apical and basal cell poles as shown previously [ 25 ]. This identical trend but different extent of modification of apico-basal polarity might be due to differences between Ishikawa and RL95-2 cells in the initial subcellular distribution of RhoA found before JAR contact: in Ishikawa cells, large amounts of RhoA were detected in the membrane fraction as well as in the cytosol but in RL95-2 considerable quantities of RhoA were only seen in the membrane fraction [ 24 ]. These differences may even indicate that distinct signalling pathways are involved which comprise a specific subset of RhoA-specific guanine nucleotide exchange factors (GEFs), trigger GTP loading, and thus activation of RhoA [ 38 , 39 ]. Indeed, RhoA and its GEFs have been reported to be present in caveolae and lipid rafts. These compartmentalised membrane signalling domains are believed to confer specificity to the complex mechanisms of RhoA signalling [ 39 , 40 ]. The fact that Ishikawa cells are not quite as adhesive for JAR spheroids as RL95-2 cells may be based on such differences [ 21 ]. After treatment with toxin A which inhibits the family of small Rho GTPases, F-actin decreases significantly in apical and basal poles of Ishikawa cells while RhoA increases significantly. In addition, adhesiveness of Ishikawa cells to JAR spheroids is not affected. Regarding this, one could argue that toxin A uptake is insufficient in Ishikawa cells but marked changes in F-actin staining with significant decrease in fluorescence intensity as well as changes in distribution of RhoA clearly contradict this. A hypothetical possibility might be that Ishikawa cells use alternative signalling cascades which compensate inhibition of small Rho GTPases. This may include an increased synthesis of Rho proteins at a level which cannot prevent morphological changes but may allow to maintain special signalling cascades resulting in preserved adhesiveness for JAR spheroids. Additionally, Ishikawa cells may be able to replace small GTPases of the Rho family by other members of the superfamily of GTPases. Indeed, it is established that different Rho GTPases can influence each other's activities [ 38 - 41 ]. It has been even proposed recently that Rho signalling may have profound effects on Rap signalling, the latter known to modulate the organisation of the actin cytoskeleton as well [ 42 - 44 ]. The response of Ishikawa cells to toxin A is in contrast to the previously described behaviour of RL-95-2 cells which show a different distribution of RhoA in Western blotting after treatment with toxin A as compared to Ishikawa cells [ 24 ]. Furthermore, adhesiveness for JAR spheroids is lost in RL95-2 cells after toxin A treatment. These differences may be due to alternative signalling cascades which may also be responsible for the different degree of apico-basal polarity in both cell lines. Indeed, Ishikawa cells have preserved major aspects of apico-basal polarity typical for simple epithelia as shown here. Nevertheless, the modified composition of junctional complexes as well as the generalised membrane localisation of E-cadherin and integrins imply a slight downregulation of the polar epithelial phenotype [ 19 ]. These modifications may be a prerequisite for the fact that also Ishikawa cells do react to JAR cell binding with molecular reorganisation in the apical as well as in the basal regions. In further investigations, it would be interesting to use a model system that mimics the in vivo situation even closer. Immortalised endometrial cells may be an appropriate experimental model in this respect [e.g. [ 45 ]]. Our findings indicate that the host cell response is indeed a complex process involving a reorganisation of the whole cell architecture [ 17 , 18 ] and not just the apical cell pole as suggested by concepts focussing on the apical plasma membrane [ 12 ]. It remains to be seen whether and to what extent this process may be governed by the same type of regulating genes and cellular mechanisms as involved in epithelial fusion processes during development and epithelial-mesenchymal transitions (EMT) [ 2 , 17 ]. In particular, the postulated master genes and specific signalling cascades studied in connection with complete EMT processes [ 46 - 50 ] should be of interest here. Conclusion Data presented here support the hypothesis that the actin cytoskeleton and the small GTPase RhoA play an important role in embryo implantation. As toxin A cannot prevent attachment of JAR spheroids to Ishikawa cells, these cells seem to be able to modify signalling pathways in a way not elucidated so far in endometrial cells. This ability may be linked to the peculiar polar epithelial phenotype observed in Ishikawa cells. This is consistent with the notion that the extent of polar organisation of uterine epithelial cell lines influences their responses to JAR cell contact implying an essential role of cell phenotype modification in apical adhesiveness of uterine epithelium for trophoblast. Competing interests The author(s) declare that they have no competing interests. Authors' contributions CH conceived of the study and carried out the confocal laser scanning microscopical and electron microscopical investigations. MS provided expertise in small Rho GTPases and conducted biochemical experiments. HWD participated in the design of the study and helped to draft the manuscript. MT conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

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1079929

Unaltered TNF-α production by macrophages and monocytes in diet-induced obesity in the rat

Background Recent findings have established an association between obesity and immune dysfunction. However, most of the studies investigating the effects of obesity on immune function have been carried out in genetically obese rodent models. Since human obesity is mostly due to intake of a high fat diet and decreased energy expenditure, we asked whether immunological defects also occur in diet-induced obesity. Specifically, we focused on the function of monocytes and macrophages, as these cells are thought to be involved in the low-grade inflammation present in obesity. Methods Male Sprague-Dawley rats were fed a high-fat or a standard chow diet for either 2 or 10 weeks. At the end of the intervention period animals were anaesthetised, blood collected for determination of plasma mediator concentrations and lipopolysaccharide (LPS) stimulated production of TNF-α by monocytes. LPS stimulated production of TNF-α in alveolar macrophages was also determined. Results High-fat feeding for either 2 or 10 weeks resulted in significant increases in fat mass and serum leptin. Although increased serum leptin has previously been linked to modulation of innate immunity, we found no significant difference in the LPS stimulated production of TNF-α by either blood monocytes or alveolar macrophages between the dietary groups. Furthermore, we failed to find a significant increase in circulating TNF-α concentrations in obese animals, as reported for genetically obese animals. Conclusion Our data suggest that defects in innate immune function observed in genetically obese animals are not mimicked by dietary obesity, and may more likely reflect the gross abnormality in leptin function of these models. Further work is required delineate the effects of dietary obesity on inflammatory state and immune function.

Background Obesity is a very common chronic disease that poses significant health risks such as diabetes, cardiovascular diseases and hypertension. This pathological condition is characterized by complex neuroendocrine changes in the brain as well as in the periphery, involving mediators such as neuropeptide Y (NPY) and leptin [ 1 ]. Additionally, there have been several reports demonstrating that obesity is associated with altered immune function and a chronic low-grade inflammatory status [summarized by [ 2 , 3 ]]. Specifically it has been reported that obese individuals have a higher incidence and severity of infectious diseases [ 4 ]. These defects also include disturbances in macrophage mediated phagocytosis and pro-inflammatory cytokine production [ 5 ] as well as increased sensitivity to endotoxin-induced lethality [ 6 ]. To date, experimental approaches to the investigation of this novel link between obesity and immune function have been predominantly carried out in genetic models of obesity that either lack leptin ( ob/ob mouse) or the long form of the leptin receptor ( db/db mouse). However, leptin mutations only account for a small fraction of obesity in humans with the majority of obesity linked to overnutrition and reduced energy expenditure [ 7 ]. With leptin resembling several aspects of a cytokine and exerting various immunological functions [reviewed by [ 8 ]], it is unclear whether these models examine the effects of obesity in general or rather the effects of a defective leptin system on immune function. This question needs to be addressed by investigating immune function in diet-induced models of obesity. Monocytes and macrophages are major cellular components of the innate branch of the immune system. With their ability to produce cytokines, e.g. tumour necrosis factor-α (TNF-α), in response to bacteria and bacterial fragments, such as LPS, monocytes and macrophages are essential to the first line of defence at contact sites between the interior and the exterior, such as the mucosa of the lungs or the gastrointestinal tract. Notably, increasing evidence suggests that macrophages also play an important role in the development of the low-grade inflammation that is present in obesity. Recent work demonstrates that macrophages infiltrate the adipose tissue and that these cells are integral to the low grade-inflammation [ 9 ]. However, many questions remain unanswered regarding the precise role of monocytes and macrophages in the course of obesity. For example, it is of great interest to examine whether the functional changes described within the adipose tissue are intrinsic to the macrophages, or whether these defects result from the interaction with the local microenvironment in the adipose tissue. If intrinsic macrophage defects are responsible for the described alterations in obesity, similar defects should also be present in other macrophage compartments. Therefore, the aim of the present study was to investigate macrophage and monocyte function in compartments other than the adipose tissue of obese animals, specifically the lungs and the blood. In order to examine monocyte and macrophage function in diet-induced obesity, we subjected male Sprague Dawley rats to a cafeteria-style diet lasting either 2 weeks (short term) or 10 weeks (long term). In this way we could examine the effects of diet per se and of established obesity. Litter mates received normal rodent chow diet. Upon completion of the dietary intervention, blood monocytes and alveolar macrophages were collected and stimulated with LPS in vitro . Under these conditions LPS induces strong production of the pro-inflammatory mediator TNF-α [ 10 ]. As leptin and sympathetic activation impact on immune function [ 11 , 12 ], plasma concentrations of NPY, a marker for sympathetic nervous system activity [ 13 , 14 ], and leptin, as well as TNF-α were also determined. Materials and methods Animals Male Sprague-Dawley rats were kept under controlled light (06.00–18.00 h) and temperature (20 ± 2°C) conditions with ad libitum access to water. Five week old rats (n = 18) were randomly divided into two groups. The control group ("controls", n = 9) was fed standard laboratory chow (12.5% calories as fat) and the second group ("high-fat diet", n = 9) was presented with a highly-palatable high-fat cafeteria-style diet (35% calories as fat), consisting of meat and pastry pies, pasta and cake and supplemented chow. Two different sets of experiments were conducted. The first series ("long term diet") was maintained for 10 weeks, whereas a second series ("short term diet") was only fed for 2 weeks. Both experimental sets consisted of control animals and diet-induced obese animals that were assigned to groups of similar starting weights. Body weight and caloric intake of all rats was monitored weekly. All procedures were approved by the Animal Experimentation Ethics Committee of the University of Melbourne. Collection of tissues At the completion of the dietary period, the animals were anaesthetized with pentobarbital (Nembutal, 100 mg/kg, Merial Australia Pty Ltd, Australia). Cardiac puncture (3 ml) was performed using a heparinised syringe to collect blood for full blood stimulation, and to allow preparation of plasma for determination of plasma mediator concentrations. Retroperitoneal white adipose tissues and the spleen were removed and weighed. Bronchoalveolar lavage To obtain alveolar macrophages, anaesthetized rats were subjected to bronchoalveolar lavage (BAL). The lungs were rinsed with 10 ml of cold, sterile PBS via a cannula placed into the trachea. The lungs were washed twice and total cell counts and viabilities were determined by ethidium bromide/acridine orange (Molecular Probes, Oregon, USA) fluorescent viability stains using a Neubauer hemocytometer. Cytocentrifuge preparations (Shandon Cytospin 3) using 100 μl of BAL were differentiated according to standard morphological criteria counting at least 500 cells (DiffQuik, Zeiss, Germany). BAL fluid contained between 97–99% alveolar macrophages. Alveolar macrophages were adjusted to 500,000 cells/250 μl and stimulated for 3 h at 37°C in the presence of various concentrations of LPS (0.001–10 μg/ml, E. Coli Serotype 026:B6, Sigma). Supernatants were collected and stored at -80°C for measurement of TNF-α. Full Blood stimulation Various concentrations of LPS (0.01–10 μg/ml) were added to 250 μl full blood and incubated for 3 h at 37°C. Upon completion of the incubation, samples were centrifuged and supernatants were stored at -80°C for measurement of TNF-α. Detection of TNF-α, NPY and Leptin All reagents were endotoxin-free to ensure that TNF-α was not artifactually induced, except where LPS was deliberately used. The concentration of TNF-α in the supernatants and plasma samples was determined by a commercially available ELISA kit (Pharmingen, Merckville, Australia) with standard concentrations ranging from 4–1000 pg/ml. Plasma leptin concentrations were measured using a commercially available radioimmunoassay kit (Linco, Missouri, USA) while NPY was measured using an in house assay utilising a rabbit antibody and 125 I-NPY (2000 Ci/mmol, Amersham, Australia) as previously described [ 13 ]. Statistics Student's unpaired t-test was used to determine significant differences for organ masses and concentrations of NPY, leptin and TNF-α. Data from BAL and full blood stimulation was analysed using one-way ANOVA and body weight data was subjected to ANOVA for repeated measures with subsequent LSD if p-values were below p < 0.05. Differences where p-values were <0.05 are considered significant. Statistics were performed using GraphPadPrism 3.0 for Windows. Results Effect of high-fat diet on caloric intake, body weight and organ mass Exposure of animals to the high-fat diet led to significant increases in caloric intake (p < 0.05; Table 1 ) and body weight from 3 weeks (p < 0.05; Fig. 1 ). Animals on the high-fat diet continued to gain weight and at the completion of the 10 week dietary intervention weighed 23% more than their respective controls. Even though body weight was not different, retroperitoneal white adipose tissue was already significantly increased after 2 weeks on the diet (p < 0.05; Table. 1 ). Continued exposure to the high-fat diet lead to progressive increases in caloric intake, and adipose tissue mass, which was 2.8 fold higher than the control animals at 10 weeks of diet (Table 1 ). Net spleen weight was significantly depressed (p < 0.05) after 2 weeks of high-fat diet. Although there was a tendency for reduced spleen mass after 10 weeks of dietary intervention, this did not reach statistical significance (Table 1 ). Table 1 Parameters of the model of diet-induced obesity. Short term diet (2 weeks) Long term diet (10 weeks) Chow High Fat Chow High Fat Caloric intake (cal/day) 95.6 ± 3.0 178.6 ± 19.1* 97.6 ± 11.3 229.9 ± 8.9* Body weight (g) 287.5 ± 2.2 302.3 ± 5.0 515.6 ± 9.1 635.3 ± 12.3 White adipose tissue (g) 1.4 ± 0.1 2.7 ± 0.2 * 4.5 ± 0.4 12.4 ± 1.5* Spleen (g) 0.88 ± 0.03 0.79 ± 0.02* 0.97 ± 0.04 0.89 ± 0.05 TNF-α (pg/ml) 5.9 ± 0.3 7.2 ± 0.9 ND ND * p < 0.05 (high fat vs. chow fed); n = 9 for all groups ND = not detectable; detection limit 5.6 pg/ml Figure 1 Body weight (g) of diet-induced obese (grey squares) and control (black squares) Sprague-Dawley rats following exposure to a cafeteria-style high fat diet or standard laboratory chow. Results are expressed as mean ± SEM (n = 9 diet-induced obese rats, n = 9 control rats). Data were analysed by ANOVA for repeated measures and significant differences (p < 0.05) are indicated by asterisks. Diet-induced effects on plasma leptin, NPY and TNF-α Consumption of a high fat diet was associated with significant increases in plasma leptin concentrations (p < 0.05; Fig. 2 ). Even after 2 weeks on diet, leptin concentrations had more than doubled, at a time when body weight was not significantly elevated (Table 1 ). The chow fed rats also showed an increase in leptin concentrations from 2 to 10 weeks (Fig. 2 ), reflecting their increase in body weight and fat mass over time (Table 1 ). When plasma NPY concentrations were compared, consumption of the high-fat diet led to a significant increase (p < 0.05) in the short-term, whereas no change was observed after 10 weeks on diet (Fig. 2 ). There was no age-related change in plasma NPY concentrations in chow fed animals, indicating the absence of age-related effects on plasma NPY concentrations over this time period. Figure 2 Plasma leptin (Fig. 2A) and NPY (Fig. 2B) concentrations after both types of dietary intervention: short term chow and fat diet for 2 weeks (STC/STF) and long term chow and fat diet (LTC/LTF). Results are expressed as mean ± SEM (n = 9 diet-induced obese rats, n = 9 control rats). Data were analysed by t-test: * p < 0.05, *** p < 0.0001. Despite other reports of increased plasma TNF-α concentrations in obesity [ 15 ], under the conditions used in this study, we failed to detect a significant difference between the chow and high-fat fed animals. In the older age group TNF-α levels were below the detection limit of the assay. LPS induced TNF-α production in full blood preparations To examine whether the high-fat diet modulates the ability of blood monocytes to produce TNF-α in response to LPS, full blood preparations from both chow and high-fat fed animals were compared. Ex vivo LPS-stimulation of full blood preparations resulted in a dose-dependent increase in the production of TNF-α (Fig. 3 ). However, the response to LPS did not differ significantly between animals fed chow and the high-fat diet at both time points examined (short term and long term diet, Fig. 3A and 3B ). Figure 3 Stimulation of full blood preparations obtained from high-fat fed rats (grey bars) and control animals (black bars) after 2 weeks (short term) and 10 weeks on diet (long term) with increasing concentrations of LPS. Results are expressed as mean ± SEM (n = 9 diet-induced obese rats, n = 9 control rats). Stimulation of alveolar macrophages with LPS In order to examine whether the dietary intervention had any effect on functional parameters of tissue-borne macrophages, alveolar macrophages were stimulated with LPS in vitro . Increasing concentrations of LPS resulted in a dose-dependent, significant increase of the production of TNF-α by alveolar macrophages (Figure 4 ). There was no significant difference in the degree of stimulation by LPS in animals fed the high-fat diet for 2 or 10 weeks. Even though the basal production of TNF-α under these circumstances was not significantly different, high fat fed animals tended to have higher basal TNF-α levels, thus the proportional increase in the high-fat animals, expressed as percent change from basal, is suppressed in comparison to chow fed animals, particularly after long-term high fat feeding (13,747% versus 19,589% at 10 μg/ml LPS in fat and chow fed rats respectively). Figure 4 LPS stimulation of isolated alveolar macrophages after 2 weeks (short term) and 10 weeks on diet (long term) of high-fat fed rats (grey bars) and control animals (black bars). Results are expressed as mean ± SEM (n = 9 diet-induced obese rats, n = 9 control rats). Discussion We have previously extensively characterised the model of dietary obesity used in the current study [ 16 , 17 ]. Animals increase caloric intake on presentation of the diet, and show significant weight gain within 3 weeks. Reproducible increases in adiposity and plasma leptin concentrations occur within 2 weeks of high fat feeding, as demonstrated in the present study. The majority of studies seeking to investigate the link between obesity and the immune system have carried out in genetic models of obesity. For example, defects in specific immunity, such as reduced lymphocyte numbers in spleen, thymus and the peripheral blood have been reported in ob/ob or db/db mice, and Zucker rats [ 2 ]. Furthermore, innate immune function seems also to be affected in genetic animal models of obesity. Specifically, it has been reported that macrophages from genetically obese animals have a reduced ability to eliminate Candida albicans and to produce proinflammatory cytokines [ 18 ]. While few studies have investigated immune function in diet-induced obesity, some changes in cellular and humoral immunity have been shown [ 19 , 20 ], however there is still no information on inflammatory immune function. Leptin has also been demonstrated to modulate several functional immune parameters [ 6 , 8 ], and a recent study in humans demonstrated that leptin activates neutrophils indirectly by stimulating monocytes to release TNF-α[ 21 ]. We therefore asked whether diet-induced obesity, which is associated with significantly increased leptin levels and more closely resembles the most common form of human obesity than genetically modified models [ 22 ], would have an impact on innate immune functions. However, in the current study we found no alteration in the ability of macrophages and monocytes to release TNF-α to an LPS challenge. We focused on blood monocytes and lung alveolar macrophages, as these cells are primary components of the innate branch of the immune system. Furthermore, with the current suggestion of a role for macrophages in driving the low-grade inflammation present in the adipose tissue [ 9 ], this approach also allowed us to evaluate whether intrinsic macrophage defects are present in obesity, as such defects would also occur in tissues other than the adipose tissue. Macrophages and monocytes produce TNF-α in response to innate immune stimuli such as LPS, which is essential for host defence against bacterial and other pathogens [ 23 ]. Our results demonstrate no obvious changes in the production of TNF-α by blood monocytes after 2 or 10 weeks of dietary intervention. It is possible that even though obesity had no influence on blood monocyte function, that the complex changes associated with obesity exert a functional influence on mature tissue-borne macrophages. However, using BAL-derived alveolar macrophages, we found no statistical difference in the net TNF-α response of alveolar macrophages upon LPS stimulation when comparing obese and control animals. Interestingly, when we analysed the percentile increase above basal TNF-α production at 10 weeks of diet, the high-fat fed animals appeared to have a blunted response to LPS, suggesting that alveolar macrophages from high-fat fed animals cannot be stimulated as strongly as the cells from the control animals. More recently, obesity itself has been viewed as an inflammatory process [ 3 , 24 , 25 ] and studies in humans have demonstrated that weight loss can reduce inflammatory markers [ 26 ]. Thus recent attention has been focused on cytokines such as TNF-α and IL-6 [ 27 ]. TNF-α, formerly known as cachexin [ 28 ], has been studied in both animal models and human obesity. Some studies have shown that in humans increasing concentrations of leptin are correlated with soluble TNF-α receptors, suggesting the development of a pro-inflammatory state as body weight increases [ 29 ]. Adipose tissue itself is capable of producing TNF-α, and increased TNF-α concentrations in elderly subjects are correlated with truncal fat mass [ 30 ]. Several investigators have also reported increased plasma levels of TNF-α in genetic models of obesity. For example, plasma TNF-α concentrations were doubled in mice that were obese due to a defect in the growth hormone gene [ 15 ]. In our hands plasma TNF-α was not dramatically affected by the high fat diet, however this may be partly due to the fact that the values were very close to the detection limit of the assay. It is also probable that this discrepancy may highlight species differences, but it could also indicate that genetic obesity and diet-induced obesity impact differently on the regulation of TNF-α levels. Changes in TNF-α may be more consistent when there is a predominant genetic basis to the obesity where the level of obesity is usually more extreme [ 28 ]. Alteration in TNF-α may be tissue specific as shown by a recent study that proposes macrophage-related inflammatory activities in adipose tissue play a role in obesity-related insulin resistance [ 31 ]. Interestingly, we also found a significant increase in the concentration of circulating NPY after 2 weeks of dietary intervention. As peripheral NPY is predominantly derived from sympathetic nerve terminals, plasma NPY concentrations can be considered a marker of sympathetic nervous activity [ 13 , 14 ]. Thus, based on the present findings and previous reports of postprandial sympathetic activation [ 32 ], we propose that short term dietary excess increases sympathetic nervous activity. Increased sympathetic activity increases energy expenditure [ 33 ], and might therefore represent an endogenous mechanism to counteract weight gain. However, the question arises as to why plasma NPY levels are not different after 10 weeks of diet. Previous studies have shown that the changes in sympathetic nervous activity may be bed specific [ 34 ], with a higher renal and lower cardiac noradrenaline spillover in obese individuals [ 34 ]. Overall whole body sympathetic nervous activity in obese subjects was normal, which may explain why we do not see a change in plasma NPY levels after long-term diet exposure. Conclusion Since specific gene defects only account for a small proportion of obesity in humans [ 7 , 35 ], this study was designed to investigate whether the functional defects in the monocyte/macrophage system described in genetically obese animal models are also present in an animal model of diet-induced obesity, that more closely resembles human obesity. The absence of any significant effects of diet-induced obesity on critical functional parameters of the monocyte/macrophage system used here raises the important question as to whether the changes in the production of pro-inflammatory cytokines observed in genetically obese animals actually result from the complex pathophysiology of obesity or are rather a consequence of the leptin defect present in these models. Our results favour the latter notion since our animals exhibit all the characteristics of obesity, yet do not display a comparable defect in the monocyte/macrophage system. Our results also show that monocyte and macrophage function in extra-adipose compartments is normal, suggesting that the chronic inflammatory state present in the adipose tissue during obesity is not a consequence of functional defects in the monocyte/macrophage system. One remaining possibility for our finding is that the period of overnutrition used does not reflect the changes observed in more chronic obesity. While our results do not support a major effect of obesity on the markers of innate immune function used here, it does not rule out effects in other immune competent tissues, such as the endothelium. Clearly further work is required to delineate the possible effects of obesity on immune function, in light of the escalating burden of this disease. Competing interests The authors declare that they have no competing interests. Authors' contributions SaB participated in experimental design, carried out most experimental procedures and put together the manuscript. EV carried out the radioimmunoassays and helped to draft the manuscript. StB participated in sample collection and designed the LPS protocol. JEJ helped with sample collection. GPA participated in experimental design. MJM conceived of the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

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1079930

Wearable kinesthetic system for capturing and classifying upper limb gesture in post-stroke rehabilitation

Background Monitoring body kinematics has fundamental relevance in several biological and technical disciplines. In particular the possibility to exactly know the posture may furnish a main aid in rehabilitation topics. In the present work an innovative and unobtrusive garment able to detect the posture and the movement of the upper limb has been introduced, with particular care to its application in post stroke rehabilitation field by describing the integration of the prototype in a healthcare service. Methods This paper deals with the design, the development and implementation of a sensing garment, from the characterization of innovative comfortable and diffuse sensors we used to the methodologies employed to gather information on the posture and movement which derive from the entire garments. Several new algorithms devoted to the signal acquisition, the treatment and posture and gesture reconstruction are introduced and tested. Results Data obtained by means of the sensing garment are analyzed and compared with the ones recorded using a traditional movement tracking system. Conclusion The main results treated in this work are summarized and remarked. The system was compared with a commercial movement tracking system (a set of electrogoniometers) and it performed the same accuracy in detecting upper limb postures and movements.

Background This work deals with the development of an innovative measuring system devoted to the analysis of the human movement. Our main aim is to provide a valid alternative comfortable instrumentation useful in several rehabilitation areas. In particular we focused our attention on the remote treatment of post-stroke patients [ 1 ]. The analysis of human movement is generally performed by measuring kinematic variables of anatomic segments by employing accelerometers, electrogoniometers, electromagnetic sensors or cameras integrated in finer equipment as stereophotogrammetric systems. In remote rehabilitation tasks, several disadvantages derive from the use of these technologies, which are mainly applied in the realization of robotics or mechatronics machines (such as MIME or MIT-MANUS [ 2 ]) which result invasive, complex and often unable to satisfy safety requirements for the presence of mechanical parts in movement. In literature, several studies are devoted to realize electric devices with properties of hight wearability [ 3 - 5 ]. The main drawbacks of wearable sensing systems available on the market are their weight, the rigidity of the fabric which they are made of, the dimension of the sensors used, and all the other properties which make them obtrusive. In particular, conventional sensors often require the application of complex and uncomfortable mechanical plug in order to position the sensors on garments. In the present work, we focused our efforts in the realization of a new system for the measurement of the human upper limb kinematic variables based on a sensorized garment, the Upper Limb Kinesthetic Garment (ULKG). Lightness, adherence and elasticity have been privileged in the ULKG realization as fundamental requirements for its unobtrusivity. These guidelines have led us to choose an elastic fabric (Lycra) to manufacture it as a sensorized shirt. In order to equip the lycra shirt with a sensing apparatus, sensors have been spread on the fabric by employing an electrically conductive elastomer (CE). CE deposition does not change the mechanical characteristics of the fabric. It preserves the wearability of the ULKG and it confers to the fabric piezoresistive properties related to mechanical solicitations. This property has been exploited to realize many other sensorized garments as gloves, leotards, seat covers capable of reconstructing and monitoring body shape, posture and gesture [ 6 ]. Furthermore, by using this technology, both sensors and interconnection wires can be smeared by using the same material in a single printing and manufacturing process. This is a real improvement in terms of comfort performed by the device because no metallic wires are necessary to interconnect sensors or to connect them to the electronic acquisition unit. In this way no rigid constraints are present and movements are unbounded. Methods Materials CE composites show piezoresistive properties when a deformation is applied and can be integrated into fabric or other flexible substrate to be employed as strain sensors. Integrated CE sensors obtained in this way may be used in posture and movement analysis by realizing wearable kinesthetic interfaces [ 7 ]. The CE we used is a commercial product by WACKER Ltd (Elastosil LR 3162 A/B) [ 8 ] and it consists in a mixture containing graphite and silicon rubber. WACKER Ltd guarantees the non-toxicity of the product that, after the vulcanization, can be employed in medical and pharmaceutical applications. Kinesthetic Wearable Sensors In the production process of the ULKG, a solution of Elastosil and trichloroethylene is smeared on a lycra substrate previously covered by an adhesive mask. The mask has been designed according to the desired topology of the sensor network and cut by a laser milling machine. After the CE deposition, the mask is removed and the treated fabric is placed in an oven at a temperature of 130°C to speed up the cross-linking process of the mixture. In about 10 minutes the sensing fabric is ready to be employed to manufacture the ULKG. Sensor Characterization The main aim of the CE sensor characterization has been the determination of the relation between the electrical resistance R ( t ) of a treated fabric sample and its actual length l ( t ). Moreover, an analysis of the thermal transduction properties and aging of the fabric has been executed [ 5 ]. In terms of quasi-static characterization, a sample of 5 mm width shows an unstretched electrical resistance of about 1 kΩ per cm, and its gauge factor (GF) is about 2.8 , where R is the electrical resistance, l is the actual length, R 0 is the electrical resistance corresponding to l 0 which represents the rest length of the specimen). The temperature coefficient ratio is 0.08 K -1 . Capacity effects showed by the sample are negligible up to 100 MHz. Dynamic Characterization Electrical resistance behavior of the examined CE samples during a deformation has been fundamental to allow us to employ them as sensors. Two different issues had to be addressed to use CE as strain sensors. The first one concerns the length of the transient time, which can take up to several minutes. It is obvious that these physical systems cannot describe human movement without a signal processing devoted to compensate the slowness of this phenomenon. Moreover, electrical trend of the analyzed specimen shows some non linear phenomena which are not negligible under certain working conditions, in particular when fast deformations are applied. In this work the following results will be introduced. The typical electrical behavior of this system, when deformations in length are applied, will be described. The results of our study will lead to the formulation of a mathematical model which approximates the sensor electrical behavior. This model will be used to implement an algorithm devoted to the system regulation which consents the sensor length determination in real time. Finally, two simplified and faster versions of this sensor length determination technique will be presented and applied in posture reconstruction. The analysis of the electrical trend of CE sensors, when deformations are applied, has been performed by using a system realized in our laboratories which can provide controlled deformations and at the same time can acquire the resistance value performed by the specimen. A wide description of this instrumentation and its performances can be found in [ 5 ]. By using this device, several deformations, which differ in their forms versus time, amplitudes and velocities have been applied to CE specimens. Figure 1 , which has been reported as an example of this analysis, shows the output of a sample stretched with trapezoidal ramps in deformation having different velocities ( t ) (where l ( t ) is the length of the sample). The main remarks on sensor electrical behavior are summarized in the following: Figure 1 Response of a CE sensor solicited by trapezoidal ramps in deformation. • Both in case of deformations which increase the length of the specimen and in case of de formations which reduce it, two local maxima greater than both the starting value and the regime value are performed. • If the relationship between R ( t ) and l ( t ) were linear, one of the extrema described in the previous point would be a minimum. • The height of the overshoot peaks increases with the strength velocity ( ( t )). • The relaxing transient time, which lasts up to several minutes, is too long to suitably code human movement. Nonlinearity in the functional which relates R ( t ) and l ( t ) suggested us to choose an approximation containing a quadratic term in the strain velocity ( ( t )). Let us consider: where a 1 , a 2 and a 3 are three nonzero real numbers. By using experimental data, we have verified that when the specimen is motionless, i.e. ( t ) = 0, the signal deriving from the sensor is representable by a linear combination of exponential function: and the values ω i do not depend on the amplitude and velocity for a wide range of the solicitation previously applied (0 – 50 per cent of the rest length and 0 – 0.1 m/s), but they vary only according to the shape and the dimensions of the specimen and on the percentages of the components in the mixture used to realize it [ 9 ]. By considering g ( t ) as the input function of the differential linear system where , we have obtained encouraging results in signal modelling [ 9 ]. In particular we have approximated the sensor behavior as the solution of a second order linear system based on equation (3): with where ω 1 and ω 2 are the two poles of the linear system (4). This relation provides an obvious (almost theoretically) method to calculate g ( t ). Since equation (3) contains only R ( t ) and its derivatives, it s simple to determine the value of g ( t ). So to obtain l ( t )in real time it is necessary to integrate the differential equation (1) (in which the three parameters a 1 , a 2 and a 3 have been identified through the values of peaks excursions in the responses of the sensor). Unfortunately, equation (1) is not generally integrable when g ( t ) is unknown and its solution l ( t ) has to be numerically computed. This is not a simple issue because the acquired data are affected by noise and sample errors. Good results have been obtained off-line by using a wide digital filtering which used the average value of a large number of sample to reduce the noise, but introduced a signal delay [ 9 ]. Next developments will be aimed at implementing the length detection in real time during a motion. Conversely, the problem has been already addressed when the system is motionless, i.e. ( t ) = 0 and g ( t ) = a 1 l ( t ), and will be treated in the next section. Transient Time Reduction After a mechanical solicitation, CE sensor resistance changes according to equation (2). Unfortunately, the values determined for ω i and the resulting transient time do not allow to directly employ the acquired signals for our applications. On the other hand, by using equation (2) it has been possible to regulate the sensor response by calculating the coefficients c i (and in particular c 0 , which represents the final value of the signal) early with respect to the transient time duration. Since the pole values are invariant with the deformation, in order to apply relation 2, they have to be calculated only once, during the system parameter identification. If the ω i are known only the c i remain undetermined and have to be computed in real time after each deformation. The parameter identification is realized by an utility package which performs a minimization of the quantity over a lattice L which spans the variables c 0 ... c p , ω 1 ... ω p and where y is a k-dimensional vector containing the acquired data during the transient time after a solicitation. The choice of k is due to the noise which affects the signal. The precision in the parameters identification increases with its value. Practically this procedures is repeated several times and the values obtained for the ω i are the average response evaluated on all the trials. When we have determined the pole values, after each solicitation coefficients c 0 ... c p have to be re-calculated to return the steady-state response and the related sensor length. We have developed two different procedures to calculate them. The first one consists in considering the iterate p derivatives of function (2) with respect to t . If k ≥ p , the set of these equations evaluated on k samples and compared with the numerical derivatives of the signal stored in vector y constitutes a welldimensioned linear system in the variables c i , which can be calculated with low computational cost. Although this methodology is clear and elegant, it presents a serious disadvantage. The computation of the numerical derivatives of the signal y is corrupted by the noise which affects the signal. Moreover the sampling noise due to the analog-digital converter in the electronic acquisition system is amplified by its derivation. Practically, this strategy is inapplicable in this form. Results remarkably improve if analogical derivators are used. This solution addresses the problems introduced by the noise, but dramatically increases the dimension of the electronic acquisition system, because in addition to the derivators, each signal and its derivatives have to be individually acquired, and the number of the acquisition channels increases according to derivative order we use [ 10 ]. To address this issue and attenuate noise components due to the coupling between high impedence front-ends to the connecting wires embedded in the garment and powerlines [ 10 ], we developed an algorithm based on iterative integrations of equation (2). Coefficients { c i } i = 0... p are in this case the solution of an over-dimensioned linear system n × p , obtained by integrating n times equation (2) on the interval [ t 0 , t k ]. It is trivial to prove that the obtained system is consistent for n ≥ p and k ≥ p by computing the jacobian matrix of the system in its parametrical form. The choice of n > p produces a filtering (based on a least square evaluation of redundant data) of signals while the coefficients are calculated. A further stabilization is due to the integration on all the interval where eq. (2) holds, by collecting all the information previously stored. No particular disadvantages arise from this methods. All the calculation is digitally computed with neither increasing the dimension of the electronic acquisition system nor introducing or amplifying further noise. The main shortcoming of this approach is that it requires that one detects each movement because equation (2) holds when the specimen is motionless, only, and the numerical integration has to be reset after each solicitation. Results are reported in Figure 2 Figure 2 Output of a CE sensor (Voltage vs. Time) for three different deformation steps imposed (above) and treated signal (below). The transient time has been reduced. Realization of the Upper Limb Kinesthetic Garment The sensing fabrics described above can be employed to realize wearable sensing systems able to record human posture and gesture, which can be worn for a long time with no discomfort. In order to realize the ULKG, we have integrated sensors into a shirt connected to an electronic unit which operates a pre-filtering process. The very innovative goal we obtained consists in printing the set of sensors and the connecting wires directly on the fabric by using CEs (in the earlier prototypes the interconnections were realized by means of metallic wires [ 5 ], which might bound movements and create artifacts). In order to realize a sensorized shirt able to monitor the kinematics of the upper limb, we have to determine position and orientation of sensors attached to the considered joints. A crucial point here is based on the observation that a redundant number of sensors (i.e. a number of sensors bigger than the number of the degrees of freedom to of the system under consideration) distributed on a surface can provide enough information to infer the essential features concerning the posture of a subject, neglecting the precise sensor location. We borrow this approach from biological paradigms [ 6 , 7 ]. A theoretical approach has been tried, by searching an optimization criterion to maximize the global content of information collected by the sensor system [ 11 ]. Unfortunately, this technique is very onerous in terms of required computational resources. The optimization of this calculation is at the present under study. Finally, an heuristic approach has been adopted. By realizing a sample of sensorized fabric and by placing it around the considered joints during the execution of natural movements we have determined the set of position which produces meaningful outputs in terms of movement reconstruction. ULKG Electrical Model and Electronic Implementation of the Acquisition Technique All the remarks and trials exposed in the previous section lead us to design the adhesive mask used to smear sensors and wires reported in Figure 3 . The sensorized prototype shirt, realized by using this mask, is showed in Figure 4 . The bold black track of Figure 3 represents the set of sensors connected in series ( S i , and covers the joints of the upper limb (shoulder, elbow and wrist). The thin tracks ( R i , Figure 3 ) represent the connection between the sensors set and the electronic acquisition system. Since the thin tracks are made of the same piezorestive CE mixture, they undergo a not negligible (and unknown) change in their resistance when the upper limb moves. Therefore the analog front-end of the electronic unit is designed to compensate the resistance variation of the thin tracks during the deformations of the fabric. The electric scheme is shown in figure 3 . While a generator supplies the series of sensors S i with a constant current I, the acquisition system is provided by an high input impedance stage realized by instrumentation amplifiers and represented in Figure 3 by the set of voltmeters. Thanks to this configuration, only a little amount of current flows through the connecting wires, which have resistance values R i , and so the voltages which fall on R i are negligible if the current I, which flows in the series of sensors, is big enough. In conclusion, the voltages measured by the instrumentation amplifiers are equal to the voltages which fall on the S i that is related to the resistances of the sensors. In this way, the thin tracks perfectly substitute the traditional metallic wires and a sensor, consisting in a segment of the bold track between two thin tracks, can be smeared in any position to detect the movements of a certain joint. Figure 3 The electronic acquisition scheme (on the left) and the mask utilized for the realization of the ULKG (on the right). Figure 4 The UKLG prototype. The ULKG Working Modes: Reconstruction of Kinematic Configurations In order to clarify how posture detection can be done by using a kinesthetic garment, some remarks are necessary. First, in order to formally define a posture, it is necessary to develop a geometrical model of the kinematic chain under study. This can be easily done by fixing a certain number of cartesian frames, one for each degree of freedom considered and relating them with the segments which compound the kinematic chain. A kinematic configuration consists in the set of the mutual positions of the cartesian frames. Obviously, the entire set of the mutual positions is not necessary to reconstruct a posture exactly, and a minimal set can be chosen in many different ways. The Denavit-Hartemberg formalism [ 12 ] is an example of a method which fixes the exact number of relations between frames and gives a standard method to write their positions in terms of rotation and translation affinities, for rotational and translational joints. When the ULKG is worn by a user which holds a given position described by the geometrical model, the set of sensors assumes a value strictly related to it. If the number of sensors is large enough and if the sensor locations are adequate, the values presented by them uniquely characterize the considered position. Let be the sensor space, i.e. the vectorial space whose elements contain the values presented by sensors and where k is equal to the number of sensors in the ULKG and let be the space containing the kinematic configurations, i.e. the space of the lagrangian coordinates that define mutual segment positions in an upper limb kinematic model, where n is equal to the number of degrees of freedom considered. To execute a reconstruction of the kinematic configuration, by knowing the sensor status, a function F which maps S into Θ has to be defined. We have implemented F both by a clusterization of the space S via a clustering norm technique into the space Θ and by the interpolation of the discrete map produced by the clusterization. In the present application the first solution has been applied by using the norm as a clustering function, where ∈ S is a k -dimensional vector which represents a center of the clusterization lattice and s ∈ S is a k -dimensional vector representing the real values assumed by the sensors. Each points s * whose distance from a certain point of the lattice * is less than a previously fixed threshold ε is related to the value that the map assumes in *. The values which the function holds in the points of the clusterization lattice is experimentally acquired. The other implementation of F is described in [ 7 ] and will be summarized in section The ULKG as Posture Detector. Kinematic Models of Human Joints – The Upper Limb Model In many disciplines as biomechanics, robotics and computer graphics, geometric hierarchical structures are used in articulated body modeling for robots, human or other creatures representations. An articulated body can be thought as a series of rigid segments connected by joints. A biological kinematic chain is exactly an articulated body. In the present work we implement an upper limb kinematic model by employing ideal joints in order to maintain a practical parameterization of movements without trivializing human motion. From a macroscopic point of view, a complete upper limb model would have at least 7 DOFs, corresponding to rotational movements. These ones, described by kinesiology [ 13 ], are reported in Table 1 . In the model we have developed, the gleno-humeral joint of the shoulder has been parameterized as a ball and socket joint, whereas elbow and wrist consist in two successions of two rotational joints. This choice has been made in order to have an intuitive kinematic reconstruction in terms of practical mathematical characterization. Three different parameterization techniques are usually considered to describe orientations between frames: Table 1 Upper limb model DOFs shoulder elbow wrist flexion-extension abduction- adduction intra-extra rotation flexion-extension pronation-supination flexion-extension abduction- adduct ion • the Euler's angles; • the exponential map; • the unit quaternion representation. There is not a general criterion to prefer one parameterization with respect to the others. The choice depends on the particular application; however, a good comparison can be found in [ 14 ]. The crucial point, as a classic control problem, is the presence of singularities. Euler's angles describe the orientation of a cartesian frame with respect to another by using three parameters, but have two singularities, known as gimbal-lock [ 15 ]. The exponential map introduces a new parameter with respect to Euler's angles but solves only one singularity. To address both the singularities, unitary quaternions can be used. The set of quaternions is a non-commutative algebra of iper-complex numbers created in 1843 by Sir R. Hamilton. The unitary quaternions constitute a subgroup in of the quaternions which have unitary cartesian norm. A clear summary of their geometric properties as vectors and their algebra can be found in [ 16 ]. We have developed our model by using both Euler's angles and unitary quaternions. This choice is due to the simplicity of the first parametrization which allows to calculate posture with low computational cost, and the necessity to realize graphic animations which interpret human movements. In [ 16 ] a methodology capable to perform fluid and biomimetic movements by using unitary quaternions is explained. We have applied Shoemake's results to represent the transition of our geometrical model and to animate an avatar piloted by the signals recorded by the ULKG. The ULKG as Posture and Movements Recorder Using the ULKG, it is possible to detect if two postures are the same or not with a certain tolerance, and it is possible to record a certain set of postures coded by the status of the sensors. In the same way, movements can be recorded as transitions from one posture to another, and they are coded by the evolution of the sensor values. In particular, we have tested this capability on a set of functional relevant postures. The ULKG showed good capabilities of repeatability, even if it is removed and re-worn. An ad-hoc software devoted to recognize recorded postures has been developed. The software is able to: • record a set of defined postures of the upper limb in a calibration phase, • recognize the recorded postures during the user's movements, • represent the movement by using a graphical representation given by the avatar. In the calibration phase the user which wears the ULKG holds a set of position θ i ( i = 1 ... p , where p is the number of positions to be recorded) and the sensor status s c i is acquired and stored in the k × p calibration matrix In the recognition phase, while the user moves the upper limb, the kinematic configurations are detected by acquiring the sensor outputs s and comparing them with the p columns of the calibration matrix. If the distance induced by the norm as defined in equation (7) between the actual sensor values and a column of the matrix is smaller than a certain threshold, the ULKG returns the position related to the selected column. In this application, it is not necessary that the entire space of the sensor values is mapped into the configuration space, so any other norm, instead of the one defined by equation(7) can be used. The system has also been tested by implementing the euclidean norm, and it has led the same results. When a posture is recognized, the visualization software performs an animation from the old position to the actual one. This transition is interpolated by using quaternions algebra: orientations acquired during the calibration in terms of Euler's angles are translated into unit quaternions and the movement from the old position d to the arrival one a are defined through the spherical linear interpolation algorithm [ 16 ] which provides the interpolated quaternion q int at each time t . Moreover, the absence of singularities in unit quaternions permits the execution of each arbitrary trajectory in the configuration space. In other words, the possibility of executing and representing each movement allowed by the physical constraint is ensured. The ULKG as Posture Detector According to the previous sections, the ULKG is able to record the sensor status in a finite number of positions in the configuration space. These data can be associated to corresponding positions to define a discrete map between subsets in the two spaces. An example of this map is the function which relates the centers of the clusters in the lattice introduced in section The UKLG Working Modes with the corresponding geometrical configuration. If the set of the points considered in the configuration space satisfy some particular requirements [ 7 ], this map can be extended by interpolation techniques to all the configuration space. A complete treatment of the requirements necessary to extend the function to all the configuration space is beyond the purpose of this paper. In [ 7 ] it is proved that the choice of a lattice having the same dimension of the space Θ ensures the possibility to extend the discrete map to a continuous one, F to all the space. Moreover a piecewise linear interpolation technique based on the decomposition of Θ into a lattice compounded by hypertetrahedra has been presented to construct F in the same work. The choice of the PL interpolation is due to the necessity to invert (or more generally, compute a pseudoinverse, F † , in case the dimensions of Θ and S do not match). PL functions are linear applications expressed by matrix, almost locally, and are invertible with low computational cost. If F † is available and the set of configurations is coded by a parametrization, we know the position with a precision that depends on the interpolation used and the choice of the lattice used to compute the value corresponding to the sensor status of any acquisition. Moreover the procedure for the determination of the position consists only in the detection of the piece of F † which holds for the particular sensor values s and multiplication F † × s . The determined value for the position in the configuration space, can be continuously represented by the avatar, which in this case does not require interpolation techniques to represent an animation. A crucial point in the building of F is the choice of a parametrization for Θ. An additional subsidiary measurement system (constituted by a set of electrogoniometers produced by Biometrics Ltd.) has been employed to parametrize the configuration space Θ relating position to numerical values. The construction of F correspond to the identification of the parameters of the entire system, being defined by a field of matrices on Θ. The ULKG as a part of a post-stroke service As mentioned in the introduction, the proposed technology is under testing in the field of post-stroke patients' rehabilitation. The main institution involved in the research and experimentation of the system to be employed in a medical environment is the S. Maugeri Foundation, in Pavia, Italy. This unit is responsible for the drawing up of a post-stroke rehabilitation protocol for hemiplegic patients according to the guideline contained in [ 17 ]. The most frequent damage in the adult stroke population concerns body district controlled by the brain areas depending on posterior and medial cerebral artery, causing plegia first and then spasticity to the upper and lower limb. More precisely, movement dysfunctions arise from a complex interaction among positive symptoms (spasticity, released flexor reflexes), negative symptoms (loss of dexterity and weakness) and changes in the physical properties of muscle tissues. These patients show clinical deficits that may include impairment of sensation, perception, cognition and motor control: together, these impairments contribute to functional limitations in mobility, posture maintenance, cares, comfort and many activities of daily living, such as to pick up a glass or to turn the pages of a book. Thus, the principal objective of rehabilitation in these patients is to improve daily functions. For our prototype, we chose to consider long term rehabilitation therapy of upper limb; in particular, we considered the shoulder and the arm. In this section we introduce the entire health care service including all the support structure of data management and communication required to improve the patients treatment both in the hospital and at home. The clinical pathway that a person affected by Stroke experiences after the event comprehends multiple healthcare environments, and depends also on the national healthcare system. In the following we refer to the Italian setting. The first step is admission in a unit for acute care for about 8–12 days. Then most of the patients, and particularly hemiplegic ones, are admitted to an Intensive Rehabilitation unit for about 30–45 days. Subsequently, if needed, patients are admitted to an Extensive Rehabilitation unit (in-patient unit where treatment lasts for no more than one-two hours a day) for about 30–40 days. Otherwise, they go home, or they enter the so called long-stay units, which host patients that, mainly for family reasons, cannot stay at home. During this intensive rehabilitation period, patients perform physical exercises with the help of physiotherapists, up to three hours each day. It is very important to continue such exercises after this period, even if with a lower intensity. According to the discharge conditions, physicians decide a personalised protocol: patients must repeat some exercises one or more times a day for a certain number of days, usually one-two months. These exercises are illustrated to the patient before discharge, but physicians could decide to update them later on, according to the patient's status modification. However, after discharge, several problems may arise, impairing the continuity of care: • patients that go back to home, without an healthcare professional stimulating them, are poorly motivated to do regular exercise • home caregivers may be not prepared adequately to give the intended support • patients admitted to long-stay units or long term care units often worsen their psychological state, and this in turn decreases disposition to do exercise • long-term care units and extensive rehabilitation settings often do not comply to evidence based rehabilitation protocols, and they have no link with the medical team that cared for the patient during the intensive rehabilitation period We think that providing the patient with a virtual trainer for his rehabilitation could help to overcome these problems. In the following, the patient is intended to be at home, or in a long-stay unit, or in an extensive rehabilitation unit. The basic idea about this application is that when the patient logs on, the system prompts him with the current status of the rehabilitation protocol, and proposes the schedule of the day. The patient wears the sensorized garment and performs the exercise with the help of a movement tracker on the PC screen. At the end of the exercise, a global error measure is given to the patient in such a way that he can decide to repeat the task to improve his performance. Thus, the device facilitates the patient in performing in the correct manner the rehabilitation exercise. But, when a new technology is proposed, mainly in the outpatient care context, great attention must be devoted to the user interface. Technologically advanced devices may fail because of scarce usability or compliance. This is a crucial issue when dealing with elderly people, as in the case of the majority of post-stroke patients. Thus, the patient must be provided with a system that is as much easy to use as possible, to allow facing multiple problems through the same interface, without requiring an extensive learning effort. In our case, this means that the sensorized shirt must be not only a means for collecting data for further analysis, but it also must be integrated into a service able to: • act as a patient-tailored support system, providing an immediate feedback about the patient's performance on a specific exercise, high-lighting, if any, the incorrect movements, • show the patient's trend (i.e. improving, stationary, etc) in a given time interval, through easy-to-understand metaphors, such as a plant that grows up or that slows down, • provide educational material, such as post-stroke rehabilitation guidelines, or movies illustrating the correct (and incorrect) movements for the specific patient's disability, • allow communication between patient and health care providers. From the health care provider side, it is important for the new service to be smoothly integrated into the clinical work-flow and take into account organizational issues. Thus, different functionalities are needed: • providing an overview of patients enrolled in the rehabilitation treatment, • following multiple patients in real-time, • retrieving an exercise and send comments to the patient, • allowing to send new exercise protocols to patients, • maintaining the control of the service flow. To support these functionalities, we developed a database, whose Entity-Relationship model lead to several tables that will store • personal data of both patients and health care professionals, • the objectives of the rehabilitation, • the description, planning and execution of the exercises, • the garment details, • the messages between patients and hospital team. From the communication infrastructure point of view, the system will be made by three main stations, located at different sites, and interconnected among them. The three sites, are • the Patient Site, physically located near the patient, who wears the sensitive garments. The Patient Site computer is connected both with the Server Site, and with the electronics which interfaces to the garments. • the Physician Site, from which the physician can monitor the patient's exercises. As mentioned above, the monitoring can happen both in real time (on-line) and on the stored sessions (off-line) • the Server Site, where a firewall-protected central server hosts the database described above and all the necessary software to serve web pages dynamically generated to provide easy access to the system. Results All the patient management system, work-flow and health-care service described in the previous section are currently under test for a clinical validation and no results on the matter is reported in the following. In the near future, we plan to collect all the achievements deriving from the clinical experimentation of the integration of ULKG in the health-care service. Here, only technical results deriving from the prototype validation, are reported. In our laboratories, the ULKG has been submitted to a series of trials in order to check the real capability of the instrument to recognize and detect gestures, postures and movements. The ULKG Performances as Posture and Movements Recorder The first basic working mode which has been tested is the ULKG functionality as posture recorder. The system has been used to record postures for the upper limb which have been related to the corresponding configurations in the model represented by the avatar. After having stored all the data concerning fifty different postures in the upper limb workspace, the same ones have been held again several times. The output of the ULKG was visualized on a computer screen, where the avatar replicated the subject's posture (Figure 5 ). The graphical representations has been performed by the avatar according the quaternion interpolation algorithm presented in section The ULKG as Posture and Movements Recorder with good animation quality. The system recognized 100% of the postures recorded, and no further re-calibration was thought to be necessary even if the ULKG had been removed and re-worn. Postures used to test the prototype included generic positions typically seen in the workspace. This trial tested both the hardware of the prototype and the clusterization and reconstruction algorithms described in section The ULKG Working Modes. Figure 5 Posture recognition trials performed by the user and represented by the avatar. The Performances ULKG as Posture Detector According to section The ULKG as Posture Detector the prototype was tested through several trials to evaluate its performances in dynamic working conditions and during the detection of unknown posture. The main powerful demonstration gathered from these trials is that the ULKG is able to reconstruct postures never recorded or held before. In each trial the ULKG was worn by a subject and a set of electrogoniometers was positioned on the user. The goniometers were adequate to detect only flexion-extension (and adduction-abduction) executed by the joints under study and they were used only to have a description of the movements performed. Torsions are not relivable by using this instrumentation. The theoretical resolution provided by the producer is 0.5 degree. No interactions between the ULKG and the goniometers were allowed. The subject was invited to perform a set of movements which involve the gleno-humeral joint, the elbow and the wrist, like flexions-extensions, abductions-adductions and circling of the body segments. Signals deriving from the ULKG and from the set of goniometers were simultaneously acquired. The outputs of the ULKG was processed according to section The ULKG as Posture Detector and the results obtained in terms of angles were compared with the goniometers output. Data obtained from these experiments are showed in two different presentation. The first one is a classical representation of the angle values versus time. In the plots, both the ULKG output and the values presented by the goniometers are shown and compared. In the other representation, we have considered some planes contained in the configuration space 0 and we have plotted the trajectories performed by some joints on them, both for goniometers and for the ULKG. This presentation is very powerful to detect divergences between the two responses. In Figures 6 , 7 the analysis of a wrist rotation is reported. Figures 6a and 6b show the flexion and abduction angles (which compound the movement) versus time. The red line is the goniometer output, while the blue one represents the ULKG response. Figure 7 composes the two angle evolutions in a trajectory which meaningfully explain the motion. Flexion is reported on the abscissa axis, while the abduction is reported on the axis of ordinates. The colors used for goniometers and ULKG are the same of Figure 6 . Figure 6 Flexion (a) and abduction (b) angles of the wrist versus time. The red line is the goniometer output, while the blue one represents the ULKG response. Figure 7 Composition of the flexion angle (in abscissa) and abduction angle (in ordinates) of the wrist. The red line is the goniometer output, while the blue one represents the ULKG response. The same scheme has been adopted to report a movement for the shoulder in Figures 8 , 9 . Extension is reported in Figure 8a (versus time) and on the y-axis of the Figure 9 . Conversely, Figure 8b and the x-axis of Figure 9 represent the evolution of the shoulder flexion. Figure 8 Extension (a) and flexion (b) angles versus time of the shoulder. The red line is the goniometer output, while the blue one represents the ULKG response. Figure 9 Composition of the flexion angle (in abscissa) and extension angle (in ordinates) of the shoulder. The red line is the goniometer output, while the blue one represents the ULKG response. Finally, an elbow flexion is shown in Figures 10 . Both shoulder rotation and elbow pronationsupination have performed qualitative results in terms of sensor signal trends but these responses have not yet been analyzed because the electrogoniometers we used are not capable to detect such responses and an identification of the ULKG along this movement direction has not been possible. Figure 10 Flexion angle of the elbow. The red line is the goniometer output, while the blue one represents the ULKG response. The results are similar to the ones demonstrated in [ 7 ]. The only difference resides in the statical character of the trials in our previous work, while in this case movements are described by the ULKG output. Two kinds of divergence between the ULKG behavior and the goniometer responses are pointed out by the introduced diagrams. The first error we can note is a real divergence between the information deriving from the two measurement systems. Estimated trajectories differ for a certain quantity and this phenomenon can be observed both in the plot showing "angle versus time" and in the one showing trajectories. It is clearly pointed out in Figure 7 in the range [-12°, -8°] for flexion angle and [4°, 8°] for abduction angle. Evaluated in cartesian norm, the error estimated is anyway smaller than 5 per cent, if compared with the dimension of the entire workspace. The other artifact we can note is a difference between signals in the "angle versus time" plots, that is not detectable by watching the other representation. This phenomenon is due to a lack of synchronization between the two measurement system and it is manifest in Figure 8a and 8b in the [0, 0.5] second range, without corresponding to an effective difference in terms of trajectory, as demonstrated in Figure 9 . The two systems lead to the same results at different time. A refinement of the movement detection algorithm may avoid these two errors and will be studied in the future. Conclusion In this manuscript, an upper limb kinesthetic garment for gesture, posture and movement detection has been presented. The main advantage introduced by this prototype is the possibility to wear it for long periods of time thus allowing clinicians to monitor patients without causing any discomfort. Several issues, deriving from the employment of the new technology which has allowed the realization of the unobtrusive device, are addressed. In particular a modeling for the physical behavior of the sensor employed was proposed. An algorithm of signal analysis derived from the model was implemented to allow the use of conductive elastomers as sensors. Moreover both the implementation of the sensing prototype and its performances as posture recorder and posture detector were introduced. We used particular care in explaining all the algorithms necessary to reconstruct or estimate posture and movement. We discussed both the application of a the classical biomechanical methodology as well as some innovative techniques whose development we deemed necessary to ensure good results in the garment employment. An application of the upper limb kinesthetic garment as useful instrumentation in post-stroke rehabilitation was described, together with a complete description of the clinical service where the garment is integrated. Finally, results on the performances of the sensing system were reported.

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1079931

The "Statinth" wonder of the world: a panacea for all illnesses or a bubble about to burst

After the introduction of statins in the market as effective lipid lowering agents, they were shown to have effects other than lipid lowering. These actions were collectively referred to as 'pleiotropic actions of statins.' Pleiotropism of statins formed the basis for evaluating statins for several indications other than lipid lowering. Evidence both in favour and against is available for several of these indications. The current review attempts to critically summarise the available data for each of these indications.

Recently while browsing through the internet, we came across a webpage [ 1 ] that reads as follows: "Statin drugs should probably be in the water, like fluoride. These cholesterol fighting wonders have been proven to prevent heart attacks..... with only rare side effects............... The hitch is that statins cost more than fluoride. A lot more. The drug industry's statin sales surpassed US $15 billion last year. The cholesterol fighting power of products like Pfizer's Lipitor and Merck's Zocor have won them the title 'Superstatins' and made them supersellers. Lipitor brought in US $9.2 billion in 2003 sales for Pfizer, making it the biggest prescription drug in the world." In 2001, we reviewed the statin literature for Medscape and were able to enlist about seven indications[ 2 ], the major one being dyslipidemia with associated coronary disease (CAD). The 1993 National Cholesterol Education Programme (NCEP) guidelines [ 3 ] were cautiously optimistic about the future of statins but subsequent publication of 3 landmark trials [ 4 - 6 ], greatly tilted the balance in their favour and since then they haven't looked back: a large number of trials and guidelines added new intensity to cholesterol lowering with the low density lipoprotein cholesterol (LDL-C) targets going for a free fall (<70 mg/dl in some situations) [ 7 - 12 ]. Although this approach of more intense lipid lowering has met with considerable criticism, this is not the topic of this review. We intend to discuss the other novel, upcoming uses of statins. In contrast to the post-hoc analysis of the Scandinavian Simvastatin Survival Study (4S) [ 4 ] in which the benefit provided was related to the magnitude of change in the LDL-C levels, some other studies have shown benefits that could not be accounted for by reduction in LDL-C alone [ 13 - 16 ]. A large number of studies showing pleiotropism of statins followed and diverse mechanisms were then proposed to explain this pleiotropism including anti-inflammatory, immunomodulating, and effects on apoptosis [ 17 - 22 ], making them potentially suitable candidates for the treatment of a wide variety of pathological conditions in many of which they are already being investigated. This article attempts to summarize the available evidence for the proposed (other than lipid lowering) indications of statins. Arrhythmias Several actions of lipid lowering therapy like reduction in myocardial ischemia, improvement of autonomic function, changes in protein channel function and inhibition of cardiac remodelling make them prospective agents for the treatment of arrhythmias[ 22 , 23 ]. Chronically administered pravastatin was shown to reduce the incidence of ischemia-induced ventricular tachyarrhythmias in experimental models [ 24 , 25 ]. Early use of pravastatin in patients with acute myocardial infarction (MI) reduced the incidence of in-hospital ventricular arrhythmias irrespective of the lipid levels [ 26 ]. The Anti-arrhythmia Versus Implantable Defibrillators (AVID) Study showed that lipid lowering therapy decreased the recurrence rate of ventricular arrhythmias in patients implanted cardioverter-defibrillator [ 27 ]. Statins have also been shown to have a role in the treatment of atrial arrhythmias. Inflammatory changes have been shown in atrial biopsy specimens of patients with lone atrial fibrillation (AF) [ 28 ]. Furthermore, serum levels of C-reactive protein (CRP), a sensitive marker of systemic inflammation, were increased in patients with AF. Not only that, CRP levels were higher in patients with persistent rather than paroxysmal AF, and persistent AF is less likely to spontaneously revert to sinus rhythm [ 29 , 30 ]. These studies suggested that inflammation may induce, provoke and promote the persistence of AF. Statins may be potent anti-inflammatory agents [ 31 ] and have also been shown to reduce CRP levels [ 32 ]. Not surprisingly, statins were subsequently shown to prevent AF recurrence in patients with lone AF after successful cardioversion [ 33 ] and in patients with CAD[ 34 ]. Both these studies were retrospective. However, it is well known that results obtained in retropsective studies may not be replicated in clinical trials [ 35 ]. Accordingly, in an open, controlled multicenter study, pravastatin did not reduce the recurrence rate of AF after electroversion[ 36 ]. Moreover, there has been an isolated case report of AF due to simvastatin[ 37 ], which further limits their role in the management of arrhythmias. The evidence available for the beneficial role of statins is largely from observational and experimental studies which is clearly insufficient to recommend them as primary or even adjunctive antiarrhythmic agents. Moreover, their role in prevention as well as treatment of arrhythmias remains to be clearly defined. Heart failure Initial experimental evidence indicated towards both potential harm and benefit of statins in heart failure. Statins modulate a variety of inflammatory and immune responses [ 38 - 40 ]. In animal models of heart failure, statins moderate abnormal collagen and β-myosin expression, attenuate increased matrix metalloproteinase activity, improve ventricular remodelling and systolic function, normalize sympathetic responses and improve survival [ 41 - 43 ] Given the relation of systemic inflammation to morbidity and mortality in heart failure patients, it was hypothesised that statins may benefit patients with heart failure separately from or in addition to effects on cholesterol and coronary disease[ 44 ]. In a report of 551 patients with systolic heart failure, statin use was associated with improved survival in patients with ischemic and non-ischemic heart failure[ 45 ]. After risk adjustment for age, gender, CAD, cholesterol, diabetes, medication, hemoglobin, creatinine and NYHA functional class, statin therapy remained an independent predictor of improved survival. Furthermore, in a randomised trial in 63 patients with heart failure, statin use improved NYHA class and ejection fraction when compared with placebo [ 46 ]. Also, statin therapy reduced new onset heart failure in the 4S Study [ 47 ], but this may have been related to effects on recurrent myocardial infarction. Using data from the Prospective Randomised Amlodipine Survival Evaluation (PRAISE) trial, association of statin therapy with total mortality among 1,153 patients with severe heart failure was evaluated [ 48 ]. Statin therapy was associated with a 62% lower risk of death. However, only 12% patients were receiving statin therapy. Moreover, the study results cannot be generalised as these patients participated in a clinical trial at a time when β blockers and spironolactone were not commonly used in severe heart failure. There also is some evidence to the contrary; lower serum cholesterol predicts worse outcomes in heart failure [ 49 ], raising concerns regarding use of lipid lowering agents. Statins also reduce ubiquinone (enzyme Q-10) [ 50 ], which may adversely affect mitochondrial and cardiac muscle function. Therefore, in lieu of conflicting experimental and clinical data, the routine use of statins in congestive heart failure will be premature. Cardiomyopathy (CMP) In initial experimental studies, simvastatin was shown to induce regression of cardiac hypertrophy and fibrosis and improve cardiac function in a transgenic rabbit model of human hypertrophic CMP [ 51 ]. Based on the knowledge that statins improve endothelial function [ 39 ] and suppress systemic inflammation [ 31 ], it was hypothesized that statins may improve cardiac function in patients with nonischemic dilated CMP [ 46 ]. Fourteen weeks of treatment with simvastatin was shown to improve left ventricular ejection fraction, reduce plasma concentration of tumour necrosis factor-alpha, and brain natriuretic factor in patients with idiopathic dilated CMP[ 52 ]. The effect on patient outcomes was however not evaluated. Again as in case of heart failure, although some evidence is available for the beneficial effect of statins in CMP, evidence to the contrary is also available. Lovastatin has been shown to significantly increase mortality in hamsters with cardiomyopathic heart due to reduction in ubiquinone supply[ 53 ]. Statins have been shown to decrease coenzyme Q levels in humans [ 54 ] and this coenzyme is indispensable for cardiac functions [ 55 ]. In wake of such conflicting evidence, their use in ishemic/nonishemic CMP cannot be advocated. Diabetic dyslipidemia In addition to microvascular complications, patients with type 2 diabetes are at an increased risk of developing CAD [ 56 ] Over a 7-year period, in patients with no history of CAD, the incidence of first MI was over five times greater for patients with diabetes compared with non-diabetic controls [ 57 ]. Diabetes is now considered to be a cardiovascular disease and all diabetics, irrespective of history of CAD, are considered within the category of secondary CAD prevention. Diabetic dyslipidemia may exist in the absence of raised total serum cholesterol due to an increased proportion of the more atherogenic LDL particles. Moreover, dyslipidemia often exists with a number of other atherogenic co-factors in patients with diabetes (e.g. abdominal obesity and hyperinsulinemia) as a part of metabolic syndrome [ 58 ]. The updated Adult Treatment Panel (ATP)-III guidelines have advocated the use of statins for diabetes with or without CAD [ 12 ]. LDL lowering treatment when LDL-C is >100 mg/dL in diabetices without CAD and >70 mg/dL in diabetics with CAD has been recommended. Since the appearance of the first report on the efficacy of statins in lowering lipid concentrations in patients with type 2 diabetes [ 59 ], clinical trial evidence has accumulated in their support as the primary lipid-lowering drugs for these patients. Subgroup analyses [ 60 ] of diabetic patients in the Antihypertensive and Lipid Lowering Treatment to Prevent Heart Attack Trial (ALLHAT-LLT) [ 9 ], the MRC/BHF Heart Protection Study (HPS) [ 15 ], and the Anglo-Scandinavian Outcomes Trial-Lipid Lowering Arm (ASCOT-LLA) [ 10 ] showed variable results of lipid lowering therapy on cardiovascular outcomes in diabetic patients. In ALLHAT-LLT [ 9 ] pravastatin did not reduce the incidence of non-fatal MI and CAD deaths in patients with diabetes. In the HPS trial [ 15 ] simvastatin significantly reduced the risk of CAD and total cardiovascular events in patients with diabetes, whether they already had CAD or not. In the ASCOT-LLA trial [ 10 ] atorvastatin did not reduce the risk of non-fatal MI and CAD death in patients with diabetes and hypertension who had no pre-existing CAD. Collaborative Atorvastatin Diabetes Study (CARDS) was carried out to evaluate the efficacy and safety of low-dose atorvastatin treatment in primary prevention of CAD in patients with type 2 diabetes at high-risk of CAD [ 61 ]. CARDS Investigators conclude that statins should be used in all patients with type 2 diabetes unless the patient has sufficiently low risk of coronary heart disease. However, generalization of CARDS results is debatable. For example, the risk of statin therapy might be increased in people older than 75 years of age in patients with chronic renal insufficiency or organ transplantation and in patients with very high triglyceride concentrations who are on fibrates [ 60 ]. Moreover, the number needed to treat will be very high in patients in whom the baseline risk is low like those with type 2 diabetes who are younger than 40 years; in premenopausal women; and in those without any CAD risk factors [ 60 ]. Development of diabetes Lipid lowering therapy with bezafibrate had earlier shown to improve plasma glucose levels and insulin response to 75 g oral glucose loading associated with hyperinsulinema [ 62 ]. An analysis of patients enrolled in the WOSCOPS study had shown a 30% reduction in the hazard of becoming diabetic [ 63 ]. The analysis was done post hoc and the levels of statistical significance was modest (p = 0.04). Additionally, by reducing the risk of CAD, the need for β-blocker use (and perhaps thiazides) was reduced. There is some evidence that β-blockers [ 64 , 65 ] and thiazides [ 66 ] may be associated with an increase in the incidence of diabetes. Although no effect of pravastatin on glucose levels was shown in another study, [ 67 ] the authors proposed that pravastatin might reduce the incidence of diabetes by a reduction in triglyceride (TG) levels. However, even this is unlikely because the effect of pravastatin on TG levels is only modest [ 68 ]. A recent case control study from the UK based General Practice Research Database failed to show reduced incidence of development of diabetes [ 69 ]. Diabetic maculopathy There has been interest in link between serum lipids and retinal exudates for 40 years [ 70 ]. A number of cross-sectional studies suggest that serum lipids may have a causative role in the formation of macular exudates [ 71 - 74 ]. A cross-sectional study of Age-related Macular Degeneration (AMD) suggests that statin therapy does have a protective role against the development of macular degeneration [ 75 ]. Few studies have evaluated statins in diabetic retinopathy [ 76 , 77 ]. In one of these, an improvement in hard exudates was noted in all patients on statins [ 76 ]. In another study, simvastatin was shown to improve fluoroscein angiographic picture and led to maintenance of visual acuity in all patients [ 78 ]. These data, though important, do not permit us to draw a final conclusion as these studies were inadequately powered. Claudication Claudication occurs when blood flow to the extremity fails to meet the metabolic demands of the skeletal muscle during exercise. It was hypothesised that statins, by improving endothelium dependent vasodilation at the arteriolar and capillary level [ 79 ], by their proangiogenic response independent of cholesterol reduction [ 80 ], and by inhibition of MMP-9 secretion by peripheral monocytes [ 81 ], could be beneficial in reducing claudication in patients with peripheral arterial occlusive disease (PAOD). Studies with lipid modifying therapies have demonstrated desirable effects in patients with PAOD [ 82 , 83 ]. A post-hoc analysis of the 4S data showed that new or worsening claudication was reduced in the group of patients receiving statins [ 84 ]. High-dose, short-term therapy with simvastatin has been shown to improve walking performance, ankle-brachial pressure indices, and symptoms of claudication in hypercholesterolemic patients with PAOD [ 85 ]. One-year treatment with atorvastatin improved pain free walking time and participation in physical activity in patients with intermittent claudication [ 86 ]. However, maximal walking time did not change significantly. Similar benefit was shown with simvastatin on treadmill exercise time until the onset of intermittent claudication [ 87 ]. Despite the evidence from these studies suggesting benefit, well-designed long-term studies assessing primary and secondary prevention of PAOD with defined endpoints such as amputation or number of vascular events are required. Multiple sclerosis (MS) In an experimental model of encephalomyelitis, lovastatin treatment was shown to block disease progression and induction of inflammatory cytokines [ 88 ]. Lovastatin treatment also attenuated the transmigration of mononuclear cells by downregulating the expression of leukocyte function antigen- 1 (LFA-1), a ligand for intercellular adhesion molecule (ICAM), in endothelial-leukocyte interaction [ 88 ] and mononuclear cell infiltration into the CNS has been implicated in MS [ 89 ]. Atorvastatin was shown to promote Th2 bias and reverse paralysis in a CD4(+)Th1-mediated experimental model of MS [ 90 ]. Therefore, statins were recognised as potential agents for future pharmacotherapy of MS [ 91 ]. In the first clinical trial of statins in MS, 80 mg oral simvastatin for 6 months significantly reduced the number and volume of gadolinium enhancing lesions [ 92 ]. However, immunological expression of surface markers on leukocyte cells or inflammatory cytokine profile showed no changes. Moreover, it was an uncontrolled, open label, small study with a baseline versus treatment comparison. Therefore, its results must be interpreted with caution. For instance, it is possible that reduction in the disease severity as measured with MRI could be due to regression to the mean. Moreover, since patients were included on the basis of the presence of gadolinium enhancement, this might have led to selection of patients with active disease who may subsequently have shown spontaneous reduction in disease activity anyhow. Additional factors like steroid use and unblinded assessment of MRI scans may have influenced the results. The exploratory immunological data in this study were also not found to be supportive. Due to the paucity of evidence from adequately powered good quality clinical trials demonstrating the benefits of statins, any conclusive statement would be rather premature. Several trials are currently underway to address this question and we are also conducting a Double-blind, Randomised Evaluation of Atorvastatin in Multiple Sclerosis (DREAMS) trial in our institution. Stroke Although cholesterol lowering is well known to decrease the risk of CAD, its association with decreased risk of stroke was demonstrated later [ 93 ]. Meta-analyses done recently have shown statin use to be associated with reduced risk of stroke by 12 to 24% [ 94 , 95 ]. Analysis of data from nine cohort studies showed a 15% decrease in thromboembolic stroke but a 19% increase in hemorrhagic stroke for a 1.0 mmol/l decrease in LDL concentration. The risk in those without a known cardiovascular risk factor was shown to be the same (6%) in clinical trials as that seen in cohort studies [ 91 ]. Though the overall risk of non-fatal strokes was reduced, the risk of fatal strokes was not [ 96 ]. Also, these results were obtained from studies which had stroke as their secondary endpoint. Moreover, in most of the included studies, incidence of stroke was very low, especially for primary prevention, reducing the power of comparison. Alzheimer's disease (AD) Addition of lovastatin to human HEK cells transfected with the amyloid precursor protein (APP) was shown to reduce intracellular cholesterol/protein ratios by 50%, and to inhibit cleavage of APP by beta-secretase [ 97 ]. Non-demented individuals with heart disease have increased prevalence of AD-like beta-amyloid deposits in the neuropil and within neurons [ 98 ]. In a cohort of patients taking lovastatin and pravastatin (but not simvastatin), a lower prevalence of diagnosed probable AD was noted [ 99 ]. A case control study has also shown a lower risk of dementia among users of statins [ 100 ]. However, in a review done by the Cochrane Group, it was pointed out that no evidence in the form of controlled clinical trials was available to recommend the use of statins in AD [ 101 ]. In a subsequent randomised, placebo controlled, double-blind trial, 26-week treatment with 80 mg simvastatin did not show any significant alteration in the cerebrospinal fluid levels of A-beta 40 and A-beta 42 [ 102 ]. Though the body of evidence for the beneficial effect of statins for AD is growing, due to the paucity of randomised controlled trials, no conclusions can yet be drawn [ 103 ]. Moreover, excessive lipid lowering may be detrimental as too little cholesterol in neural membranes has been shown to increase the vulnerability of neural membranes to dysfunction [ 104 ]. Low serum cholesterol concentrations have been shown to be associated with cognitive decline in prospective studies of aging American twins [ 105 ] and elderly Finns [ 106 ]. Depression Two observational studies showed that long-term statin use is associated with a reduced risk of depression in patients with CAD [ 107 , 108 ]. After an average follow up of 4 years, comparison of psychometric scores between users and nonusers of statins showed that statin use was associated with lower risk of abnormal scores for depression, anxiety and hostility [ 107 ]. Authors have attributed the findings to a possible direct effect of statins on psychological well being. Similar reduced risk of depression was noted with statins in patients with hyperlipidemia [ 108 ]. A more plausible possibility of reduced risk of depression due to an improvement in the overall quality of life was suggested in this study. On the other hand, lowering of serum cholesterol may be associated with an increased incidence of depression and suicides [ 109 - 113 ]. To sort of neutralize the evidence, some randomised, placebo controlled trials of statins have shown that depression was neither more nor less common among those taking active treatment [ 114 - 116 ]. Rheumatoid arthritis (RA) Statins were shown to inhibit LFA-1, which is known to play an important role in the pathophysiology of inflammatory and autoimmune diseases [ 117 ]. Statins also led to significant suppression of collagen-specific Th 1 humoral and cellular immune responses, reduction of anti-CD3/anti-CD28 proliferation and IFN-gamma release from mononuclear cells derived from peripheral blood and synovial fluid [ 118 ]. Based on these findings, a putative role for statins in RA was suggested. A preliminary study done in 15 patients with RA who were receiving methotrexate as a single disease modifying agent with no satisfactory responses, showed improvement after eight weeks of treatment with 40 mg simvastatin [ 119 ]. Recently, in a randomized placebo controlled trial [ 120 ], atorvastatin 40 mg was shown to significantly improve disease activity score after 6 months of therapy although the effects were modest. The use of disease modifying anti-rheumatic drugs was rather heterogeneous among the treatment groups in this study, with more patients receiving methotrexate in the atorvastatin group. Other limitations were a small study group and a direct effect of statins on hepatic CRP synthesis, which could exaggerate the impression of disease modification. Osteoporosis The biologic effects of statins on bone metabolism have been reported in literature [ 121 ]. Statins were shown to be potent stimulators of bone formation in vitro . Statins were shown to stimulate the bone morphogenic protein-2 (BMP-2) promoter in an immortalized osteoblast cell line [ 121 ]. BMP-2 is known to enhance osteoblast differentiation [ 122 ]. Further supporting evidence for its beneficial role came from osteoporosis observational studies [ 123 - 126 ]. However, in these studies, no adjustment for weight was made and part of the protective effect of statins could be because of reduction in weight. By contrast, the Women's Health Initiative Observation Study found no relationship between statins and hip/wrist/arm/non-spine fracture rates after adjusting for weight and other potential confounders [ 127 ]. Lack of benefit of statins in reducing hip and non-spine fracture was also reported in a case control study from the General Practice Research Database [ 128 ]. In the first placebo-controlled trial specifically designed to assess bone turnover, statin treatment did not show any difference in rates of bone formation [ 129 ]. Other uncontrolled studies have been conflicting; both increased [ 130 ] and decreased [ 131 , 132 ] rates of bone formation have been reported. In spate of high optimism, it has been suggested that increasing the bioavailability of statins to the bone may lead to better results [ 133 ]. As of now, keeping in mind lack of a consistent response with statins in various studies, it will be inappropriate to conclude that statins have a meaningful benefit for patients with osteoporosis. Cancer Similar to most of the above mentioned indications, the action of statins in cancer has been bi-fold with arguments and evidence both in favour and against having been published. It was suggested, nearly a decade ago, that cholesterol inhibition could inhibit tumour cell growth and possibly prevent carcinogenesis [ 134 ]. Recently, statin use was shown to be associated with a reduced risk of breast [ 135 ] and colorectal [ 136 ] carcinoma. However, these findings need confirmation as they were based on a small number of events. Statin use has been associated with a 20% reduction in colon cancer, if used for more than 4 years and if more than 1350 defined daily doses were taken [ 136 ]. Evidence to the contrary has also grown simultaneously. Epidemiological studies in the early 1990s had shown a rise in non-cardiovascular mortality, particularly cancer deaths in people with low cholesterol concentrations [ 137 ]. Similar conclusions have been drawn from results of early trials of cholesterol lowering [ 138 ]. Some researchers have shown that lipid-lowering drugs, including statins, increase the occurrence of several types of cancer in rodents [ 139 ]. In the CARE trial [ 6 ], incidence of female breast cancer and in the PROSPER trial [ 8 ] in elderly, incidence of all cancers increased in patients given pravastatin. With such conflicting evidence available there is a need for exercising cautious scepticism for a potential beneficial role of statins for cancers. Acquired Immune Deficiency Syndrome Hyperlipidemia induced by antiretroviral treatment is observed frequently and can cause an increase in cardiovascular risk in HIV patients [ 139 ]. Moreover, HIV infection itself induces pro-atherogenic lipid changes, which may lead to an increased cardiovascular risk but are partly reversed by antiretroviral regimens [ 140 ]. Statins, given to patients with HIV infection and hyperlipidemia, effectively reduced total cholesterol (27%) and triglycerides (15%) [ 141 ]. In the first double-blind, placebo-controlled study of the effects of statin therapy on lipids, lipoprotein subfractions, and endothelial function in HIV patients taking protease inhibitors, pravastatin reduced concentrations of atherogenic lipoproteins [ 142 ]. Similar beneficial effects of statins were shown in a cohort of 245 patients [ 143 ]. However, in all these studies the decrease in total cholesterol, LDL and triglycerides was only modest, and a significant number of patients did not achieve their NCEP goals. Moreover, the risk of rhabdomyolysis with concomitant use of statins in patients receiving highly active anti-retroviral therapy needs to be carefully evaluated in future studies. Statins have been shown to have a direct effect on HIV infection itself [ 144 , 145 ]. In in vitro studies, 9 days after viral loading, lovastatin inhibited both sterol synthesis and viral multiplication in Human H9 lymphocytic cell line [ 144 ]. Rho-guanosine triphosphatase (GTPase) activity is required for HIV infectivity into the cells [ 145 ]. Statins block Rho-A activation induced by HIV-1 binding to target cells and also inhibited entry of HIV-1 pseudotyped viruses. These data are only experimental and considerable work will need to be done before any speculations for anti-retroviral potentials of statins are made. Other indications Some of the other uses for which statins are being evaluated are drug-induced dyslipidemia following transplantation [ 146 , 147 ], for causing immunosuppression in patients undergoing organ transplantation [ 148 ], promotion of fracture healing in vascularised bone allograft [ 149 ], sickle cell anemia [ 150 , 151 ], idiopathic pulmonary fibrosis [ 152 , 153 ], sensorimotor recovery after experimental intra-cerebral haemorrhage [ 154 ], sepsis [ 155 - 157 ], and glomerulonephritis [ 158 ]. However, only limited, preliminary data are available to support routine use of statins in most of these indications and no recommendations can be made at present. Safety issues One cannot ignore the safety concerns with statin use; besides the well known side effects of myopathy, procarcinogenesis potential [ 159 , 160 ], nerve damage [ 161 , 162 ], short temper [ 163 ], cognitive decline [ 164 ], memory loss [ 165 ], teratogenic potential [ 166 , 167 ], and more recently loss of libido [ 168 ] are some of the other concerns. The rise, plateau and fall (?) of statins There is no doubt that statins have become one of the most commonly utilized drugs in cardiac patients not only in developed [ 169 ] but also in developing nations [ 170 ]. It is also obvious that their use will be intensively promoted in many non-cardiac conditions discussed above although the tremendous promise seen in some experimental and initial clinical studies failed to be sustained in clinical trials or if it did the effect was only modest. For others the initial conflicting results continue to exist. Recent years have shown a kind of contagiousness being demonstrated in research. Foremost among these have been the case of COX-2 inhibitors. After the discovery of COX-2 isoenzyme, almost every pathophysiological process showed involvement of COX-2 [ 171 , 172 ]. Selective inhibition of COX-2 was thought to be the answer to a number of problems in therapeutics. A large number of studies giving evidence to the contrary or addressing adverse effects of COX-2 inhibitors got overshadowed (or were suppressed) in the hype created over COX-2 inhibitors [ 173 , 174 ]. Rofecoxib and some other selective COX-2 inhibitors are being withdrawn for their adverse effect profiles as their discoverer companies gear up for payments of compensation claims made by sufferers. Many other molecules have suffered similar fate and we hypothesized that statin research may also be on decline. To test this hypothesis we searched Medline using the MeSH term "statins", "statins AND cancer (as well as other indications one by one)" for overall and yearwise extraction of citations. A total of approximately 11,000 citations were found out of which about 50% have appeared in only the past 4 years (since our last review [ 2 ]). An analysis of yearly trends showed some interesting details. The first study on statins was reported in 1975 [ 175 ]. Subsequently, there was a steady increase in the publications until pleiotropism of statins was suggested in the mid-90s [ 176 ] and since then (especially since 2000), a steep rise in publications for various indications with a peak around 2002–2003 can be noticed. It is interesting to note that a trend towards a decline in the number of these studies can already be seen for statins in general (Fig 1 ) and in many indications specifically (Fig 2 ). This declining trend is probably due to failure to establish any definite benefit in majority of the indications for which their use was proposed. Figure 1 Number of statin publications in each year from 1974 to 2004. The numbers depict the citations obtained from Pubmed on entering the MeSH term 'Statins' Figure 2 The trend in the number of published research articles in Pubmed, categorized according to the various pathological conditions discussed in the text. Therefore, our hypothesis which appeared quite implausible initially may not have been altogether wide of the mark. Consequently, it remains to be seen whether statins can withstand the test of time or will sink into oblivion like many of the other molecules. Conclusion If we take an overview of the evidence available for each of the above indications of statins we notice that it is rather weak even for the indications in which there are controlled trials available. Moreover, these trials are either inadequately powered or have measured only soft endpoints or have been of short duration to be conclusive. And lastly, a considerable number of contradictory studies make their utility in most of these diverse conditions doubtful.

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1079932

The Classics of Immunology

Medical Immunology will be publishing invited Reviews and Commentaries from investigators who are at the forefront of their fields, to up-date our readers as to the current state of their art. These Reviews and Commentaries will be accompanied by Editorials that place the current work into the perspective of the first contribution in an area, which resulted in a "Classic" paper. Where possible, links will be provided to the original publication, so that the modern student of immunology can read the original and draw their own conclusions as to the value of the "Classic" contribution, and its relationship to our contemporary views as to how the immune system functions. To begin this process at the very dawn of immunology, we highlight Sir Edward Jenner's first descriptions of the use of cowpox to immunize individuals against the dread disease smallpox.

Editorial With this editorial, Medical Immunology embarks on a new endeavor. One of the advantages of the electronic/digital age is that so much of the scientific literature is now available to everyone, not only to those with access to the specialized scientific or medical libraries. However, unless one knows which author/investigator/topic to seek out, it is still difficult for the student or layperson to know where to begin and which key words to use in the search engines. Accordingly, Medical Immunology now is devoted to an historical development of the field of immunology, much of which was organized chronologically in my first editorial[ 1 ], which attempted to justify why we needed a new on-line journal devoted to Medical Immunology. So much of the more recent scientific literature is difficult for one who is uninitiated in the science to be able to just pick up and read outright, because each contribution usually describes only a segment of a larger work by the authors and many others, and each individual contribution is but a very small part of a much larger area of investigation. As well, if one is not schooled in the vocabulary, it is difficult, if not impossible to begin to understand experiments performed and the significance of the findings reported. I have developed a method of inquiry of the scientific literature when embarking on a new subject that works for me, and allows me to organize the experimental findings and to rank them in importance one with another. Thus, I take a chronological approach, beginning with the very first report in a particular field, and then tracing the papers forward in time. This approach has the advantage of starting way in the past, at a time when often the concepts and methods of investigation were quite simple and therefore more easily understood, so that as one studies more recent reports it is much easier to appreciate the significance of a new approach or fresh insight into an old problem. Therefore, with this as background, we begin our course in immunology with Sir Edward Jenner's "An Inquiry into the Causes and Effects of the Variolae Vaccinae, a Disease Discovered in some of the Western Counties of England, particularly Goucestershire, and known by the name of The Cow Pox" , which was published in 1798 [ 2 ]. Reading through this detailed account of Jenner's experiments, which he conducted over several years, one is struck by the meticulous care this country doctor made in documenting his efforts, as well as his powers of observation, which allowed him to identify the unique characteristics of the cowpox lesions, so that he could transfer only the liquid from cowpox pustules from one individual to another. Of course, unbeknownst to Jenner at the time were the myriad of microorganisms that could mimic such pustules, such as the pyogenic bacteria such as staphylococci and streptococci. If he had not been so careful to detail and identify the characteristics of the cowpox lesions, he could have missed the observations that started the science of immunology, and that led to his worldwide fame and his Knighthood, as well as his lifetime pension from the Crown. It is perhaps ironic that we find ourselves 207 years later still concerned with smallpox, and concerned because of the idea that this devastating organism could again be used as a weapon of biological warfare. The stories of the English soldiers, who gave smallpox-infected blankets to the North American Indians during the French and Indian Wars, are familiar to us. Now we are afraid that new foreign terrorists may do the same to us! Even though Jenner's experiments showed us the way to effective vaccination, and even though smallpox was declared eradicated from the world in 1980, we still have not understood how to make an effective and safe vaccine. All of the vaccines that we have, including those developed empirically during the 1950's and 1960's, were developed before our present understanding of how the immune system functions, which has occurred only since 1970. Unfortunately, because of the threat of bioterrorism, we are now hard-pressed to invent, as rapidly as possible, new vaccines to protect our population against smallpox, as well as many of the very pathogenic microorganisms for which we do not have safe and effective vaccines. Accordingly, the questions as to how to construct new vaccines for smallpox, as well as many other infectious diseases are paramount. In this regard, how to test for the efficacy of vaccines is central to both the problem and the solutions. Of course, Jenner monitored a successful "take" of the cowpox inoculation as the development of a localized lesion that resembled the cowpox lesions on the hands of the milkmaids, and that resembled the skin lesions that became generalized after a smallpox infection. Accordingly, because the cowpox virus actually replicates in humans, it generated a localized self-limiting infection that could not only be detected with the naked eye, but by so doing, a very strong immunological stimulus resulted, which left the host with long-lasting immunological memory. Now that we know much more than did Jenner more than 200 years ago about how the immune system functions, one might imagine that it should be relatively easy to construct a safe and effective smallpox vaccine. Therefore, to begin our discussions in this regard, I have asked Mark Slifka, from the Vaccine and Gene Therapy Institute at the Oregon Health & Sciences University, to write a Commentary on the current state of the art of immunity to smallpox that can be detected by our most modern methods of monitoring the immune response, and to identify where we are, and where we need to go. Mark is especially suited to educate us, in that his group has published one of the most extensive analyses of the immunologic memory of individuals vaccinated against smallpox, using modern up-to-date methods [ 3 ]. This effort in Classics in Immunology should be construed as an open invitation for investigators to send us contributions that deal with new approaches to smallpox vaccines, as well as the use of smallpox vaccines as vectors for other microbes. As well, it is an invitation to all readers to write to the editors with questions or comments.

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1079933

Anthrax vaccine design: strategies to achieve comprehensive protection against spore, bacillus, and toxin

The successful use of Bacillus anthracis as a lethal biological weapon has prompted renewed research interest in the development of more effective vaccines against anthrax. The disease consists of three critical components: spore, bacillus, and toxin, elimination of any of which confers at least partial protection against anthrax. Current remedies rely on postexposure antibiotics to eliminate bacilli and pre- and postexposure vaccination to target primarily toxins. Vaccines effective against toxin have been licensed for human use, but need improvement. Vaccines against bacilli have recently been developed by us and others. Whether effective vaccines will be developed against spores is still an open question. An ideal vaccine would confer simultaneous protection against spores, bacilli, and toxins. One step towards this goal is our dually active vaccine, designed to destroy both bacilli and toxin. Existing and potential strategies towards potent and effective anthrax vaccines are discussed in this review.

Anthrax as a biological weapon Bacillus anthracis , the etiologic agent of anthrax, is an appealing biological weapon due to its high lethality and the ease of production and dissemination [ 1 , 2 ]. B. anthracis is a Gram-positive, aerobic, facultatively anaerobic, rod-shaped bacterium 1–1.5 μm wide and 3–10 μm long. An important aspect of B. anthracis is its ability to form dormant spores. Anthrax spores are naturally present in soil throughout the world, can remain viable for decades, and are highly resistant to adverse conditions such as heat, drought, ultraviolet light, gamma irradiation, and many disinfectants [ 3 ]. Anthrax spores are about 1–2 μm in diameter, optimal for inhalation and deposition in the alveolar spaces [ 4 ]. Inhalation of spores is almost always fatal, even with aggressive antimicrobial therapy [ 5 ]. Robust enough to withstand bomb detonation and small enough to aerosolize, anthrax spores may be easily dispersed over large populations by missiles, bombs, and aerosolization from flying aircraft. An aerosol release of odorless, invisible anthrax spores could travel far downwind from the point of release and cause catastrophic loss of life. Natural cases of human inhalational anthrax infection are rare in recent history. In the century preceding the anthrax attack in 2001, only 18 cases of inhalational anthrax were reported in the U.S., with the most recent one in 1976. However, several lethal anthrax attacks have been documented. In 1979, anthrax spores were accidentally released from a military laboratory in the former Soviet city of Sverdlovsk. Sixty-four people were reported dead, although U.S. intelligence sources claimed the toll might have reached 1,000 [ 1 ]. In 2001, through deliberate delivery of anthrax spores in mailed letters, 10 confirmed cases of inhalational anthrax and 12 confirmed or suspected cases of cutaneous anthrax in humans were reported in the U.S. [ 6 ]. If handled less competently, this attack could have killed many more people. For example, the letter sent to Senator Daschle reportedly contained 2 g of B. anthracis powder, equivalent to 200 billion to 2 trillion spores [ 2 ]. The human LD 50 (dose sufficient to kill 50% of infected persons) is estimated to be 2,500 to 55,000 inhaled spores, and as few as 1 to 3 spores may be sufficient to cause infection. Hence, an envelope full of anthrax spores, properly distributed, could kill many people. Disease and treatment Anthrax can affect a wide variety of wild and domestic animals as well as humans [ 7 ]. Humans may become infected by anthrax spores through skin abrasions, ingestion, or inhalation [ 6 ]. Cutaneous anthrax is mild and can be treated with antibiotics. Gastrointestinal or inhalational anthrax, if left untreated, usually leads to fatal systemic disease with a mortality rate approaching 100 percent [ 6 ]. Inhalational anthrax is the most lethal form. When anthrax spores are inhaled, they are deposited in alveolar spaces and ingested as inert particles by local macrophages [ 4 , 5 ]. The spores are then transported by the infected macrophages to mediastinal and peribronchial lymph nodes, where they germinate into vegetative bacilli. The bacilli escape from the macrophages and begin unimpeded extracellular multiplication within the lymphatic system, causing regional hemorrhagic lymphadenitis. The bacilli then spread into the bloodstream and continue rapid replication, reaching as many as 10 9 organisms per milliliter of blood [ 8 ]. All the while, the bacilli secrete high levels of exotoxins that intoxicate the host. The initial symptoms, such as malaise, fatigue, and cough, are nondescript and resemble those of influenza and other common upper respiratory infections, which makes early specific diagnosis difficult. After 2 to 5 days, there is a sudden onset of acute symptoms, which may include fever, chills, subcutaneous edema of the cheek and neck, widening of the mediastinum and pleural effusions, and hemorrhagic meningitis [ 4 , 5 ]. Death usually occurs within 24 hrs due to respiratory failure, with overwhelming bacteremia often associated with meningitis and subarachnoid hemorrhage [ 9 ]. Theoretically, there are several ways to approach anthrax: (i) vaccination to prevent disease development, (ii) elimination of spores, (iii) antibiotics to kill vegetative bacilli before the disease reaches a systemic stage, and (iv) conjunctive antitoxin therapy against anthrax toxin. Currently, treatment for anthrax relies mainly on antibiotics. A combination of antibiotics and aggressive hospital supportive care may succeed in the prodromal stage, but because the bacteria produce massive amounts of toxins that rapidly flood the blood and lymph system and send the patient into shock, the disease is often beyond treatment and inevitably fatal once the symptomatic stage has been reached [ 4 , 10 ]. Antitoxin therapy is also attractive, but if the bacteria continue to grow, will not be sufficient to stop anthrax. Full recovery from anthrax requires timely administration of antibiotics and antitoxic remedies, but timing is difficult due to non-specific initial symptoms. Hence, a prophylactic vaccine that prevents infection or stops infection at an early stage would be highly desirable. Vaccine against spores? Critical steps in B. anthracis infection include spore entry and germination, bacillar multiplication and dissemination, and toxin production. Destroying the spores either before or after they enter host cells is an attractive strategy to prevent disease development. However, such an approach aimed either at control or treatment has thus far not been developed. This is partially due to our almost complete lack of molecular understanding of anthrax spores and the inability of our immune system to disable anthrax spores. Our knowledge about spores is essentially limited to descriptive electron-microscopic morphology dating back mostly to the 1960s. The anthrax spore consists of several morphologically distinct layers, from outside to inside: exosporium, spore coat, cortex, spore membrane, and core [ 11 ]. These structures jointly provide a highly protective "lockbox", protecting the core which houses the spore's genetic material [ 12 ]. Because of this robust architecture, anthrax spores are long-lived and extremely resistant to adverse environments. Once inside the host, the same structures allow anthrax spores to survive the host immune defense and to germinate. It is not clear which type of immune response will be effective against spores. On the humoral immunity side, one could develop vaccines that elicit specific antibodies to opsonize spores. Several spore surface proteins have been identified recently [ 13 - 16 ]. However, since ingested, un-opsonized spores germinate in and are not killed by macrophages, would opsonized spores be taken up by macrophages or other phagocytes and be killed [ 17 ]? Would complement be able to lyse the spores? On the cellular immunity side, could cytotoxic T cells destroy the dormant or germinating spores inside macrophages? Consequently, whether a conventional vaccine design with the goal of eliciting antibodies or T cell immunity would be effective against spores is still an open question. By contrast, development of vaccines targeting the anthrax exotoxin and extracellular bacilli has been much more promising. Two major virulence factors The virulence of B. anthracis is attributable to two major factors: a poly-γ-D-glutamic acid (PGA) capsule and a secreted tripartite protein complex toxin. Fully virulent strains of B. anthracis carry two large extrachromosomal plasmids, pXO1 and pXO2, which encode for the toxin and the PGA capsule [ 18 , 19 ], respectively. Absence of either plasmid results in a marked reduction in virulence [ 7 , 20 ]. The first description of the B. anthracis capsule dates back to 1903 and M'Fadyean's discovery of the relationship between in vivo formation of the capsule and virulence [ 21 ]. The importance of the capsule was further emphasized in the 1950s when Bail demonstrated that strains that had lost the ability to produce a capsule were avirulent [ 7 ]. Wild-type anthrax bacillus is capsulated in animal hosts, but not in culture unless suitable conditions are provided [ 7 ]. Factors that influence capsule formation are important for determining the outcome of infection. In susceptible animals, the bacilli remain encapsulated, whereas in resistant animals, the capsule is shed [ 22 ]. PGA is an anionic, poorly immunogenic polypeptide that disguises the bacteria from the host immune surveillance and, by virtue of its negative charges, inhibits bactericidal activity by the host [ 7 ]. Thus, the PGA capsule allows virulent anthrax bacilli to grow virtually unimpeded in the infected host. Anthrax toxin, the other major virulence factor of B. anthracis , was discovered in 1954, when Smith and Keppie demonstrated that sterile plasma from experimentally infected guinea pigs was lethal when injected into other animals [ 8 , 23 ]. Anthrax toxin includes lethal toxin and edema toxin, which are binary complexes formed, respectively, by lethal factor (LF) and edema factor (EF) with protective antigen (PA) [ 24 , 25 ]. These proteins are released discretely as nontoxic monomers. The characteristic edema observed in cutaneous anthrax is produced by edema toxin [ 10 , 22 ]. EF is a calmodulin-dependent adenylate cyclase and catalyzes the production of intracellular cyclic AMP from host ATP [ 26 ]. Increased cellular levels of cyclic AMP upset water homeostasis and can cause massive edema. The most severe symptoms of anthrax, such as hypotension, shock, and death, are caused by lethal toxin [ 10 , 22 ]. LF is a zinc metalloprotease that inactivates mitogen-activated protein kinase kinase and acts specifically on macrophages [ 27 - 29 ]. Lethal toxin, when given intravenously, is relatively weak compared with other bacterial toxins [ 9 ]. Both lethal and edema toxins are thought to be important in the establishment of disease by impairing the host defenses. Overall, the capsule and toxin act jointly to maximize the survival of bacilli in the host. While the PGA capsule passively protects the bacilli from host defense, the toxin actively impairs the host to further ensure a favorable environment for bacillar growth. Veterinary vaccine: live attenuated organisms Studies on the vaccination of animals against anthrax date back to the end of the 19th century. In the 1870s, Robert Koch established B. anthracis as the etiologic agent of anthrax. In 1881, Pasteur demonstrated protective immunization against anthrax using a heat-attenuated strain, which was later recognized as an encapsulated strain with reduced virulence [ 7 , 21 , 30 ]. In the 1930s, Sterne developed an attenuated, toxigenic, but non-encapsulated strain that proved to be remarkably effective as a vaccine for domestic animals and is now used worldwide [ 7 ]. Although effective, the attenuated spore vaccines developed by Pasteur and Sterne suffer from declining potency and troublesome variations in virulence that led occasionally to the death of animals [ 21 , 30 ]. Due to residual virulence, the attenuated spore vaccines are not used in humans. Existing human vaccine: crude preparation of PA The development of anthrax vaccines for human use began in the 1940s, motivated by fear of the use of anthrax as a biological weapon. In 1970, the protective antigen (PA)-based cell-free subunit vaccine, designated "anthrax vaccine adsorbed" (AVA) or Biothrax, was licensed and recommended for use by a small population of mill workers, veterinarians, laboratory scientists, and others with risk of occupational exposure to anthrax [ 30 , 31 ]. Increased concern about the use of anthrax in warfare led the Department of Defense to vaccinate U.S. military personnel in the 1990s. AVA is prepared from microaerophilic cultures of the attenuated, non-encapsulated strain V770-NP1-R of B. anthracis [ 9 , 31 ]. Downstream processing begins with filtration, which removes the bacterial cells along with some EF and LF. The cell-free culture filtrate, thought to contain predominantly PA, is then adsorbed to aluminum hydroxide [ 32 ]. Small amounts of formaldehyde and benzethonium chloride are added as preservatives [ 9 ]. Although PA is by itself an effective immunogen, it is not clear whether the small amounts of LF and EF that may be present in some lots contribute to the vaccine's effectiveness. A vaccine licensed in the UK is prepared by alum precipitation of the sterile culture filtrate of a derivative of the attenuated Sterne strain. It contains higher amounts of LF and EF than AVA [ 33 , 34 ]. The efficacy of AVA and highly purified PA preparations have been tested in various animal models, including mice, hamsters, guinea pigs, rabbits, and monkeys [ 31 , 32 , 35 ]. Interestingly, protection varies widely among species. AVA does not protect hamsters at all [ 36 ]. In mice, the PGA capsule appears to be the primary virulence factor, and PA-based vaccines confer only limited protection [ 37 ]. There is no direct correlation between anti-PA titers and protection in mice and hamsters [ 36 , 37 ]. In guinea pigs, AVA provides partial protection [ 35 , 38 , 39 ], but AVA appears to be more effective in rabbit and macaque models [ 35 , 40 - 42 ]. It is noteworthy that "efficacy", as defined in these studies, is relative. When macaques were exposed experimentally to doses of up to 900 times the LD 50 , 88–100% of the animals were protected [ 31 ]. However, simulation studies suggest that a person opening a letter filled with anthrax spores and standing over it for 10 min could inhale up to 3,000 times and perhaps as much as 9,000 times the LD 50 for humans [ 2 ]. A controlled human trial was conducted in the 1950s with a vaccine similar to AVA but derived from a different attenuated, non-encapsulated strain of B. anthracis grown aerobically. In a susceptible population of textile mill workers in the northeastern states of the US who processed occasionally contaminated goat hair, vaccination provided 92.5% protection against cutaneous anthrax [ 43 , 44 ]. However, no assessment of inhalational anthrax could be made, because cases were too few. There are several concerns regarding AVA: (i) It does not protect all animal hosts against different strains of B. anthracis . AVA or PA-based vaccines in general induce toxin-neutralizing antibodies. The mechanism underlying the protective action of PA-based vaccines is unclear. It is thought that anti-PA vaccines protect the host from intoxication and thus allow the immune system to deal with the organism. However, evidence that primates vaccinated with AVA or PA vaccine develop transient episodes of bacteremia suggests that vaccination does not prevent the growth of bacilli. (ii) The administration of AVA is burdensome, requiring subcutaneous injections at 0, 2, and 4 weeks and 6, 12, and 18 months with subsequent yearly boosters [ 31 , 32 ]. In a field trial of a vaccine similar to AVA, one case of cutaneous anthrax occurred 5 months after the initial 3-dose series and just before the scheduled 6-month booster [ 9 ]. This case suggests that immunity is not long-lasting and that frequent boosters may be necessary. (iii) The preparative processing of AVA is crude and lacks consistency. Furthermore, there are relatively high rates of local and systemic adverse reactions, likely due to residual toxicity in AVA or other contaminants. Improvement: highly purified recombinant PA The limitations of AVA have raised widespread interest in developing improved anthrax vaccines consisting of well-characterized components. A new generation of vaccines based on highly purified recombinant PA is currently being developed and evaluated. There have been numerous attempts to establish high-level PA expression systems based on a variety of organisms, including attenuated strains of B. anthracis , B. subtilis , B. brevis , Salmonella typhimurium , E. coli , viruses, insect cells, and plants [ 45 - 50 ]. In addition, genetic immunization with DNA encoding for PA is being explored [ 51 ]. The highly purified PA vaccines are expected to induce essentially the same immunity as AVA. While some disadvantages of AVA due to its "dirty" preparation may be overcome, limitations in protection and lack of an immune memory response may be intrinsic to PA itself. New strategies are needed for further improvement. One possibility is that other antigens or cellular immunity in addition to PA-specific antibodies are required for full protection in different animal species. This is supported by studies showing that the live veterinary vaccine provides significantly greater protection against anthrax in experimental animals than does AVA, despite the fact that it frequently induces lower levels of antibodies to PA [ 33 , 39 , 42 , 52 , 53 ]. After a naturally acquired infection, and depending on when samples are taken, 68–93% of cases develop antibodies to PA, 42–55% of cases develop antibodies to LF, and antibodies to EF are less frequently detected [ 34 , 54 - 56 ]. Interestingly, antibodies to the capsule are detected in 67–94% cases [ 55 , 56 ], whereas no response to the capsule is expected in the vaccinees who have been vaccinated with AVA or non-encapsulated live vaccines. Further improvement: two-in-one postexposure antitoxic therapy/vaccine Post-exposure vaccination is the most likely scenario, given the rarity of natural inhalational anthrax infection. However, the use of PA as a postexposure vaccine may be limited. Since PA is a natural component of anthrax toxin and may contribute to toxin formation, it may not be safe to administer a PA-based vaccine to persons who have been or are suspected of having been exposed to anthrax. We recently proposed the replacement of PA in vaccines with a dominant-negative inhibitor (DNI) of anthrax toxin [ 57 ]. DNI is a translocation-deficient mutant of PA carrying double mutations of K397D and D425K and has been demonstrated to interfere with the intoxication process, providing immediate therapeutic protection against anthrax toxin in vivo [ 58 , 59 ]. Furthermore, when used as a vaccine, DNI is more immunogenic than PA [ 57 ]. The symptoms and incubation period of human anthrax vary depending on the route of transmission. The reported incubation period of inhalational anthrax, the most lethal form, ranges from 1 to 43 days [ 60 ]. Data from animal studies suggest that anthrax spores persist in the host for several weeks after infection and that antibiotics can prolong the incubation period for developing disease. Studies in nonhuman primates indicate that inhaled spores do not immediately germinate within the alveolar recesses but reside there, possibly for weeks, until taken up by alveolar macrophages [ 61 ]. Development of anthrax disease can be prevented as long as a therapeutic level of antibiotic is maintained to kill germinating bacilli. After antibiotics are discontinued, disease will develop if the remaining spores germinate in the absence of a protective immune response. In previous animal studies, treatment with antibiotics for 5 or 10 days, beginning one day after anthrax spore aerosol challenge, was protective during drug therapy, but animals died after the antibiotic was discontinued [ 61 ]. Longer antibiotic treatment, e.g., for 30 days, might be necessary to ensure full recovery. However, antibiotic treatment cannot protect against relapse or subsequent exposure to anthrax. Long-term protection was achieved only by combining antibiotic therapy with post-exposure vaccination [ 62 ]. In such situations, conjunctive antibiotic treatment and vaccination with DNI would be ideal, whereas administration of PA would be potentially dangerous because it may combine with trace amounts of LF or EF and cause toxicity. During the anthrax attack in the U.S. in 2001, some groups of individuals underwent a 60-day regimen of antibiotic prophylaxis. Statistical analysis shows that this preventative measure may have saved many lives [ 63 ]. In the event of an anthrax attack, the incubation period will vary among individuals. The timely post-exposure administration of DNI as both a conjunctive therapy to antibiotics and a prophylactic vaccine would be expected to be superior, both for individual and public health perspectives. Enhanced antigen processing through "endosomal trapping"? The increased immunogenicity of DNI not only recommends it as a potential new anthrax vaccine but may also point to a potentially important general strategy for future vaccine design. We proposed an "endosomal trapping" mechanism that rationalizes more efficient antigen processing of DNI over native PA [ 57 ]. As illustrated in Figure 1 , PA molecules undergo several transformations during the intoxication process [ 24 ]. Upon release, the 83-kD PA molecule first binds to a receptor present on most mammalian cells [ 25 , 64 - 66 ]. The cell-bound PA is then cleaved by furin or a furin-like protease into two components [ 67 ]. The dissociation of the N-terminal 20-kDa fragment PA 20 exposes a binding site for LF or EF to the cell-bound 63-kDa fragment PA 63 and also enables PA 63 to assemble into a heptameric, ring-shaped prepore (PA 63 ) 7 [ 68 ]. LF or EF bind competitively to (PA 63 ) 7 with very high affinity ( K d ~ 1 nM) [ 69 , 70 ]. The complex is internalized by receptor-mediated endocytosis and trafficked into an acidic endosomal compartment. (PA 63 ) 7 undergoes a major conformational rearrangement following the pH change and forms a membrane-spanning β barrel that enables its penetration into the cytosol [ 71 , 72 ]. By means of (PA 63 ) 7 , EF and LF translocate into the cytosol, where they modify substrates and exert toxic effect (see above). However, the two mutations in DNI inhibit the required conformational change of (DNI 63 ) 7 or chimeric DNI/PA heptamers from a ring-shaped core to a β barrel and thus prevent the heptamer from inserting into the endosomal membrane [ 59 ]. Consequently, these mutations inhibit the translocation of LF or EF into the cytosol and prevent cytotoxicity [ 58 ]. Figure 1 Models illustrating the different cellular fates of PA and DNI. Top: Upon binding cellular receptors (step 1), PA is cleaved (step 2). The small fragments diffuse away, and the cell-bound fraction self-assembles into heptameric cores termed (PA 63 ) 7 (step 3). The heptamers then undergo receptor-mediated endocytosis (step 4). Once inside the acidic endosomal compartment, the PA heptamers change conformation and insert into the endosomal membrane (step 5). Bottom: DNI, similar to PA, enters the endosome (steps 1–4). However, DNI heptamers do not undergo the necessary conformational changes for insertion into the membrane and therefore remain trapped inside the endosome. Protein antigens are commonly processed in the endosomes of antigen-presenting cells and elicit T-cell help in antibody production. The peptides from degraded protein antigens are loaded onto MHC class II molecules and transported to the cell surface. Thus, the insertion of PA pores in the endosomal membrane may disrupt the integrity of the endosome and thus compromise its function, perhaps leading to altered vesicular trafficking of endocytic PA molecules. Altered vesicular trafficking of PA may affect its delivery to specialized endosomal compartments, where antigenic processing of PA occurs. It is also possible that PA pore formation might simply change the pH or ionic environment inside the lumen of the endosome and thus affect the processing/presentation machinery. Furthermore, PA may disrupt or otherwise "leak" out of the endosome and simply enter the cytosol (as is the case for the LF and EF subunits) and thus escape efficient endosomal processing. In contrast, DNI heptamers do not insert into the endosomal membrane and block translocation of LF and EF into the cytosol. Thus, DNI is expected to be trapped in the endosome and undergo normal vesicular trafficking. Hence, the translocation-deficient mutant DNI may concentrate in the endosome; that is, DNI may be more prone to "endosomal trapping" than PA, and this difference in localization may increase the generation of processed DNI peptides suitable for binding to MHC class II molecules. If such a mechanism indeed explains the enhanced immunogenicity of DNI, similar strategic mutations might be introduced into other toxin immunogens to enhance their immunogenicity via this "endosomal trapping" mechanism. New-generation vaccines: dual protection against bacilli and toxin Despite the direct toxicity of anthrax toxin, systemic anthrax disease is amplified by the massive extracellular replication of the bacilli that produce it. Directly targeting B. anthracis at the extracellular stage is both appealing and feasible. It should be reiterated that it is the bacilli that are replicating and secreting toxins, and eliminating bacilli would abrogate toxin production at the source. A major virulence factor of B. anthracis is its anti-phagocytic PGA capsule [ 7 , 73 ]. The role of a capsule in virulence has been well established for numerous bacterial species, such as Streptococcus pneumoniae and type b Haemophilus influenzae (Hib) [ 74 , 75 ]. In these bacteria, the capsule also confers resistance to phagocytosis by host cells. The immunological role of the B. anthracis PGA capsule very much resembles the role of capsular polysaccharides in other bacteria. The weakly immunogenic capsules do not favor an immune response but rather enable encapsulated bacteria to evade the host immune defenses. Vaccines based on capsular polysaccharides have been highly successful against pathogens such as Hib. We hypothesized that PGA-based vaccines could have similar success in protection against B. anthracis infection. Although capsular PGA is a promising antigen for a new anthrax vaccine, PGA alone has limited use because of its weak immunogenicity. Fortunately, similar to polysaccharides, the immunogenicity of PGA can be significantly enhanced by conjugation to a strongly immunogenic protein carrier [ 76 , 77 ]. Functional studies have demonstrated that anti-PGA antibodies do indeed confer protection by mediating opsonophagocytosis of B. anthracis [ 78 , 79 ]. Because the pathogenesis of anthrax is largely attributable to replication of bacilli and release of toxin, we constructed a new generation of anthrax vaccines by chemically conjugating PGA and PA, the two virulence components of B. anthracis . This dually active anthrax vaccine (DAAV) is capable of inducing high levels of specific antibodies to both capsule and toxin [ 76 ]. We envision that a PGA-directed antibody response will achieve protection against anthrax by eliminating bacteria early in the sequence of infection, well before the onset of bacteremia and toxemia. The fact that the very early stages of anthrax disease can be treated by antibiotics also supports the notion that an anti-bacillar vaccine may be effective, or at least a valuable addition to vaccines based on PA alone. In addition, antibodies to PA provide a parallel line of defense against residual toxin. DAAVs embody the paradigm of combining both antibacterial (i.e., prophylactic) and antitoxic (i.e., therapeutic) components into a single vaccine. Authors' contributions Julia Y. Wang and Michael H. Roehrl jointly wrote the paper.

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1079934

Actinomycetes as host cells for production of recombinant proteins

Actinomycetes ( Actinobacteria ) are highly attractive as cell factories or bioreactors for applications in industrial, agricultural, environmental, and pharmaceutical fields. Genome sequencing of several species of actinomycetes has paved the way for biochemical and structural analysis of important proteins and the production of such proteins as recombinants on a commercial scale. In this regard, there is a need for improved expression vectors that will be applicable to actinomycetes. Recent advancements in gene expression systems, knowledge regarding the intracellular environment, and identification and characterization of plasmids has made it possible to develop practicable recombinant expression systems in actinomycetes as described in this review.

Review As a result of the sequencing of entire genomes of more than 100 organisms, the number of open reading frames with unknown functions has increased. To characterize such proteins biochemically and structurally, it is important to obtain substantial amounts of recombinant proteins. Escherichia coli , a Gram-negative γ-proteobacterium, is the most commonly used host bacterium for the large-scale production of recombinant proteins. However, the expression and isolation of all the proteins in E. coli is difficult on account of the problems of insolubility, cytotoxicity, post-translational modifications, or inefficient translation. In order to overcome these problems, host-vector systems other than E. coli have also been developed in both prokaryotic and eukaryotic cells. Bacillus spp. (e.g., B. brevis and B. subtilis ) and Lactococcus lactis are Gram-positive bacteria with low G + C content and are often used to secrete expressed proteins into the culture media [ 1 - 5 ]. Secretion prevents the local accumulation of the recombinant proteins and this occasionally aids in correct protein folding [ 6 ]. Bacillus brevis has an extremely high capacity of protein secretion and is being used for the expression of prokaryotic and eukaryotic proteins on an industrial scale [ 2 ]. Lactococcus lactis can also be used for the secretion of recombinant proteins and these proteins can be used directly in food applications [ 5 ]. Eukaryotic cells such as yeast cells, insect cells, or immortalized cell lines are particularly useful for the expression of proteins that undergo post-translational modifications [ 7 ]. In actinomycetes (Gram-positive bacteria with a high G + C content), the genera Streptomyces , Rhodococcus , Corynebacterium , and Mycobacterim have received an increasing amount of attention, particularly in the industrial fields. They exhibit potential advantages in the synthesis of secondary metabolites of industrial and medical importance, in the production of amino acids by fermentation, and in bioconversion processes. There have also been several host-vector systems developed for actinomycetes [ 8 , 9 ], although further improvements were needed to provide highly inducible and tightly regulated promoters, broad-host range vectors, and high producibility of recombinant proteins. Recently, improvements in the host-vector systems in this class of bacteria were reported, thereby making it possible to obtain significant amounts of recombinant proteins under strictly regulated promoters [ 10 - 12 ]. Here, we review the host-vector systems, particularly expression vectors, in actinomycetes and also describe the benefits and future possibilities of the system. Why actinomycetes? We can highlight two striking characteristics of actinomycetes as host cells. First, they exhibit a unique metabolic diversity and enzymatic capabilities. The compounds they produce as secondary metabolites are valuable for industrial and pharmaceutical purposes [ 13 ], and the enzymes themselves are also valuable. For example, Streptomyces spp. produce various types of antibiotics [ 14 ] and some Rhodococcus spp. are being used for the industrial production of acrylamide [ 15 ]. Historically, the host-vector systems in actinomycetes have been developed to obtain such enzymes in large quantities and/or to manipulate the metabolic pathway involved in the production of antibiotics [ 8 , 9 ]. Second, actinomycetes are expected to have different intracellular milieu as compared to conventional host cells such as E. coli . Until recently, no host cell from which all the proteins can be universally expressed in large quantities has been found. Therefore, it is important to provide a variety of host-vector systems (expression systems) in order to increase the opportunities to screen for the most suitable expression conditions or host cell. It is important to select an appropriate promoter for high-level protein expression, and generally, an inducible promoter is more preferable than a constitutive promoter [ 8 , 10 ]. Several reports used well characterized promoters of the nitrilase gene [ 10 ], acetoamidase gene [ 16 ], and tipA [ 11 , 12 , 17 ]. In S. coelicolor , the expression vector containing act I/ act III promoter was induced during the transition from the growth to the stationary phase to successfully produce polyketido synthases [ 18 ]. A derivative of this vector can be used with other actinomycetes [ 19 ], thereby expanding the application of the expression system. In M. smegmatis , novel strong constitutive promoters were identified using a genomic library fused to promoterless green fluorescent protein in combination with a fluorescence-associated cell sorting technique [ 20 ]. On the other hand, the mutagenesis of the repressor gene is another possible strategy in which the constitutively expressed temperature-sensitive repressor protein is unable to repress the target gene expression at a high temperature [ 21 , 22 ]. In this review, we have focused on the systems using heterologous promoters to drive strong expression and some examples of such vectors of actinomycetes are summarized in Table 1 . In the next sections, several recent topics are discussed in detail. Streptomyces species Among the Streptomyces spp., S. lividans has been extensively utilized as a potential host for both cytoplasmic and secreted protein production because it lacks restriction systems that are generally present in other Streptomyces and prevent the genetic manipulation of host cells [ 23 ]. In addition, S. lividans also exhibits a very low endogenous extracellular proteolytic activity [ 23 ]. Recently, Herai et al. reported an expression vector that functions in several Streptomyces spp. [ 10 ]. The vector carries the nitrilase gene promoter (P nitA ) originating from the nocardioform actinomycete Rhodococcus rhodochrous J1 [ 15 ]. The expression is tightly regulated and strongly induced only in the presence of the inducer ε-caprolactam. These researchers expressed several bacterial genes and estimated that up to ~40% of all soluble protein comprised a target protein and that up to 396 mg of the protein per liter of culture media was produced (e.g., the Streptomyces inducible expression system had thus far expressed a maximum of 38 mg of the protein per liter of the culture media [ 10 , 24 ]). However, the report does not refer to secreted proteins or the production of proteins originating from sources other than bacteria. This hyperinducible expression vector can be further improved to enable protein secretion and can be used to express higher eukaryotic proteins. This vector may also enable rapid progress in genome mining and the production of natural-product gene clusters such as those identified for enediyne antibiotics [ 25 ]. An increasing number of studies over the past years have reported Streptomyces as an ideal host for the production of secreted proteins. Signal peptides are an important factor for improving the efficiency of secreted protein production, and are extensively studied via mutagenic approaches [ 26 ]. Several Streptomyces secretion systems have successfully produced eukaryotic proteins [ 27 - 29 ]. The soluble form of human CD4 was efficiently produced using S. lividans as a host cell. Over 300 mg of protein was produced per liter culture by using pLTI-CD4 containing S. longisporus serine protease inhibitor gene promoter and secretion signals [ 27 ]. Rhodococcus species In Rhodococcus , several cloning vectors have been developed since the first report on E. coli - Rhodococcus shuttle vector [ 30 ], and recently, versatile expression vectors have been constructed [ 11 , 12 ]. The expression levels of proteins from these vectors are not as high as that from the expression system in E. coli (a maximum of 10 mg of protein per liter of culture media). However, in Rhodococcus , proteins can be expressed over a wide range of temperatures – from 4°C to 35°C [ 11 , 12 ] – because some Rhodococcus cells are psychrotrophic. When the thiostrepton inducible tipA promoter was used, several milligrams of mouse protein per liter of culture media could be expressed even at 4°C [ 11 ]. Most of the recombinant protein expression systems established until recently can only be used within the range of 10°C to 37°C. For example, E. coli is a mesophilic bacterium that grows at temperatures ranging from 18°C to 37°C, and recombinant protein expression has been carried out in a similar range of growth temperatures [ 31 ]. It is commonly known that a lower temperature is often more preferable for the production of recombinant proteins [ 32 ]. Some mouse proteins that could not be expressed in E. coli could be expressed in Rhodococcus at 4°C [ 11 ]. The expression at lower temperatures is expected to be effective in producing proteins that damage the host cell, because enzymatic activities of such proteins can be suppressed. In the case of R. erythropolis , the mycolic acid composition of the host cells makes it difficult to disrupt the cell walls and necessitates an approach for the modification of the host cells to simplify recombinant-protein extraction procedures [ 33 ]. The authors reported lysozyme-sensitive mutants that can be lysed by the addition of 12.5 μg ml -1 lysozyme, while the wild type is resistant to over 1 mg ml -1 lysozyme. Rhodococcus spp. are tolerant to various organic solvents and toxic chemicals. A highly efficient bioconversion process can be achieved by the combination of the expression system and this characteristic feature of Rhodococcus spp. Corynebacterium and Mycobacterium species Corynebacterium and Mycobacterium spp. are phylogenetically closely related to Rhodococcus spp. [ 34 ]. C. glutamicum is used for the industrial production of L-glutamate, while C. diphtheriae is the causative agent of diphtheria [ 35 ]. Among the Mycobacterium spp., the fast-growing non-pathogenic M. smegmatis is widely used as a model species [ 36 ]. Microorganisms such as M. tuberculosis and M. leprae , which are highly virulent human pathogens, are also well characterized species [ 8 ]. Spratt et al. identified strong expression promoters and demonstrated that one of them enabled the production of approximately 125 μg protein per milligram cell lysate [ 20 ]. The techniques developed by the authors for identifying the above mentioned novel promoters may be useful, although the technique is only applicable to constitutive promoters. As shown in Table 1 , some other expression vectors in these species are used to express homologous and/or heterologous proteins. Conclusion When expressing recombinant proteins, it is often recommended to use host cells that are phylogeneticaly closely (ideally, identical) related to the origin of the protein of interest. This is due to the similarity in frequency of codon usage, compatibility with machineries of translation and molecular chaperones, and/or redox states of the cells. Hence, when expressing higher eukaryotic proteins, in principle, using higher eukaryotes as hosts is ideal but it often results in low yields, and furthermore, is expensive and time consuming. The host-vector systems of actinomycetes are suitable for expressing proteins of actinomycetes and proteins from closely related organisms as well as from higher eukaryotes. However, further development of host-vector system in actinomycetes is required, particularly with respect to the modification of host cells. This includes improvement in stability and easy maintenance of foreign genes (e.g., integration of plasmids), removing host proteins that hamper production (e.g., knock-out of proteases) either at the gene level or during the extraction of proteins.

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1079935

Screening for novel enzymes from metagenome and SIGEX, as a way to improve it

Metagenomics has been successfully applied to isolate novel biocatalysts from the uncultured microbiota in the environment. Two types of screening have been used to identify clones carrying desired traits from metagenomic libraries: function-based screening, and sequence-based screening. Both function- and sequence- based screening have individual advantages and disadvantages, and they have been applied successfully to discover biocatalysts from metagenome. However, both strategies are laborious and tedious because of the low frequency of screening hits. A recent paper introduced a high throughput screening strategy, termed substrate-induced gene-expression screening (SIGEX). SIGEX is designed to select the clones harboring catabolic genes induced by various substrates in concert with fluorescence activated cell sorting (FACS). This method was applied successfully to isolate aromatic hydrocarbon-induced genes from a metagenomic library. Although SIGEX has many limitations, it is expected to provide economic advantages, especially to industry.

Review More than 99% of bacteria in the environment cannot be cultured using conventional methods [ 1 , 2 ]. To study and use the genomes of such uncultured microbes, metagenomics has been in the spotlight since the 1990s [ 3 ]. Many studies have constructed metagenomic libraries to search for novel biocatalysts or molecules for biotechnological and pharmaceutical applications. To date, metagenomics has uncovered a variety of novel genes ranged from small genes conferring enzymes to complex gene clusters encoding proteins involved in antibiotic production, using different kinds of vectors such as plasmids, cosmids, fosmids and bacterial artificial chromosomes [ 4 ]. However, the efficiency of searching for novel catalysts from metagenome can still be improved. Screening for desired traits needs improvement because this step is still labor-intensive and time-consuming. This mini-review discusses the strategies that have been used in metagenome screening, particularly the recently introduced screening strategy, SIGEX. The characteristics of the discussed strategies are summarized in Table 1 . Functional- and sequence based screening Two strategies are generally used to screen and identify novel biocatalysts or genes involved in the production of antibiotic from metagenomic libraries: function-based and sequence-based screening. In function-based screening, clones expressing desired traits are selected from libraries, and aspects of molecular biology and biochemistry of active clones are analyzed. Many enzymes of industrial importance have been discovered using this strategy (Table 1 ). This approach enables the rapid acquisition of clones that have potential of direct application in industry. Moreover, this screening method can detect genes with completely novel DNA sequences, which may have functions distinct from known biocatalysts. However, function-based screening has several limitations. This method requires expression of the function of interest in the host cell (e.g. Escherichia coli ) as well as clustering of all of the genes required for the function. In addition, efficient and economical screening methods for desired traits must be established to facilitate high-throughput-screening of vast libraries. Conversely, sequence-based screening is not dependent on the expression of cloned genes in heterologous hosts. Generally it is based on the conserved DNA sequences of target genes. Hybridizations or PCR are performed based on the deduced DNA consensus. However the limitations of sequence-based screening are that DNA consensus must be analyzed and determined, which cannot be applied to many biocatalysts, and that it does not guarantee acquisition of full-length genes or full gene clusters that are necessary for the production of the desired product. Moreover, the sequence-based screening never screens desired genes with completely different sequences, and easy expression or correct folding of the screened gene is not assured. In metagenomics, several novel enzymes of industrial importance have been screened successfully using this strategy, but the typical application of sequence-based screening is to obtain ribosomal RNA genes for phylogenetic surveys. As a form of sequence-based screening, shotgun sequencing of metagenomic libraries has recently provided vast amount of data, including phylogenetic relationships, millions of novel genes, and deduced metabolic pathways of uncultured bacteria [ 5 - 7 ]. Some of the novel genes might be of industrial importance. However, shotgun sequencing is extremely expensive and labor intensive, especially when one aims to discover genes of desired traits. Moreover, since the data from shotgun sequencing are analyzed in sequence-similarity searches based on constructed database, this method is not free from the limitations of sequence-based screening. Although both of function- and sequence-based screening strategies have been applied to isolate novel biocatalysts from metagenome, both approaches are laborious due to the low frequency of clones with desired traits (e.g. 4 from 930,000) [ 8 ]. To improve the frequency of screening, several strategies have been developed. For example, to overcome the difficulties with the heterologous expression of secondary metabolites, Streptomyces lividnas or Pseudomonas putida have been used in addition to E. coli [ 9 - 11 ]. In addition, enrichment steps for uncultured microorganisms containing the desired traits have been used successfully before library construction [ 12 - 15 ]. This approach is also advantageous because it overcomes the cloning difficulties due to the contaminants in environmental samples. Yet, the biased selection of metagenome argues against enrichment. SIGEX, the third screening method In an effort to improve the frequency of screening hits, Kazuya Watanabe and colleagues proposed substrate-induced gene expression screening (SIGEX), and its utility was evaluated for the screening of aromatic hydrocarbon-induced genes from a groundwater metagenome library [ 16 ]. To design of SIGEX is based on the facts that the expression of catabolic genes is generally induced by substrates or metabolites of catabolic enzymes, and that the expression of catabolic genes is controlled by regulatory elements located proximately in many cases. SIGEX screens the clones harboring desired catabolic genes that are expressed in the presence of substrates but are not expressed in the absence of substrates. The procedure is described in Figure 1 . To make SIGEX a high-throughput process, an operon-trap vector (p18GFP) was constructed, in which the cloning site divides the lac promoter and the gfp structural gene. Metagenomic libraries are constructed using p18GFP (Step 1). Self-ligated clones and the clones expressing gfp constitutively are removed by IPTG induction in the absence of the substrate (Step 2). The expression of catabolic genes in cloned metagenomic DNA is determined by gfp expression in the presence of the substrate (Step 3), and then the positive clones are separated on agar plates and characterized (Step 4). Fluorescence-activated cell sorting (FACS) is applied to the sorting and separation of GFP-expressing clones, i.e . the clones with desired catabolic genes. Watenabe and colleagues constructed a metagenomic library using groundwater sample and successfully applied SIGEX to isolate 33 clones induced by benzoate and two clones induced by naphthalene from 152,000 clones [ 16 ]. In addition these researchers showed that enzyme Bzo71-8 P450 from the metagenomic library is novel. These data demonstrate the practice of SIGEX for screening catabolic genes using appropriate inducers or substrates, and the possibility of SIGEX to yield more active clones than conventional screening methods. Advantages and disadvantages of SIGEX SIGEX has many advantages in metagenome screening. It provides an efficient and economic way of high throughput screening, because it allows for semi-automation thereby saving time, labor and expenses. This is particularly important for industrial applications. SIGEX is also advantageous because it can detect catabolic genes for which colorimetric or other on-plate screening methods are not established. Using this strategy, the Watanabe group screened hydrocarbon-induced genes, which are difficult to screen using conventional methods [ 16 ]. In addition, SIGEX does not require the modified substrates that are often used in colorimetric screenings, which are occasionally toxic, cause side-effects, and are generally more expensive than unmodified substrates. Moreover, SIGEX enables the deduction of the substrates for an unknown enzyme from the induction substrate used in the SIGEX screening. This helps to increase scientific knowledge about the genetics of previously unknown and hypothetical genes. However, the application of SIGEX has limitations. First, SIGEX is sensitive to the structure and orientation of genes with desired traits. SIGEX misses catabolic genes that are expressed constitutively. In addition, SIGEX cannot detect any active clones in which the desired catabolic genes are cloned in the direction opposite gfp . Moreover, it misses the active clones that have a transcription terminator between catabolic genes and the following gfp . In these cases, conventional function-based screening methods have been successfully applied to detect active clones. Particularly for the last reason, SIGEX is not suitable for applying to metagenomic libraries harboring large insert DNA due to the abundance of transcription terminators [ 17 ]. Because the probability of finding a screening hit using function-based screening increases exponentially with DNA insert size [ 18 ], the application of SIGEX should be considered carefully, especially when large pieces of environmental DNA are readily prepared. Second, substrates that do not migrate to the cytoplasm cannot be used with SIGEX. Many enzymes, such as amylases, proteases, lipases, cellulases and xylanases target macromolecules that do not migrate to the cytoplasm. To date, such enzymes have been detected by the incidental natural secretion of intracellular proteins or artificial cell disruption. Since many of these enzymes are of industrial importance, this drawback cannot be overlooked. Finally, the gate setting in FACS and the media conditions containing the inducer are critical for discriminating false-positive and false-negative results. Therefore, when SIGEX is applied, these drawbacks should be considered carefully. In conclusion, under the conditions where SIGEX is applicable, i.e . when appropriate substrates and target genes are selected, and the gate-setting of FACS is optimized, SIGEX can be a very powerful tool, especially to industry, for screening genes involved in antibiotics production or biodegradation induced by small molecules. Conclusion Metagenomics has proven effective for isolating novel biocatalysts from the environment as well as to acquire ecological data. Its scale and scope have been expanded since its concept was first introduced. For example, robotic automation has been developed to construct and screen metagenomic libraries, and large corporations have provided substantial funding for metagenomics. However the major problems in constructing metagenomic libraries remain to be solved. No standard protocol exists for isolating sufficiently purified metagenomic DNA from environmental samples. In addition, the heterologous expression system of genes from metagenome requires further improvement. An efficient expression system other than E.coli should be developed and settled (trials have recently begun). Moreover, since the conventional screening system is costly and time-consuming despite the recent improvement of automation, it remains necessary to develop more effective and economic strategies. SIGEX is a good way to overcome this bottleneck. Therefore, to exploit the enormous genetic resources in the environment in more efficient ways, these problems should be solved, and improved technologies should be developed.

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