The objective of our study was to investigate the use of the interrupter technique as a method to measure the response to provocation and to compare it with the conventional PD100Rint, EI) as well as by percent dose response ratio (DRR).We studied 170 normal subjects, 100 male and 70 female , non-smoking from healthy families. These subjects had no respiratory symptoms, rhinitis or atopic history. A dosimetric cumulative inhalation of methacholine was used and the response was measured by the dose which increases baseline end interruption resistance by 100% , with relatively narrow limits of agreement at -1.39 μmol and 1.27 μmol methacholine, respectively, but the interrupter methodology proved more sensitive than FEV1 in terms of reactivity (DRR).BHR at a cut-off level of 0.8 mg methacholine exhibited 31 (18%) of the subjects (specificity 81.2%), 21 male and 10 female, while 3% showed a response in the asthmatic range. The method was reproducible and showed good correlation with PDInterrupter methodology is clinically useful and may be used to evaluate bronchial responsiveness in normal subjects and in situations when forced expirations cannot be performed. In addition, a deep inspiration, as it is required during an FEViculties  and henciculties .The interrupter method has been shown to be a simple and non-invasive technique of measuring airway mechanics in children or patients with limited co-operation . It is aAn official statement by the ATS (American Thoracic Society) on methacholine provocation indicated that the interrupter method may be useful in testing patients who cannot perform acceptable spirometry manoeuvres but its use should be restricted to laboratories with expertise in their application and interpretation . Further20FEV1 method, and therefore clinically useful.We hypothesized that, since normals present with lower levels of airway obstruction during challenge, the interrupter technique in this case might be suitable and comparable with the reference PDThe study was conducted in a tertiary referral centre for respiratory disease and 198 subjects were initially enrolled. All subjects were healthy with a negative history and physical examination, normal blood counts, chemistries, chest radiography and spirometry.One individual from this sample reacted to the diluent control solution (0.6%), defined as a resistance difference of > 30% baseline  and was Routine spirometry was performed according to standardized guidelines . InterruThe airflow interruptions were performed using the Bronchoscreen system  , a compu1 had fallen = 20% compared with FEV1 after an initial saline inhalation. The bronchial aerosol provocation system  was used in this procedure. The nebulizer was calibrated to draw 5 μL of solution per automatic actuation lasting 0.6 seconds. The 100 μL of aerosol bolus had a mass median aerodynamic diameter of 1.9 μm with 80% of the droplets being less than 5.5 μm at a set pressure of 1.6 bar (22.8 psi). The subjects inhaled methacholine by slow inspiratory capacity manoeuvres guided by the green colour of light emission diodes (so that inspiratory flow was <0.5l/s) and the response was assessed 1 min after each inhaled dose.Bronchial responsiveness was measured by a rapid methacholine provocation dosimetric test, as previously described by our group with the same apparatus but using histamine instead . Briefly100Rint, EI), calculated by interpolation from the last two points of the cumulative semilogarithmic dose-response diagram, and 2) the percent slope (dose-response ratio-DRR) of a line extending from the origin to the last point of the curve (DRR) [100 Rint, EI >4 mg. The DRR data were analyzed from the whole sample. The 10 day reproducibility of the PD100Rint, EI was investigated by randomly asking 39 subjects to come again after one week for a second examination. During the second visit we compared Rint, EI with FEV1 as measurements of response to provocation, the latter determined 30 s after the assessment of Rint, EI [1 variation within ± 5% were received and the highest value was used for calculating the dose producing a 20% fall in FEV1 (PD20FEV1).Data was assessed by using two different estimates: 1) provocation dose which increases Rint, EI by 100% (PDve (DRR) . PlateauRint, EI . At leas2 test, were used for statistical analysis. The relative duplicate error was used to assess test-retest reproducibility of the PD100Rint, EI , defined as a standard deviation of the differences divided by the v2 after log transformation (approximates coefficient of variation)[20FEV1 was defined and calculated according to Bland and Altman [Regression analysis and correlation, Shapiro-Wilk test for normality and the non-parametric Mann-Whitney-U-/Wilcoxon Rank Sum test with normal approximation and the xariation). Agreemed Altman . Normal d Altman .1 and maximal expiratory flow when 50% of the forced vital capacity (FVC) remains to be exhaled (Vmax50), were higher in males by 16.3%, 14, 7% and 2.5% than in females. While Rint, EI was higher in females, possibly reflecting smaller airway size, but these differences were not statistically significant. The distribution of PD100Rint, EI is shown in Figure Subjects' anthropometric data and baseline spirometry are shown in Table 2 = 1.48, p = 0.22, odds = 1.79). Furthermore, 5 of these 31 subjects  were found to show moderate bronchial hyperesponsiveness , as frequently found in current symptomatic asthmatics [100Rint, EI to baseline post-saline Rint, EI and the respective DRR. Subjects with negative reactions (> 8.8 μmol) showed DRRs that were ten times lesser compared to those with BHR . Plateau response was exhibited by 66 (38%) of the subjects,  without gender related statistical difference . They had DRRs that were 2.5 times smaller compared to the subjects with normal but measurable reactions .Twenty one males and ten females (18%) of our subjects exhibited bronchial hyperesponsiveness. These values were normally distributed , with no gender related difference  with relatively narrow limits of agreement .PD100Rint, EI was found reproducible with a duplicate error of 8.3% or 0.65 doubling doses (within 140 μg). A close correlation was found between PD100Rint, EI, is comparable to the conventional PD20FEV1 method for evaluation of BHR in a large sample of normal subjects. We have also found that this technique has a specificity of 81.2% for normal subjects and its dose response ratio is 7-fold more sensitive than the conventional FEV1 method. It is known that specific airway conductance is four times more sensitive than FEV1 as a measure of response to provocation but the use of a body plethysmograph makes assessment of bronchial challenge expensive and time consuming. The present methodology is particularly useful in children and in the elderly, since it is non-invasive, sensitive to changes in airway calibre and requires no subject co-operation. The opening interrupter technique offers the additional advantage of simplicity and ease of application, being particularly useful in subjects unable to perform forced manoeuvres.In this study we have shown that the interrupter technique, and specifically PD30Rint, EI), so we assessed the PD100Rint, EI threshold, which is above the 95 % confidence interval at one tail direction (1.96SD) observed in our normal sample at baseline  has been reported (17), data on agreement are presented for the first time in this study.The Rint, EI was measured during expiration above forced residual capacity (FRC), because resistance hardly changes above this level and since subjects performed relaxed tidal flow maoeuvres, measurements were not affected by variations in breathing. Furthermore, we did not correct Rint, EI by lung volumes because the variability formed in the FRC can reduce the benefit of standardization of Rint, EI and the correlation between respiratory resistance and FRC is not significant over the limited FRC range of healthy subjects . Problem100Rint, EI methacholine, which defines severe and moderate hyperresponsiveness compatible with asthma [20FEV1 as well as to 2.5%, which represents the proportion of subjects beyond the 2 SD of the mean on one side of a normal distribution. If the PD100Rint, EI threshold is set at 0,8 mg methacholine [1) showed that 50% of normals had no calculable PC20FEV1.If a cut-off value is set at 0,4 mg PDh asthma  then 3% h asthma  working acholine , which iacholine . In contacholine . A plateacholine , using aIn this study, by direct comparison of two methods, one involving a deep inhalation, the relative effects of maximal expiratory manoeuvres on airway calibre can be indirectly assessed. The data in Table 1 and airway conductance in a large sample of subjects but not all were normals. It is known that in bronchial challenge the dose-response curve is expressed mainly by the threshold dose indicating hyperresponsiveness and the rate and magnitude of the response . The less reactive DRRs of the FEV1 method lend support to the perturbed actomyosin equilibrium hypothesis recently described, in that with stretching there is a decrease in myosin duty cycle and the magnitude of the contractile response becomes functionally disengaged from the level of the contractile stimulus [Recently, Sundblad et al  reportedstimulus . Furthemstimulus . The fac6 FEV1. The limits of agreement between PD100Rint, EI and the classical PD20FEV1 were found relatively small at -1.39 μmol and 1.27 μmol respectively. This implies that this method may be used as an alternative to FEV1 during provocation, as it is simple and easy to perform and requires no patient co-operation.No correlation was found in this study between BHR and baseline airway calibre, although Malo et al. found a Gender differences in BHR were explored because of the smaller lung size in women. Our data are in agreement with recent studies  that in In summary, the interrupter technique as an index of response to provocation has been shown to be useful to assess bronchial responsiveness in normal subjects, when maximal efforts cannot be performed. We recommend threshold doses of 100% baseline, because they show reliable agreement with the classical PD20FEV1 method.1:Forced Expiratory Volume in 1 sec, FVC:Forced Vital Capacity, FRC:Functional Residual Capacity, PA:Alveolar Pressure, Pao: airway opening Pressure, PC20FEV1:Provocative Concentration causing a 20% fall in FEV1, PC30Rint, EI:Provocative Concentration causing a 30% increase in Rint, EI, PD100Rint, EI: Provocation dose which increases Rint, EI by 100%, PD20FEV1:Provocation Dose producing a 20% fall in FEV1, Rint, EI:Interrupter Resistance at End Interruption, VC:Vital Capacity,ATS:American Thoracic Society, BHR:Bronchial Hyperresponsiveness, DRR:Dose Response Ratio, FEVThe authors declare that they have no competing interests.PP, IK, AT, SA, DB were involved with the study conception. PP, AT, SA performed the interrupter technique and collected the data. PP did the statistical analysis. PP, AT, DB prepared the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The typical symptoms of coronary heart disease (CHD), chest pain and breathlessness, are well-known. They are considered quite dramatic, and can thus be fairly reliably mapped by a survey. However, people might have other clearly unpleasant symptoms impairing quality of life. The aim of this study is to evaluate the appearance of these complaints of working-aged people with self-reported CHD.The study consists of a postal questionnaire of randomly selected Finns in age groups 30–34, 40–44 and 50–54, a response rate of 39% . The subjects were asked whether or not a doctor had told them that they had angina pectoris or had had myocardial infarction. Four randomly selected age and sex matched controls were chosen for every patient. The occurrence of self-reported dyspnoea, chest pain during anger or other kind of emotion, palpitation and perspiration without physical exercise, irregular heartbeats, flushing, trembling of hands and voice, jerking of muscles, depression and day-time sleepiness were examined. Odds ratios (OR) with 95% confidence intervals (CI), between occurrence of symptoms and CHD with and without heart infarction, were computed by multivariate logistic regression analysis.The sample eventually comprised 319 CHD patients. Dyspnoea, chest pain during anger or other kind of emotion, palpitation, perspiration without physical exercise, irregular heartbeats daily or almost daily, trembling of hands and voice, and jerking of muscles occurred statistically significantly more frequently among CHD patients than among controls. The CHD patients also reported more depressive mood according to Beck's inventory scores and poorer sleep and more frequent day-time sleepiness than controls. In the multivariate logistic regression analysis chest pain during anger or other kind of emotion (ORs 4.12 and 3.61) and dyspnoea (ORs 2.33 and 3.81) were the symptoms most associated with CHD.Working-aged people with self-reported coronary heart disease evince a number of symptoms limiting the quality of their every day life. This aspect should be paid attention to when evaluating functional capacity of these patients. In Finland, as in most industrialised countries mortality from cardiovascular diseases has shown decreasing trends since around 1970 . The MinThe typical symptoms of coronary heart disease (CHD), chest pain and breathlessness, are well-known. They are considered quite dramatic, and thus can be fairly reliably mapped by a survey . HoweverThe diagnosis of CHD is usually based on medical examinations or register data and not on what people by themselves experience. However, people act and use health services according to what they experience to suffer from and what they experience to limit their ability to work. Since CHD patients have many other symptoms than the traditional and well-known, it is important to know what the spectrum of symptoms and complaints among working-aged people with self-reported coronary heart disease is in relation to functional capacity.The Health and Social Support study (HeSSup) is a prospective etiological follow-up study on the psychosocial health of the Finnish working-aged population. The HeSSup population consisted of a random sample of 39,563 individuals drawn from the Finnish Population Register in three age groups: 30–34, 40–44, and 50–54. The survey was carried out by postal questionnaire. Forms were returned by 15,477 individuals , a response rate of 38.9% .The sample was subjected to a thorough analysis of non-response . The anaThe subjects were asked whether or not a doctor had told them that they had angina pectoris or had had myocardial infarction. The perceived state of health was determined according to Likert's five-step scale . In order to avoid small frequencies in the analyses this was modified to a three-step scale .The appearance of dyspnoea was categorised into four degrees of difficulty according to a widely used cardiovascular survey method . PersonsParticipants were also asked whether or not they had experienced daily or weekly chest pain during anger or other kind of emotion, palpitation and perspiration without physical exercise, flushing, trembling of hands and voice, and jerking of muscles. Irregular heartbeats daily or weekly were asked, too. Depression was estimated by Beck's  depressiIn interpretation of results the coronary heart disease (CHD) patients were divided into two groups. The first comprised coronary patients not having had heart infarction (angina pectoris group) and the second patients having had heart infarction (infarction group). In order to have the best available comparison groups, four randomly selected age and sex matched controls for comparison were selected for every patient. Thus there were altogether 740 controls for the angina pectoris group and 536 controls for the infarction group. Stroke was ruled out in the control groups, but otherwise there were no differences between the CHD groups and their respective controls.The associations, odds ratio (OR) with 95% confidence intervals (CI), between symptoms and coronary heart disease with and without heart infarction, were computed by multivariate logistic regression analysis. The analyses were made using the SAS System for Windows, release 8.2/2000.The data comprised 319 patients: 185 coronary heart disease patients who had not experienced heart infarction (55.1% were men) and 134 patients who had (78.4% were men) Table .State of health was perceived as good or quite good by 37.3% in the angina group and by 24.6% in the infarction group. The corresponding figures in the control groups were 76.0% and 67.4%, the differences being statistically significant (p < 0.001). State of health was perceived as poor or rather poor by 28.1% in the angina group and by 32.8% in the infarction group.At least mild breathlessness occurred in two thirds of the angina group and three fourths of the infarction group Table . DifficuIn the multivariate logistic regression analysis chest pain during anger or other kind of emotion and dyspnoea were the symptoms most associated with CHD Table . IrregulThe principal finding in this study was that many working-aged coronary heart disease patients experience unpleasant symptoms such as dyspnoea, chest pain during anger or emotion, irregular heart beats, perspiration without physical exercise, and jerking of muscles. In addition, the frequency of most of the self-reported symptoms among the study population is higher also in respect of those symptoms, which would not be expected at least among those CHD patients whose disease is in good balance. Working-aged CHD patients may be regarded as a special group compared with the main part of CHD patients who are already by age entitled to a pension. It is likely that working-aged CHD patients have experienced the most widespread and intense exposure to risk factors, which thus has caused them this disease among the first ones within their age group. As working-aged they are wished, however, to return back to normal life and work as soon as possible. According to our study they still have a lot of symptoms concerning their every day life, which harm their recovery and rehabilitation. It is also noteworthy that many of the working aged CHD patients are still in working life. Although chest pain and dyspnoea do not prevent them to work at customer service, many of the symptoms such as trembling of hands interfere their normal jobs while appearing mostly in rest. Trembling of hands and voice are also very irritating symptoms, and they may be considered shaming. Thus they may interfere social life and reduce the quality of life.The study material may be considered representative of the Finnish working-aged population, although the response rate was only 39%. Careful non-response analysis indicated that respondents and non-respondents were comparable in respect of the most important demographic variables . It is pOur method to determine the existence of CHD is based on the patients' report on whether a doctor had told them that they suffered from this particular disease. Thus, we cannot know for sure the accuracy of the information reported. Nowadays, CHD is rare among young people ,2. In ouThere are validated instruments, such as the generic The Short Form 36 Health Survey  (SF-36),The occurrence of dyspnoea and chest pain even during anger or other kind of emotion may be considered a finding that was expected. In an American study on the care of coronary heart patients at the emergency department the most frequently reported symptom was chest pain (70% among men and 71% among women) and dyspnoea (30% men and 29% among women) . The typIt was suggested as far back as 1987 that palpitations are not an independent risk factor for increased cardiac morbidity or mortality . Our finThe high occurrence of trembling of hands and voice, and jerking of muscles may be considered an unexpected finding. Most CHD patients use beta-blocking medicines, which in addition to protecting the heart muscle also reduce the adrenergic stimulation and thus relief the symptom of trembling.Concerning depression our findings support those of previous studies. One out of four of symptomatic coronary heart disease patients have namely been found to have a probable depressive disorder, but none of them had previously been identified as suffering from depression or been treated for this reason . In primIt was also of noteworthy that daytime sleepiness was connected with coronary heart disease. A cross-sectional study of 5,419 Finnish adult men found a higher prevalence of diagnosed myocardial infarction among those who slept more than nine hours, whilst those sleeping less than six hours per night had more symptomatic coronary disease . In a SwFrom previous research we know that despite having survived a life-threatening clinical event, CHD patients appear to have continued adverse behaviours such as smoking, being obese and having frequent hangovers more than the control population . The folAccording to the present findings many working-aged people with self-reported coronary heart disease perceive their state of health as poor or rather poor. They suffer from a wide range of symptoms limiting their every day life. It is noteworthy that many of these symptoms are not only irritating, but constitute a threat to health. The health related quality of life is poor among working-aged coronary heart disease patients.The author(s) declare that they have no competing interests.MPTS drafted the manuscript; SBS participated in drafting of the manuscript; MJK participated in the design of the study and the statistical analyses; LHS participated in the statistical analyses; KJM conceived of the study, and participated in its design and co-ordination. All authors have read and approved the final manuscript.


Past estimates have put the prevalence of schistosomiasis between 40% and 50% in the Malawi population overall based on studies undertaken ten years or more ago. More recent surveys in known high risk areas find similar levels. However control measures, changing ecology and migration may have led to changes in the prevalence of schistosomiasis in different parts of Malawi. A national schistosomiasis and soil-transmitted helminth (STH) survey was undertaken to measure the distribution, prevalence and intensity of infection in November 2002.Schistosoma haematobium.A school was selected randomly from a random sample of 30 Traditional Authorities stratified by six distinct ecological zones, and 1,664 year 3 pupils (9–10 year olds) were questioned about recent illnesses and "red urine". Samples of urine and faeces were examined for the presence of eggs using the standard Kato-Katz technique for soil-transmitted helminths and intestinal schistosomiasis and urine samples using the filtration technique for Schistosoma mansoni is 0.4% (95% CI 0–1.3%), S. haematobium 6.9% (95% CI 1.9 – 11.9%), hookworm 1.3% (95% CI 0.4–2.3%), Ascariasis 0.5% (95% CI 0.1–1.0%) and trichuriasis 0% in year 3 pupils . Intensity of infection is low for all infections except for 2.5% who have high intensity S. haematobium infection. The "red urine" question is 67% sensitive and 80% specific for positive S. haematobium microscopy.The prevalence of The reduction in prevalences may be real as a result of recent control measures, or false if historical results were based on surveys of high risk populations. Another explanation is that this survey used an unrepresentative sample of schools. Detailed analysis suggests this is unlikely.Recommendations include the use of a 30% positive threshold for the "red urine" screening question to be used in schoolchildren in high prevalence areas.This survey, based on a national probability sample excluding the northern region lakeside area, finds much lower overall prevalence and intensity of schistosomiasis and STHs than previous estimates based on selected surveys. Disease control featuring chemotherapy may be having a profound effect. The localised nature of the distribution of the infections means that control programmes may work best if undertaken at district level or below. "Red urine" questionnaire surveys may help identify hot spots. Chronic infections with soil-transmitted helminths and schistosomiasis are common in Malaŵi and cause considerable morbidity . The MinS. mansoni and S. haematobium were detected in 27% and 20% of schoolchildren respectively . The mean age of boys was 10.9 years and of girls 10.5 years.S. haematobium detected by urine microscopy, of which 45 had high intensity infection. The prevalence in schools ranged between 0 (11 schools) and 43.1% (a school in the southern lowlands). Twenty one children from eight different schools had visible haematuria. Only 8 children from 2 different schools had S. mansoni detected by stool microscopy and none were high intensity infections.A total of 140 children from 19 different schools had S. mansoni was found to be 0.4%. The national prevalence of S. haematobium was found to be 6.9% (95% CI 1.9–11.9%) .as Table . The higS. haematobium based on ecology and known previous prevalence and compared our survey results with these expectations. Of the 12 expected high prevalence schools seven were found to be high and 5 unexpectedly low. Of the 18 expected low prevalence schools 12 were found to be low and 6 unexpectedly high. Unexpected results were found in the expected low prevalence as much as in the expected high prevalence schools using the McNemar test for symmetry actual (p = 0.29).To assess the sampling method used to select schools we classified the selected schools into high and low expected prevalence of We also compared the historical results for Malawi districts compiled by the London School of Hygiene and Tropical Medicine for the Schistosomiasis Control Initiative in 2001  with our results. Prevalence has fallen overall by 24% . A formal paired t-test finds this reduction is true for low as well as high prevalence schools .There was also a low prevalence of STHs in all areas, though urban areas had the highest rates for any STH (6.6%) and hookworm was most prevalent in the Northern Highland area (2.6%). There were no STHs detected in the Central lakeshore schools.S. haematobium  found in the Southern Lowlands zone the positive predictive value is 49% and the negative predictive value is 89%.Total number of respondents was 1,662 Table . It is pum Table . The senS. haematobium and 6%–42% in the case of S. mansoni. The school located closest to rice fields had a high prevalence of schistosomiasis. In another recent survey carried out at the Cape McClear peninsula in the Southern Lake zone results showed markedly different S. haematobium and S. mansoni prevalence amongst adjacent villages situated on the lake shore and inland .The overall prevalence of schistosomiasis is well below expectations. Based on previous studies in Malawi the overall prevalence of schistosomiasis was thought to be between 40% and 50% in the population. Of course these were surveys of selected populations, perhaps undertaken in the season of high transmission. Certainly the disease is localised in Malawi. In the study conducted in the northern lakeshore area in 2000 by Randall et al , schoolcS. haematobium and 0.4% for S. mansoni. The finding highlights two important features of schistosomiasis in Malawi. Firstly, these infections are highly localised and secondly they are not common in general. As expected higher prevalence rates are found in the Southern lake/lowlands zone. S. mansoni does not seem to be a generalised problem in any particular ecological area in the country.Our survey, representative of all schoolchildren in the country, and undertaken just before the rainy season, suggests far lower levels of 7% for S. mansoni and S. haematobium. The differences could be due to seasonal variation (the survey in Karonga district was conducted after the rainy season). This possibility is supported by a low prevalence found in Karonga district in samples collected in November 2001, prior to the rains . Supporting evidence is seen in the prevalence rates found in urban areas where rains would not affect transmission as much as in rural areas. Similar results are found in previous studies in urban Blantyre: S. mansoni (0%), Ascaris (18%) and Hookworm (0.4%) [®, Intercep Ltd) in order to concentrate the stools and achieve high diagnostic sensitivity. Because of the dissimilar laboratory methods we have been unable to include results for the Northern Lakeland Ecological zone in the estimate of overall prevalences for the whole country. The northern lakeshore zone has tended to have high prevalences. The impact of omitting this zone is likely to underestimate the national results. We intend to survey the zone using our own laboratory methods when funds are available to complete the national picture.The overall prevalence of STHs is lower than expected. The survey conducted in Karonga District  finds am (0.4%) . Anotherm (0.4%)  was usedm (0.4%) ,18. On tAlthough this survey is designed to be representative of this age group in Malawi, it is limited in scale by the budget . This is an important limitation when one considers that the distribution of schistosomiasis in populations is likely to be highly localized . The popDrug treatment has been widely if intermittently available. The National Schistosomiasis Control Programme does not have documented evidence of where and when universal drug treatment has been offered to communities. Nor does it have details of where and when Praziquantel has been available for prescription to symptomatic individuals at health centres and hospitals. Supplies and universal treatment programmes have tended to depend on intermittent interest and funding from NGOs. However Praziquantel seems to have been widely available  and may have had an effect. In this survey of 264 people living in high risk villages in eleven randomly selected districts 71% reported receiving anti-schistosomiasis treatment.Ascaris lumbricoides (1.9%), Hookworm (24%), and S. mansoni (16%) in four schools. The last survey done in 2000 showed an overall prevalence of A. lumbricoides (0.4%) and Hookworm (1.5%) on the four schools after two consecutive courses of treatment . Similar interventions may have had an effect on the prevalence of infections in other parts of the country.Several NGOs such as Save the Children (US) in Mangochi and World Vision in Chikwawa in recent years have studied helminth infections in schoolchildren. Save the Children (US) surveyed and treated schoolchildren in the entire Mangochi District after finding a prevalence of Another possible explanation for the unexpectedly low prevalence results could be due to a sampling method that selected a low preponderance of schools in high risk areas. However the results of the analysis looking at expected and actual prevalence found in each school suggest this did not happen. Unexpected results were found in the expected low prevalence as much as in the expected high prevalence schools using the McNemar test for symmetry actual (p = 0.29).S. haematobium now is not due to asymmetric sampling of low risk schools; it is due to a marked trend of a reduced prevalence across the board. It is not just the high risk schools that have lower prevalence now. Low risk schools have lower rates as well. Compared to the historical results for Malawi districts compiled by the London School of Hygiene and Tropical Medicine for the Schistosomiasis Control Initiative in 2001, prevalence has fallen overall by 24%, and this reduction is true for low as well as high prevalence schools. Our interpretation is that the results reflect a widespread reduction in schistosomiasis prevalence and not a biased sample in the current survey which inadvertently missed schools in high risk areas. However bias may still be an explanation for the change. The widespread reduction in prevalence could be due to bias in the selection of high risk schools in the historic studies rather (than in the selection of low risk schools in our survey). Our sampling fraction of 30 of a possible 3,900 schools is sufficient to give statistical confidence in our overall prevalence result of 6.9% (95% CI 1.9 – 11.9%) for S. haematobium so long as our schools and Traditional Authorities were chosen randomly, which they were.The reason for the comparative low prevalence rate of Of the questions asked, only the red urine question is sufficiently sensitive and specific to be used as a screening test. It is a cheap way of assessing schistosomiasis prevalence and as such may be useful to identify hot spots within districts.Past control programmes, despite their fragmentary application in Malawi, seem to have achieved much in recent years. High intensity infections are now rare, and it is of course these that are thought to cause morbidity. There remain some high prevalence locations but these are uncommon now. The survey findings suggest that future approaches to control can focus on the identification of hot spots through local surveys. These should be undertaken in districts in high prevalence ecological zones, probably through "red urine" questionnaire surveys of school children. Because of the low positive predictive value of the "red urine" question, a low threshold should be used to categorise each school. The categorisation as proposed in Table The highly effective drugs now available and the control methods used in Malawi seem to have reduced the prevalence of both schistosomiasis and soil helminths to low levels.There remain some localised areas of high prevalence for which local control measures will be required. It seems that the epidemiology of these diseases makes local control at district level or below the preferred public health approach.The author(s) declare that they have no competing interests.BS conceived the survey and provided logistical advice. CB designed the survey and undertook the final analysis and first drafting. BP supervised the survey and undertook the initial analysis. PM undertook the randomization and led the survey team. MP supervised the quality control of the laboratory work. All contributed to the final report.The pre-publication history for this paper can be accessed here:questionnaire; the questionnaire in Chichewa used in the schools to find out about the health of the pupils surveyed.Click here for file


Drosophila melanogaster is extensively studied as a very powerful model of developmental biology. One main advantage of this system is the ability to study the origin and development of individual sensory cells. However, there remain several discrepancies regarding the organization of sensory organs in each abdominal segment A1-A7.The embryonic and larval peripheral nervous system of We have constructed a web site called FlyPNS  that consolidates a wide range of published and unpublished information about the embryonic and larval sensory organs. It communicates (1) a PNS pattern that solves the discrepancies that have been found in the recent literature, (2) the correspondence between the different nomenclatures that have been used so far, (3) a comprehensive description of each sensory organ, and (4) a list of both published and unpublished markers to reliably identify each PNS cell.Drosophila sensory organs.The FlyPNS database integrates disparate data and nomenclature and thus helps understanding the conflicting observations that have been published recently. Furthermore, it is designed to provide assistance in the identification and study of individual sensory cells. We think it will be a useful resource for any researcher with interest in  Drosophila abdominal larval PNS is composed of a constant number of neurons and associated cells, whose characteristics and positions are reproducible between individuals [The ividuals -5. Its sDespite the extraordinary utility of the PNS for understanding these diverse problems, a prominent current limitation is the lack of agreement in the literature of the exact number, position and nomenclature of sensory neurons. For example, many different names have been used to describe abdominal neurons, reflecting the number of cells within a cluster, their position ,7, theirWe have constructed a website, named FlyPNS, that consolidates a wide range of published and unpublished information regarding the embryonic and larval sensory organs and their associated glial cells. Motoneurons and other glial cells have not been included. The FlyPNS web site is arranged in 6 sections: General references, Nomenclature, Abdominal PNS organization, Sensory organ description, Antibodies and enhancer-traps, Gal4 lines, and a Search option.General references section provides a list of papers on various aspects of sensory organ morphology and development. Subcategories include general descriptions, descriptions of dendritic arborizations, axonal pathways, developmental changes, and functions of multidendritic neurons. Links are provided to PubMed entries for access to abstracts or full-texts of papers.The Abdominal PNS organization section, we have represented the PNS pattern that we have observed , markers, morphology, development and related references. A few unpublished observations relating to maker expression and morphology are also mentioned. A typical sensory organ description is shown in Fig. The Antibodies, enhancer-traps and Gal4 lines pages display a collection of published and unpublished data on expression patterns of antibodies, enhancer-traps and Gal4 lines, with links to the gene/insertion information contained in Flybase when available.The Search option provides a convenient link to the information about any of the organs/markers.Finally, the Drosophila investigators.Much of the discordance in the literature regarding the larval and embryonic PNS pattern seems to be rooted in variation in the precise position of sensory cells with segment, embryo, and time, and a lack of specific markers for some organs. These factors might explain why, for example, some dorsal cluster neurons were often missed in previous publications. We have attempted to resolve remaining ambiguities by (1) identifying the total number of sense organs and neurons in each abdominal segment A1-A7, (2) resolving different nomenclatures used for each organ, and (3) providing a collection of molecular markers and descriptions that provide an unambiguous guide for cell identification. This data compilation will hopefully also increase the accessibility of the fly PNS literature to non-. Comments and additions are welcomed by e-mail to VO.The FlyPNS web site is available from the link under Miscellaneous in the FlyBase Allied Data section PNS: peripheral nervous systemda neuron: neuron displaying a dendritic arborizationThe PNS pattern was established based on the observations of WBG and VO. VO created the FlyPNS web site and WBG made significant improvements and corrections to the web site. Both VO and WBG wrote the manuscript.


A cross sectional study was designed to survey the relationship between anxiety/depression and duration/cause of infertility, in Vali-e-Asr Reproductive Health Research Center, Tehran, Iran.After obtaining their consents, 370 female patients with different infertility causes participated in, and data gathered by Beck Depression Inventory(BDI) and Cattle questionnaires for surveying anxiety and depression due to the duration of infertility. This was studied in relation to patients' age, educational level, socio-economic status and job (patients and their husbands).Age range was 17–45 years and duration and cause of infertility was 1–20 years. This survey showed that 151 women (40.8%) had depression and 321 women (86.8%) had anxiety. Depression had a significant relation with cause of infertility, duration of infertility, educational level, and job of women. Anxiety had a significant relationship with duration of infertility and educational level, but not with cause of infertility, or job. Findings showed that anxiety and depression were most common after 4–6 years of infertility and especially severe depression could be found in those who had infertility for 7–9 years.Adequate attention to these patients psychologically and treating them properly, is of great importance for their mental health and will improve quality of their lives. The impact of infertility on the psychological well being of couples involved has been the object of increasing attention in recent years. It cannot be denied that infertility is a deeply distressing experience for many couples . In lattWe aimed to examine prevalence and severity of anxiety/depression in relation to duration/cause of infertility in Iranian infertile women.Several studies have demonstrated anxiety has a detrimental effect on fertility  and thatInfertility sometimes is accompanied by existential crises and emotional tensions such as anxiety, interpersonal problems, and suppressed anger, unsatisfactory in interpersonal, frustration, inferiority feeling, depression, rejected feeling and unconscious guilt feeling. Those couples with a history of failure in Assisted Reproductive Technique (ART) have shown personality maladjustment .Overall percentage of psychological problem in infertile couples ranges between 25 and 60% . One stuAnother study showed those who had 2–3 years infertility had more depression / anxiety than those who had this problem for a year or more than 6 years. Peak of depression could be seen during third year of infertility. After six years there will be a reduction in psychological symptoms in women. During first three years, infertility is accompanied by signs such as anxiety, depression, loss of self-esteem, impotence and maladjustment of marital status. After 3 years, optimistic attitude would change to despair and at last there will be some emotional changes to adopt a child or live without one, thereafter. Those who have social support, positive personal characteristics, and have a satisfactory life with their spouse show no signs of anxiety/depression .Since most literature on psychological aspects of infertility is from developed countries it was thought that a study from a developing country with a different culture might contribute to existing knowledge on the topic. We aimed to examine prevalence and severity of anxiety/depression in relation to duration of infertility in Iranian infertile women. The results of this study, which included counseling and couple-therapy, are being prepared for infertile couples.The subjects were 370 infertile women who were referred to Vali-e-Asr Reproductive Health-Research Center between January 2001 and January 2002 for treatment of their infertility problems. In this group mean (± SD) age was 28 (± 5.37) and mean (± SD) duration of infertility was 6.36 (± 4.18). Among them 293 (79.2%) women were housewives and the others were working outside.A gynecologist evaluated patients and then they were visited by a psychologist and were informed of the study purposes. Diagnosis of male factor is based on WHO criteria 1999. Menstrual history and mid-luteal progesterone level considers ovulatory factor, diagnosis of endometriosis is done by lapascopy, tubal factor is diagnosed by HSG and cervical facto is detected in Post Coital Test. After obtaining oral consent from each patient, data were collected using BDI , and CatThe test used was a translated and validated Persian version of Beck's depression Inventory. A full 21-items BDI was administered. This scale is a widely used measure for intensity of depression.Each item describes a specific behavioral manifestation of depression. Scores on each item can range from 0, indicating no depressive symptomatology, to 3, indicating a severe level of symptomatology. Total scale scores can thus range from 0 to 63. Scores of 17 or above it indicates of a clinically significant depression. The classification of depression scores involves:1. 0–16 (without depression)2. 17–27 (mild depression)3. 28–34 (moderate depression)4. 35–63 (severe depression)The Cattle inventory is a 40 items self-report measure of anxiety. This test was a translated and validated Iranian version of Cattle's Inventory. Scores can range from 0 to 80, with scores of 28 or above demonstrate anxiety. Classification of anxiety scores involves:1. 0–27 (without anxiety)2. 28–40 (moderate anxiety)3. 41–49 (neurotic anxiety)4. 50–80 (severe anxiety)For each patient the following data were recorded: age, cause and duration of infertility, education and job.Data were analyzed by using statistical SPSS. The relationship between continuous and binary explanatory variables with Beck and Cattle scores were assessed using spearman's rho and unpaired t-test, respectively. In addition, the relationship between categorical responses and explanatory variables were evaluated using chi-square test. For descriptive purposes, we presented frequency tables.Three hundred seventy infertile women were considered in this cross-sectional study. The results of Beck and Cattle inventories showed that 40.8% and 86.8% of these women had depression and anxiety symptoms, respectively Table .Mean scores of depression and anxiety by age groups are shown in figure 2 statistic for this 2 × 9 table (chi-square = 20.643 P = 0.014) showed that prevalence of depression is not equal in these categories. Same analysis for the prevalence of anxiety  in different groups of infertility causes showed no significant difference between them (chi-square = 7.491 P = 0.485)  in all categories of infertility causes. ChiThe correlation between Beck and Cattle scores based on Spearman's Rho was 0.707 (P < 0.001), which shows a significant relation between depression and/or anxiety scores. Then, we checked correlation coefficient between Beck and Cattle scores with quantitative variables like age, education (in years) and especially the duration of infertility.Duration of infertility showed a significant relation with both Beck  and Cattle  scores. Based on duration of infertility 31(29.2%), 51 (42.1%), 30 (46.8%) and 39 (49.3%) patients had depression in different groups, but there was no significant relationship between duration of infertility and depression  (Table Also 82 77.3%), 110 (90.9%), 58 (90.6%) and 71 (89.8%) of patients had different stages of anxiety and there was a significant relation between anxiety and duration of infertility  . Anxiety and depression were observed more in homemakers comparing to outside employees though we found no significant effect of women's job on Cattle scores  Table .Patients participating in this study were from different geographical areas in Iran. The finding of this study provides information about frequency and severity of anxiety/depression in order to duration of infertility in childless women.The prevalence of psychiatric morbidity specially depression and/or anxiety in infertile patients have been assessed in several countries, for example Jones et al 1993) found that, there was mild to moderate depression in 28.3% of infertile women, moderate to severe depression in 7.2% and 1.2% had most severe depression based on BDI 993 found. AnxietyThe most common age for depression and/or anxiety in our study was 21–25 years. In this study, anxiety and/or depression had negative correlation with education. In other words, with the raise of education level, anxiety and/or depression decrease.Results of different studies about relationship of age and education with anxiety and/or depression were not similar. Age and education level have no significant relationship with depression and/or anxiety . AnotherHaving a job may reduce stress from In Vitro fertilization (IVF) . In our Based on previous researches -28, infeBased on our study depression is more common in "unexplained cause" group comparing to other causes and anxiety is more common in "endometriosis" group comparing to other causes, our results is similar to other studies ,3,6,9,13Anxiety and/or depression increases with duration of infertility . A studyIn conclusion it can be suggested that psychological interventions especially in 4–9 years of infertility may prevent the surge in depression/anxiety and could presumably lead to increased pregnancy rates.FM contributed to design and management of the study.NA contributed to study design and data gathering.The initial idea of this study was by MMA.NK contributed in design, writing of the manuscript and analysis of this research.FZ contributed in design, writing of the manuscript and analysis of this research.MS contributed in edition and revision of the article.MJ contributed in edition and revision of the article.The pre-publication history for this paper can be accessed here:


Recently, secreted S100A4 protein has been shown to possess a number of functions, including induction of angiogenesis, stimulation of cell motility and neurite extension.S100A4 is a small CaCell cultures from two human osteosarcoma cell lines, OHS and its anti-S100A4 ribozyme transfected counterpart II-11b, was treated with IFN-γ and recombinant S100A4 in order to study the sensitizing effects of extracellular S100A4 on IFN-γ mediated apoptosis. Induction of apoptosis was demonstrated by DNA fragmentation, cleavage of poly (ADP-ribose) polymerase and Lamin B.In the present work, we found that the S100A4-expressing human osteosarcoma cell line OHS was more sensitive to IFN-γ-mediated apoptosis than the II-11b cells. S100A4 protein was detected in conditioned medium from OHS cells, but not from II-11b cells, and addition of recombinant S100A4 to the cell medium sensitized II-11b cells to apoptosis induced by IFN-γ. The S100A4/IFN-γ-mediated induction of apoptosis was shown to be independent of caspase activation, but dependent on the formation of reactive oxygen species. Furthermore, addition of extracellular S100A4 was demonstrated to activate nuclear factor-κB (NF-κB).In conclusion, we have shown that S100A4 sensitizes osteosarcoma cells to IFN-γ-mediated induction of apoptosis. Additionally, extracellular S100A4 activates NF-κB, but whether these events are causally related remains unknown. It has operties , stimulaoperties . Moreoveoperties , indicatoperties .Interferon-gamma (IFN-γ) is a pleiotropic cytokine secreted from activated T lymphocytes and natural killer cells. It can activate and suppress a number of target genes, leading to various effects, including inhibition of viral replication, activation of immune cells and induction of cell cycle arrest and apoptosis ,15. ApopSearching for cytokines and signal transduction modulators affecting S100A4 expression, we recently discovered that IFN-γ downregulated S100A4 transcription and induced apoptosis in the human osteosarcoma cell line OHS . In the 4 cells/cm2. After overnight incubation, the culture medium was replaced with medium in presence or absence of IFN-γ and recombinant S100 proteins, and harvested as indicated in the text. Where indicated, protease inhibitors or antioxidants were added to the cell culture simultaneously as IFN-γ.The human osteosarcoma cell line OHS and its anti-S100A4 ribozyme transfected counterpart II-11b have been described previously . Cell liHuman recombinant IFN-γ, L-NMMA, zVAD-fmk, zYVAD-fmk, zVDVAD-fmk, zDEVD-fmk, zVEID-fmk, zIETD-fmk, zLEHD-fmk, zFA-fmk and E64 were purchased from Calbiochem . N-acetylcysteine was from Sigma Chemical Co .2+-column and dialyzed against phosphate-buffered saline. A satisfactory degree of purity was confirmed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) where a single band was visualized by silver staining. Verification of the identity of the protein was performed using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI). Human recombinant S100A10 and S100A13 tagged with maltose-binding protein were kindly provided by C. S. Skjerpen .Histidine-tagged mouse recombinant S100A4 was cloned into the pQE30 vector , expressed in E. coli, purified on a Ni® AQueous One Solution Reagent (MTS)  according to the manufacturer's manual. The absorbance at 490 nm was recorded using a Wallac 1420 Victor2 Multilabel counter .Cell viability was estimated by measuring metabolic activity using CellTiter 96Protein lysates were prepared in 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl and 0.1% NP-40 with 1 mM PMSF and 2 μg/ml each of pepstatin, aprotinin  and leupeptin . Total protein lysate from each sample was separated on 8–12% SDS-polyacrylamide gel electrophoresis, and transferred onto Immobilon-P membranes  according to the manufacturer's manual. As a loading and transfer control, the membranes were stained with 0.1% amidoblack. After blockage of non-specific binding sites with 10% non-fat dry milk in Tris-buffered saline with 0.25% Tween (TBST), blots were incubated for 1 h at room temperature with rabbit polyclonal anti-poly (ADP-ribose) polymerase , mouse monoclonal anti-lamin B , mouse monoclonal anti-α-tubulin , rabbit polyclonal anti-caspase-1 , goat polyclonal anti-caspase-3 , mouse monoclonal anti-caspase-8 , mouse monoclonal anti-caspase-9 , rabbit polyclonal anti-caspase-12 , mouse monoclonal anti-NFκB p65  or mouse monoclonal anti-phospho-IκBα (Ser32/36) . After washing, the blots were incubated for 1 h at room temperature with a horseradish peroxidase-conjugated secondary antibody  diluted 1:5000. Anti-lamin B, anti-α-tubulin, anti-PARP, anti-caspase-1, anti-caspase-8, anti-NFκB p65 and anti-phospho-IκBα were diluted in TBST with 5% non-fat dry milk, anti-caspase-3 and anti-caspase-12 were diluted in TBS containing 0.05% Tween and 2% non-fat dry milk and anti-caspase-9 was diluted in TBS with 0.05% Tween, 1% non-fat dry milk and 1% BSA. Signals were visualized using the ECL chemiluminescence substrate .Cells were resuspended in lysis buffer  and incubated for 1 hour at 50°C. High molecular weight DNA was precipitated by adding 1 M NaCl and subsequently incubated overnight at 4°C. Samples were centrifuged for 30 min, 2700 g at 4°C, and supernatants collected. 95 % ethanol was added, and DNA precipitated overnight at -20°C. Samples were centrifuged for 10 min, 10 000 rpm at 4°C, and pellets were washed in 70 % ethanol and re-centrifuged. Thereafter, pellets were air-dried and dissolved in 10 mM Tris-HCl (pH 7.0), 15 mM NaCl, 1 mM EDTA and 0.2 mg/ml RNAse A, and incubated for 1 h at room temperature. DNA was separated by electrophoresis in 1.0 % agarose gel containing ethidium bromide and photographed under ultraviolet illumination.Cell culture medium from OHS and II-11b cells were harvested at 24, 48 and 72 hours, and immediately filtered using a 0.45 μm filter to discard cells in suspension. From each sample, 1.5 ml of medium was subjected to immunoprecipitation using 15 μg of rabbit polyclonal anti-S100A4  or 15 μg rabbit polyclonal anti-p300 . Primary antibody binding was carried out in cell culture medium at 4°C overnight. Thereafter, antibody was precipitated using Protein A Sepharose beads for 1 h at 4°C and eluted in loading buffer. Samples were subjected to Western blot analysis as described above using rabbit polyclonal anti-S100A4 diluted 1:300 in TBST.6 II-11b cells were transiently transfected with 10 μg of the NF-κB reporter construct using electroporation (240 V). Thereafter, cells were seeded in 96-well plates at a density of 4.5 × 104 cells/cm2 and incubated in the absence or presence of rS100A4 and zFA-fmk. Cells were harvested 48 h later and assayed for luciferase activity using the Luciferase Assay System  according to the manufacturer's manual.The NF-κB reporter construct containing three NF-κB response elements  driving luciferase expression has been described previously . 1.0 × 1All statistical analyses were performed using Student's t-test. P-values less than 0.05 were considered to be statistically significant.We have previously observed that the human osteosarcoma cell line OHS is more sensitive to IFN-γ induced apoptosis than the S100A4-ribozyme transfected counterpart II-11b . To examPreviously, we have shown that treatment of OHS cells with IFN-γ induced a significant increase in the fraction of apoptotic cells, while the cell cycle distribution remained unchanged . Thus, iWhen performing the OHS cell culture experiments, we observed that, when keeping the concentration of IFN-γ constant, decreasing the volume of cell culture medium or increasing the number of cells resulted in increased cell death (data not shown). This observation suggested that the cells might secrete a soluble factor with impact on the level of apoptosis induced by IFN-γ. Since the II-11b cells are derived from the OHS cells by transfection with an S100A4-specific ribozyme, this finding prompted us to investigate whether S100A4 is secreted from these cells, and whether extracellular S100A4 could cooperate with IFN-γ in induction of apoptosis. Fig. In order to investigate whether extracellular S100A4 could increase apoptosis induced by IFN-γ, recombinant S100A4 (rS100A4) was added to the cell culture medium of OHS and II-11b cells in addition to IFN-γ. Addition of rS100A4 alone had no effect on cell viability at concentrations ranging from 400 pg/ml to 20 μg/ml Fig. . In factCaspase-mediated apoptosis is the most important program of cell death. In order to investigate whether IFN-γ activated the caspase cascade in our cell system, we have followed two different approaches: detection of activated caspases by Western blotting and treatment of cell cultures with caspase inhibitors. Using specific antibodies detecting both the proenzyme and the active form of caspase-1, -3, -8, -9 and -12, we were not able to detect any activation of caspase-1, -3, -8 or -12 Fig. . This coThe caspase inhibitors used in the experiments above were solved in dimethyl sulfoxide (DMSO), and therefore DMSO was included as a negative control. Surprisingly, a significant inhibition of IFN-γ-induced apoptosis was observed using 0.25 % DMSO Fig. . One of in vitro [The pan-caspase inhibitor zVAD-fmk has previously been shown to bind the cysteine protease cathepsin B and to suppress cathepsin B activity in vitro . Since cin vitro , zVAD-fmin vitro ; thus, win vitro . Interes2+-binding protein S100A4 sensitizes human osteosarcoma cells to IFN-γ-induced apoptosis. Furthermore, we have shown that the IFN-γ-mediated cell death is independent of caspases, but possibly mediated by reactive oxygen species. Finally, we have demonstrated that extracellular S100A4 can activate NF-κB through increased phosphorylation of IκBα, but whether this NF-κB transactivation is required for apoptosis induction remains unknown.IFN-γ induces apoptosis in a variety of different cell types, but some cells are less susceptible or even resistant to IFN-γ-mediated apoptosis. The reasons for this variation have not been clearly elucidated, and given the biological importance of IFN-γ, a better understanding of the mechanisms of IFN-γ-induced cell death is warranted. In the present study, we have demonstrated that the Ca2+ or other divalent cations [Several of the S100 proteins are released into the extracellular space, mostly by unknown mechanisms ,31,32. T cations .Addition of 20 μg/ml rS100A4 (1.5 μM) to the cell medium of II-11b cells was required to induce apoptosis upon IFN-γ-treatment. Comparably, micromolar amounts of S100B and S100A8/A9 were needed to induce cell death ,34. Alsoet al demonstrated that S100A4 interacts with the tumor suppressor protein p53 to enhance p53-dependent apoptosis. The proposed mechanism was based on modulation of p53-dependent transactivation of target genes [et al., in preparation). These findings, in addition to the present work, suggest that secreted S100A4 could sensitize IFN-γ-signaling, and this possibility is currently under investigation. Previously identified sensitizers of IFN-γ-signaling include type I interferons (IFN-α/-β) [et al. discusses, based on their experiments using peripheral blood mononuclear cells, that other factors probably also exist to sensitize IFN-γ-signaling. Interestingly, S100A4 is strongly expressed by T lymphocytes , but whether lymphocytes also secrete S100A4 remains to be investigated.In many cases, stimulation of tumor cells with a single proapoptotic signal is not sufficient for induction of programmed cell death. On the other hand, a number of cytokines and other proapoptotic signal substances can increase the cells' sensitivity to other apoptosis-inducing agents, thus initiating the cell death program. IFN-γ induces apoptosis in synergism with a wide variety of such agents, and particularly members of the tumor necrosis factor (TNF) superfamily have been studied, e.g., TNF-α , FasL 3, LIGHT  and TRAIet genes . The ostet genes , thus, SFN-α/-β)  and IFN-FN-α/-β) . Hu et aet al. suggest that zFA-fmk has a promoter-specific NF-κB inhibitory effect [Our results suggest that S100A4-mediated NF-κB activation might be required for triggering the apoptotic process induced by IFN-γ. To support this hypothesis, we made attempts to inhibit NF-κB signaling by treatment with SN50, a peptide that contains the p50 NLS and selectively blocks nuclear translocation of NF-κB, but this lead to an increased fraction of apoptotic cells. Schotte y effect , whereasy effect .IFN-γ-induced apoptosis in OHS cells was shown to be caspase-independent by two different approaches: (i) no active forms of a number of caspases were detected; and (ii) caspase inhibitors were not able to suppress cell death triggered by IFN-γ. Even though the caspase cascade was not activated, well-known hallmarks of apoptosis were identified. DNA fragmentation and Lamin B cleavage have previously been shown to occur independent of caspase activation ,45, wherIn conclusion, we have demonstrated that extracellular S100A4 induces NF-κB transactivation and possesses proapoptotic activity through sensitization of IFN-γ-induced apoptosis, but whether activation of NF-κB is required for the observed sensitization of cell death is still unknown. Even though the precise physiological role of the apoptosis sensitizing effect of S100A4 awaits further studies, it highlights yet another area of research in the attempts to elucidate the complex biological roles of S100A4.None declared.KBP carried out the cell culture experiments, immunoprecipitations and western blots used in the detection of S100A4 in cell culture medium. In addition he did the dose-response experiments in II-11b cells, all the experiments using caspase inhibitors and NAC, western blot analysis of caspase activity, and drafted the manuscript. KA carried out dose-response experiments in OHS cells, and in addition all the cell culture experiments used in subsequent western blot analysis of caspase- and NF-κB activity and DNA fragmentation analysis. In addition she did transient transfections and all cell culture experiments using other S100 proteins and anti-RAGE antibodies. ØF participated in the design of the study. GMM conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


We have demonstrated that moderate alcohol consumption  for 8 weeks resulted in significantly increased levels of serum estrone sulfate and DHEAS in 51 postmenopausal women in a randomized, placebo-controlled trial. We now report on the relationships between serum estrone sulfate and dehydroepiandrosterone sulfate (DHEAS) levels after 4 weeks of moderate alcohol supplementation, and compare the results to the 8 weeks data to elucidate time-to-effect differences.Postmenopausal women (n = 51) consumed 0 (placebo), 15 (1 drink), and 30 (2 drinks) g alcohol (ethanol)/ day for 8 weeks as part of a controlled diet in a randomized crossover design. Blood samples were drawn at baseline, at 4 weeks and at 8 weeks. Changes in estrone sulfate and DHEAS levels from placebo to 15 g and 30 g alcohol per day were estimated using linear mixed models.At week 4, compared to the placebo, estrone sulfate increased an average 6.9% (P = 0.24) when the women consumed 15 g of alcohol per day, and 22.2% (P = 0.0006) when they consumed 30 g alcohol per day. DHEAS concentrations also increased significantly by an average of 8.0% (P < 0.0001) on 15 g of alcohol per day and 9.2% (P < 0.0001) when 30 g alcohol was consumed per day. Trend tests across doses for both estrone sulfate (P = 0.0006) and DHEAS (P < 0.0001) were significant. We found no significant differences between the absolute levels of serum estrone sulfate at week 4 versus week 8 (P = 0.32) across all doses. However, absolute DHEAS levels increased from week 4 to week 8 (P < 0.0001) at all three dose levels.These data indicate that the hormonal effects due to moderate alcohol consumption are seen early, within 4 weeks of initiation of ingestion. Epidemiological evidence consistently shows a positive association between alcohol, even low to moderate intake, and breast cancer risk . HoweverA reanalysis of nine prospective studies shows that levels of endogenous sex hormones are strongly associated with breast cancer risk in postmenopausal women . Clear eDetails of the Women's Alcohol Study (WAS) was previously published . BrieflyBlood for hormone analyses was collected after an overnight fast at weeks 4 and 8. Serum was separated, and aliquots were frozen at -70°C. In the samples taken after 4 weeks of alcohol supplementation, we only measured serum estrone sulfate and DHEAS, as they were the only two hormones that were significantly elevated at 8 weeks [Hormone concentrations were transformed using natural log. Changes in hormone concentrations from placebo to 15 g and 30 g of alcohol per day were estimated at 4 and 8 weeks using linear mixed models including a random intercept and alcohol levels as fixed effects treated as two indicator variables. Separate models used alcohol as a continuous variable to test for trend. Regressions to evaluate the effect of several baseline covariates on the precision of the parameter estimates for alcohol consumption included age, BMI, and years since menopause modeled as continuous fixed effects; assignment order, dietary period, hysterectomy, and race were modeled as indicator variables. Effect modification by race, assignment order, dietary period, age, BMI, and years since menopause were assessed by likelihood ratio tests of improvement in the model fit after addition of cross-product terms to models that included main effects for alcohol and the characteristic being evaluated. All tests of statistical significance were two-sided. Statistical analyses were performed using S-PLUS (S-PLUS version 6.1 for Windows. Seattle (WA): Insightful Corporation; 2002.)Table The effects of alcohol supplementation on serum estrone sulfate and DHEAS levels did not vary with age, BMI, race, or years since menopause. These hormone concentrations also did not differ among the three dietary periods, and the order of the assignment to the treatment regimens did not modify the associations with either of the two hormones.At 8 weeks of alcohol intake, the 15 g dose versus the placebo increased serum estrone sulfate and DHEAS by 7.5% and 5.1% respectively, whereas the 30 g dose compared to the placebo, increased levels of estrone sulfate and DHEAS by 10.7% and 7.5% respectively. When comparisons using linear mixed models are made, after controlling for alcohol intake across all doses, there was no statistically significant difference between the absolute levels of serum estrone sulfate at week 4 versus 8 (P = 0.32). However, controlling for alcohol intake absolute DHEAS levels increased between weeks 4 and 8 (P < 0.0001). The increase in DHEAS between weeks 4 and 8 did not occur in just the 15 or 30 g per day groups, however; the increase was similar in each of the three groups. In the 0 g per day group the geometric mean shifted from 55.3 ng/dL at 4 weeks to 59.4 ng/dL at 8 weeks. In the 15 g per day group the geometric mean shifted from 59.7 ng/dL to 62.5 ng/dL, and in the 30 g per day group the geometric mean shifted from 60.4 ng/dL to 63.9 ng/dL. The models did not suggest an interaction between measurement week and alcohol dose in serum estrone sulfate (P = 0.32) or DHEAS (P = 0.58).In postmenopausal women moderate alcohol consumption for four weeks resulted in statistically significant increased levels of serum estrone sulfate, the most abundant circulating estrogen, and DHEAS, the steroid hormone with the highest concentration in the blood. Compared to the placebo, serum estrone sulfate levels increased 6.9% (P = 0.24) and 22.2% (P = 0.0006) respectively among women who consumed 15 g and 30 g alcohol per day for four weeks. Serum DHEAS concentrations also increased by 8.0% (P < 0.0001) and 9.2% (P < 0.0001) respectively among women who consumed 15 g or 30 g alcohol per day for four weeks. At week 8, serum estrone sulfate levels increased 7.5% and 10.7% respectively among women who consumed 15 g and 30 g alcohol per day whereas DHEAS concentrations increased 5.1% and 7.5% respectively among women who consumed 15 g or 30 g alcohol per day compared to the placebo [For DHEAS, the absolute levels were statistically (P < 0.0001) higher at week 8 compared to week 4 in all the alcohol groups. This increase in DHEAS between weeks 4 and 8 did not differ regardless of 0 g, 15 g, or 30 g per day dose (P = 0.58). Biological reasons may explain the increase in the 15 g and 30 g per day alcohol dose, but the reasons behind the increases in DHEAS for the 0 g dose need further research. We did not find any evidence for an effect modification between measurement week and alcohol supplementation in serum estrone sulfate (P = 0.32) or DHEAS (P = 0.58).These data indicate that the hormonal effects due to moderate consumption of alcohol equivalent to one or two drinks per day are seen early, within 4 weeks of initiation of ingestion. Importantly from a study design perspective, our study also demonstrates that it may be possible to utilize shorter study periods when assessing the effects of alcohol consumption on hormone levels, at least in postmenopausal women. To understand the earliest effects of moderate alcohol intake on hormone levels in postmenopausal women, future studies will have to be designed to assess serum levels earlier than 4 weeks or possibly later than 8 weeks.Postmenopausal women with elevated levels of serum estrone sulfate ,9 and DHAlcohol has many physiologic effects and could influence breast cancer risk through non-hormonal mechanisms as well. Experimental evidence suggests that alcohol interferes with folate absorption, transport, and metabolism, potentially limiting folate stores in the tissues and may interfere with DNA methylation ,13. AlcoIn conclusion, our results provide additional evidence for a mechanism by which moderate alcohol drinking could modify breast cancer risk, indicating that this effect occurs after a short time period (as early as four weeks), and thus provides further support for a causal association.None declared.


Colon adenocarcinomas are refractory to a number of widely used anticancer agents. Multifactorial mechanisms have been implicated in this intrinsically resistant phenotype, including deregulation of cell death pathways. In this regard, the p53 protein has a well established role in the control of tumor cell response to DNA damaging agents; however, the relationship between p53-driven genes and drug sensitivity remains controversial. The present study investigates the role of the p53/p21 system in the response of human colon carcinoma cells to treatment with the cytotoxic agent doxorubicin (DOX) and the possibility to modify the therapeutic index of DOX by modulation of p53 and/or p21 protein levels.The relationship between p53 and p21 protein levels and the cytotoxic effect of DOX was investigated, by MTT assay and western blot analysis, in HCT116 (p53-positive) and HT29 (p53-negative) colon cancer cells. We then assessed the effects of DOX in two isogenic cell lines derived from HCT116 by abrogating the expression and/or function of p53 and p21 . Finally, we evaluated the effect of pre-treatment with the piperidine nitroxide Tempol (TPL), an agent that was reported to induce p21 expression irrespective of p53 status, on the cytotoxicity of DOX in the four cell lines. Comparisons of IC50 values and apoptotic cell percentages were performed by ANOVA and Bonferroni's test for independent samples. C.I. calculations were performed by the combination Index method.Our results indicate that, in the colon carcinoma cell lines tested, sensitivity to DOX is associated with p21 upregulation upon drug exposure, and DOX cytotoxicity is potentiated by pre-treatment with TPL, but only in those cell lines in which p21 can be upregulated.p21 induction may significantly contribute to the response of colon adenocarcinomas cells to DOX treatment; and small molecules that can exploit p53-independent pathways for p21 induction, such as TPL, may find a place in chemotherapeutic protocols for the clinical management of colorectal cancer, where p53 function is often lost, due to genetic or epigenetic defects or to post-transcriptional inactivating mechanisms. Colorectal cancer is the second most common cause of cancer-related mortality in Western countries, with about 1 million new cases every year diagnosed world-wide and 500,000 patients dying from the disease . In addiOver the past decade, induction of apoptosis has emerged as a major event in tumor cell response to cytotoxic agents (for a recent review see ). This vextrinsic pathway is initiated by ligation of transmembrane receptors to activate membrane proximal "activator" caspases, which in turn cleave and activate downstream "effector" caspases. The intrinsic pathway requires disruption of the mitochondrial membrane and the release of mitochondrial proteins, two events that are regulated by the opposing actions of pro- and antiapoptotic Bcl-2 family members. "Intrinsic stresses", such as those produced by DNA-damaging agents, activate the intrinsic apoptotic pathway; the multifunctional transcription factor p53 is thought to be part of a "fast track" connection between nuclear DNA damage and the intrinsic pathway machinery [p53 regulates multiple responses to genotoxic stress by transcriptional activation or repression of a number of genes encoding proteins involved in cell cycle control (p21WAF1/Cip1), DNA repair (gadd45), and apoptosis  [p53 and in the p53 pathway can produce multidrug resistance in vitro and in vivo, and reintroduction of wildtype p53 into p53 null tumor cells can re-establish chemosensitivity [p53 status is not a universal predictor of treatment response, in part because not all drugs absolutely require p53 for their apoptotic function [Two separable, although extensively cross-talking, pathways leading to apoptosis have been characterized ,8. The eachinery . p53 regurvivin) . Mutatiositivity . p53 stafunction  and in sfunction . Still, function  and the function ).N-oxyl  analysis of the relationship between p21 status and sensitivity to treatment with the cytotoxic anticancer agent doxorubicin (DOX) in p53-positive and -negative colon cancer cell lines; (b) design of treatment strategies based on the use of small molecules able to modulate p21 status; for the present study we have used a low molecular weight, stable nitroxide radical, 4-hydroxy-2,2,6,6,tetramethylpiperidne-N-oxyl  and cell culture reagents were purchased from Sigma-Aldrich srl. , unless otherwise indicated; doxorubicin (DOX) was kindly provided by Dr. A Suarato .Standard chemicals, including 4-hydroxy-2,2,6,6-tetramethylipiperidine-2 and 95% humidity.The human colon carcinoma cell lines HCT116, HT29  and HCT116 p21-negative cells (HCT116 p21-/-), kindly provided by Dr B. Vogelstein , were maintained in DMEM medium supplemented with 10% FBS (Mascia Brunelli); the HCT116-E6 cell line, obtained from HCT116 cells by transfection with pCMVneo-E6 plasmid (provided by Dr B. Vogelstein) containing the HPV16-E6 human gene, was maintained in ISCOVE medium supplemented with 10% FBS and geneticin (500 μg/ml). All the cell lines were cultured at 37°C, in an atmosphere of 5% CO25 or IC50values obtained for each cell line according to schedule (b). At the end of the treatment period, 50 μl of MTT (2 mg/ml in PBS) were added to each well at 37°C for 3 h and the reduction of MTT by viable cells was measured colorimetrically at 570 nm, using a Universal Microplate Reader EL800 (Bio-Tek Instruments). IC25 and IC50 values  were calculated according to the median effect equation and analysis of the interaction between DOX and TPL was performed as described by Chou & Talalay [The effects of DOX and/or TPL on cell growth were assessed by the MTT assay . Briefly Talalay .Transfection of HCT116 cells with the pCMVneo-E6 plasmid was performed by electroporation as described by Yanez and Porter , using aThe expression of p53 and p21, before and after 1 h exposure to DOX (1.0 and 10 μM) followed by 23 h incubation in drug-free medium, or after 24 h exposure to TPL (1.0 and 2.5 mM), was evaluated by Western blot analysis of total protein extracts . Protein concentration in the cellular lysates was determined by the BCA assay . 15 μg of protein extract/lane were loaded onto 11% polyacrylamide gels and separated under denaturing conditions. Protein samples were then transferred onto nitrocellulose membrane and Western blot analysis was performed by standard techniques. using a mouse anti-p53 monoclonal antibody  and a rabbit anti-p21 polyclonal antibody . Proteins were visualized using peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies (Amersham Pharmacia Biotech) and the ECL Plus Western Blotting Detection Reagents (Amersham Pharmacia Biotech).Densitometric analysis was performed using the Scion Image software .5 cell samples were analyzed and data were processed using the CellQuest software (Becton Dickinson). The percentage of apoptotic cells in each sample was determined based on the sub-G1 peaks detected in monoparametric histograms.The presence of apoptotic cells in HCT116, HT29, HCT116-E6 and HCT116 p21 -/-, before and after 1 h exposure to DOX (1.0 and 10 μM) followed by 23 h incubation in drug-free medium, was evaluated by flow cytometric analysis, using a Becton Dickinson FACScalibur flow cytometer. Cells were detached by trypsinization, washed in phosphate-buffered saline (PBS) and fixed in ice-cold 70% ethanol for 20 min at -20°C. After an additional wash in PBS, DNA was stained with 50 mg/ml propidium iodide in PBS in the presence of RNAse A (30 U/ml) at 37°C for 30 minutes. 5 × 1050 values and apoptotic cell percentages in the four different cell lines were performed by ANOVA and Bonferroni's test for independent samples. C.I. calculations an relative statistical analysis were performed as described by Chou and Talalay [combination index (C.I.) can be calculated from dose-response curves obtained following exposure to DOX and/or TPL as single agents and in combination. C.I. values approximating 1.0 indicate additive interactions between the two agents; C.I. < 1.0 indicate synergy and, conversely, C.I. > 1.0 indicate antagonism.Comparisons of IC Talalay . Accordi50 values calculated from these data are reported in table resistance index (R.I.) is calculated as the ratio between the IC50 value obtained for each cell line and that obtained for HCT116 cells. The HT29 cell line, which carries a mutant form of the p53 gene, is significantly more resistant to the cytotoxic action of DOX than the HCT116 cell line, carrying a wild-type p53 gene . HCT116-E6 cells are 2-fold more resistant to DOX than HCT116 cells , while HCT116 p21-/- cells are 14-fold more resistant to DOX than HCT116 cell line , in spite of the presence of a wild type form of p53.Figure M for 1 h followed by 23 h in drug-free medium), in the four cell lines tested; panels B and D report the densitometric analysis of p53 and p21 immune reactive bands, respectively. In the absence of drug treatment, it is possible to observe that HT29 cells show higher p53 but lower p21 protein levels, as compared with the HCT116 cell line. DOX treatment induces a dose-dependent increase in both p53 and p21 levels in HCT116 cells, whereas in HT29 cells p53 protein levels are not significantly modified by the treatment and p21 protein levels are only detectable when 25 μg of protein extract/lane are loaded onto the gel (instead of the 15 μg loaded for the other cell lines), and even then only at the highest DOX concentration used. As expected, HCT116 E6 cells do not show detectable p53 levels, both under baseline conditions and following DOX treatment; in contrast in HCT116-E6 cells p21 levels are increased by DOX treatment in a dose-dependent fashion, although to a lesser extent than in HCT116 cells. HCT116 p21-/- cells show higher baseline expression levels of p53 compared with HCT116 cells and DOX treatment in this cell line enhances p53 expression to a an even greater extent than in HCT116 cells line. As expected, p21 is undetectable in HCT116 p21-/- cells, and DOX treatment does not modify the intracellular levels of this protein.Figure Figure 25 and IC50 values obtained for the four cell lines after 24 h of continuous TPL exposure followed by 72 h in drug-free medium. TPL can be observed to inhibit cell growth in all four cell lines; although no significant differences can be detected among IC50 values, HCT116 p21-/- cells appear to be less responsive than the other three cell lines. Figure Table 25 and IC50 values obtained for each cell line, on DOX cytotoxicity. The cells' response to DOX is expressed as the IC50 values derived from dose/response curves obtained after 1 h exposure to DOX with or without pretreatment with TPL (24 h), followed by 72 h in drug-free medium. Analysis of cytotoxicity data shows a synergistic interaction (C.I.<1) between DOX and TPL for both TPL concentrations in HCT116 cells and in HCT116-E6 and HT29 cells at the lower concentration; only additive effects (C.I. ≈ 1) can be observed in HCT116 p21 -/- cells. IC50 values for DOX and TPL according to the three different schedules are reported in table Figure p53 status is not unequivocally related to the response to DOX: in fact, while p53-deficient cells  are indeed less responsive than the p53/wt parental HCT116 cell line, the highest resistance index was obtained for HCT116 p21-/- cells, harboring two wildtype p53 alleles. As expected, treatment with DOX leads to p53 upregulation in the cell lines expressing wildtype p53; this effect has been thoroughly documented in colon cancer cells as well as in tumor cell lines derived from other tissues, and has been attributed to phosphorylation and subsequent stabilization of p53, possibly through activation of DNA-dependent protein kinase or ATM (ataxia-teleangectasia mutated) kinase . In see e.g. ) and cansee e.g. , the rolsee e.g. ,25. In asee e.g.  or by losee e.g. ,27-30. Hsee e.g. -34 and tsee e.g. -37. The see e.g. , but stiInterestingly, the situation outlined by our results does not seem to conform to either view: in fact, while induction of p21 in p53-proficient and -deficient cell lines is associated with increased response to drug treatment, this was not accompanied by a parallel increase in apoptotic cells, as no significant differences in apoptosis were observed between HCT116 cells and the 14-fold resistant HCT-116 p21-/- cell line  p21 induction may significantly contribute to the response of colon adenocarcinoma cells to DOX treatment; and 2) small molecules that can exploit p53-independent pathways for p21 induction, such as TPL, may find a place in chemotherapeutic protocols for the clinical management of colorectal cancer, where p53 function is often lost, due to genetic or epigenetic defects or to post-transcriptional inactivating mechanisms.The author(s) declare that they have no competing interestsThe pre-publication history for this paper can be accessed here:


Erythroid nuclear cells (ENC) of the bone marrow (BM) have not previously been considered as important producers of wide spectrum of haemo- and immunoregulatory cytokines. The aim of the current work was to confirm the production of the main hemo- and immunoregulatory cytokines in human ENC from BM.+ or AG-EB+) produced a wide spectrum of immunoregulatory cytokines. Human BM ENC produce cytokines such as interleukn (IL)-1β, IL-2, IL-4, IL-6, interferon (IFN)-γ, transforming growth factor (TGF)-β1, tumor necrosis factor (TNF)-α and IL-10. They can be sub-divided into glycophorin A positive (Gl A+) and erythroblast antigen positive (AG-EB+) cells. To study potential differences in cytokine expression between these subsets, ENC were isolated and purified using specific antibodies to Gl A and AG-EB and the separated cells were cultivated for 24 hours. The cytokine contents of the supernatant were measured by electrochemiluminescence immunoassay. Quantitative differences in TGF-β1 and TNF-α production were found between Gl A+ and AG-EB+ BM ENC. Furthermore, in vitro addition of erythropoietin (EPO) reduced IFN-γ and IL-2 production specifically by the AG-EB+ ENC. Thus, Gl A+ and AG-EB+ ENC produce IL-1β, IL-2, IL-4, IL-6, IFN-γ, TGF-β1 and TNF-α. Gl A+ ENC also produce IL-10.We used native human BM ENC in our experiments. We for the first time have shown, that the unstimulated erythroblasts (Gl ACytokine production by erythroid nuclear cells suggests that these cells might be involved in regulating the proliferation and differentiation of hematopoietic and immunocompetent cells in human BM. Haematopoesis is regulated by lymphoid and non-lymphoid cells through a complex network of paracrine and autocrine mechanisms involving cytokines, growth factors and their receptors. However, although stromal -6, endotExperiments on ENC isolated from mouse spleen undergoing erythroid hyperplasia, and on cells separated from single erythroid colonies, have revealed that mRNAs for cytokines such as IL-1α, IL-1β, IL-4, IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF), TGF-β1 and IFN-γ are present ,18. Prod+ BFU-E cells) and on colony-forming units – erythroid (CFU-E) (up to 36% of AG-EB+ CFU-E cells) [+erythroblasts). Later in development, AG-EB expression decreases, and only 4% of reticulocytes carry AG-EB [The aim of the current work was to confirm previous observations by studying the production of the main hemo- and immunoregulatory cytokines, IL-1β, IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and TGF-β1, in human ENC from BM. Furthermore, we wished to determine whether different subsets of ENC (AG-EB+ and Gl A+ erythroid cells from human BM) might account for altered expression levels. Expression of both these markers is specific to erythroid cells; they are absent from the membranes of other blood-forming cells. AG-EB is expressed in a proportion of BFU-E cells (up to 4% of AG-EBE cells) . Its expry AG-EB . In contry AG-EB ,27. ShemIn this article we show which human BM ENC produce the cytokines IL-1β, IL-2, IL-4, IL-6, IFN-γ, TGF-β1, TNF-α and IL-10.+ cells, whereas Gl A+ secrete more TNF-α (p < 0.05).Erythroid cells were separated according to the presence of the surface markers AG-EB and Gl A. BM erythroid cells carrying AG-EB produce the cytokines IL-1β, IL-2, IL-4, TNF-α, IFN-γ and TGF-β1  or Glycophorin A (Gl A) . Monoclo6 cells were placed in culture medium containing 2 U/ml Epo  for a 24-h incubation.1 × 105/ml and then cultivated in MethoCult H4433 medium  for 14 days. Colonies were visually characterised as CFU-E using an inverted microscope. After cultivation, the colonies were harvested and washed twice to remove methyl cellulose. The resulting cellular suspension (1 × 106/ml) was cultivated for 24 h in RPMI-1640 with 10% horse serum, then supernatants were collected. Samples were stored at -20°C.Cells from the enriched erythroid population were maintained as described above ,20 at 2·+, AG-EB+ and CFU-E cells) were characterised as erythroblasts and were haemoglobin positive.Cell purity was assessed using Nocht-Maksimov staining of smears  and staiQuantitative determination of cytokines was performed by the electrochemiluminescent immunoassay -52 usingFor TGF-β1 detection, samples were treated with HCl to obtain intact and easily detectable proteins as described .All polyclonal and monoclonal antibodies were purchased from R&D Systems . The following antibodies were used: against recombinant IL-1β, IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, TGF-β1 polyclonal and monoclonal: anti-hIL-1β #AB-201-NA, anti-hIL-1β #MAB201, anti-hIL-2 #AB-202-NA, anti-hIL-2 #MAB202, anti-hIL-4 #AB-204-NA, anti-hIL-4 #MAB204, anti-hIL-6 #AB-206-NA, anti-hIL-6 #MAB206, anti-hIL-10 #AB-217-NA, anti-hIL-10 #MAB217, anti-hTNF-α #AB-210-NA, anti-hTNF-α #MAB210, anti-hIFN-γ #AB-285-NA, anti-hIFN-γ #MAB285, anti-hTGF-β1 #AF-101-NA, anti-hTGF-β1 #MAB240. No antibody used displayed cross-reactivity with other cytokines.3 and 0.1% BSA .Recombinant cytokines were purchased from R&D Systems : rhIL-1β #201-IL, rhIL-2 #202-IL, rhIL-4 #204-IL, rhIL-6 #206-IL, rhTNF-α #210-TA, rhIFN-γ #285-IF, rhTGF-β1 #240-B-002; and PeproTech, Inc. : rhIL-10 #200-10 was used for calibration curves. All the recombinant cytokines were diluted to 2 μg/ml in PBS, pH 7.4, supplemented with 0.05% NaNResults are presented as means ± SEM. Statistical significance was determined using the nonparametric Mann-Whitney U-test. P < 0.05 was considered to be significant.IBK, NJD, ANZ and MIL carried out the selection of donors, sternum BM sampling and analysis of myelograms. TVI, ANS and SVK carried out all of the experiments and drafted the manuscript. SVS participated in design and coordination of the research and revision of the manuscript. ANS participated in the preparation and revision of the manuscript. VAK conceived of the study. All authors read and approved the final manuscript.


In this study we have made novel observations with regards to potentiation of the tumoricidal activity of the oncolytic adenovirus, dl1520 (ONYX-015) in rat glioblastoma cell lines expressing heat shock protein 72 (HSP72) due to permissive virus replication. ONYX-015 is a conditionally replicating adenovirus that is deleted for the E1B 55 kDA gene product whose normal function is to interact with cell-cycle regulatory proteins to permit virus replication. However, many murine and rodent cell lines are not permissive for adenovirus replication. Previously, it has been reported that the heat shock response is necessary for adenovirus replication and that induction of heat shock proteins is mediated by E1 region gene products. Therefore, we hypothesized that HSP72 expression may allow for permissive replication of ONYX-015 in previously non-permissive cells. Rat glioma cell lines 9L and RT2 were transfected with a plasmids expressing HSP72 or GFP. After infection with ONYX-015, no tumoricidal activity is observed in GFP expressing cell lines despite adequate transduction. In contrast, HSP72 transfected cells show cytopathic effects by 72 hours and greater than 75% loss of viability by 96 hours. Burst assays show active virus replication in the HSP72 expressing cell lines. Therefore, 9L-HSP72 and RT2-HSP72 are ideal models to evaluate the efficacy of ONYX-015 in an immunocompetent rat model. Our study has implications for creating rodent tumor models for pre-clinical studies with E1 region deleted conditionally replicating adenovirus. Adenovirus vectors are commonly utilized in cancer gene therapy experiments. They readily infect numerous tumor cell types and are easily manipulated allowing for transgene expression. In an eThe E1 region genes of the common serotypes of adenovirus optimize their own replication in target cells and interact with many cell cycle associated gene products. The E1a region genes promote transition of cells into S phase through their interaction with the retinoblastoma protein (pRB). pRB in Various heat shock proteins have been shown to be necessary for efficient adenovirus replication. The avian adenovirus CELO requires the induction of HSP70 and HSP40 for production of viral proteins. CELO muAdenovirus E1 region genes from serotype 5 and 12 have previously been shown to induce HSP72 expression . HSP72 iOne of the difficulties with studying E1 adenovirus deletion mutants is the lack of appropriate animal tumor models which allow for permissive growth of these viruses. Common animal tumor models for glioblastoma are the cell lines 9L and RT2. Previously, it has been shown that replication competent E1 deletion mutants can transduce 9L cells and result in transgene expression but there is a block against virus replication. In thisPrior to studying the efficacy of ONYX-015 to result in tumor cell lysis, we determined the optimal dose necessary for tissue transduction. Adenovirus infection efficiency is predicated on the expression of adenovirus surface receptors. Using aThe dramatic difference in tumoricidal effect between HSP72 transfected and control tumor cells is best observed by cytopathic effect. In Figure When HSP72-transfected cells were infected with a low dose (MOI 100) of ONYX-015, there is no loss of cell viability. However, comparison of cell numbers to GFP-transfected cells or AD-GFP infected cells showed a dramatic cytostatic effect  supplemented with 10% (v/v) heat inactivated fetal bovine serum , 2 mM L-glutamine and 100 units/ml Penicillin and 1000 ug/ml Streptomycin (Invitrogen). 9L and RT2 cells were transfected with the plasmids pEGFP-N2 (Clontech) or pHSP72 using Lipofectamine (Invitrogen) according to the manufacturers protocol followed by selection in 0.2 mg/ml G418 (Invitrogen). 293 cells were obtained from American Type Culture Collection  were used for virus propagation and purification.9L and RT2 cells are rat glioma cell lines with different derivations. 9L cells are chemically induced gliosarcoma cells while RTpHSP72 was created by ligation of the DNA Polymerase blunt ended 2.3 KB Bam H1-Hind III fragment from plasmid pH2.3 (ATCC) into the blunt ended Xho1-XbaIA vector pEGFP-N2.4 cells/well in 96 well flat-bottomed tissue culture plates. Virus titer from combined supernatant and freeze-thawed cell lysate was determined by serial 10-fold dilution into different rows of the titer plate. After 10 days, wells were scored for cytopathic effect. The titer was calculated using the formula T = 101+d(S-0.5) where d is the Log 10 of the dilution and S is the sum of the ratios of positive wells in each row. A more lengthy description of this method is provided in the adenovirus application manual at .Creation of ONYX-015 has previously been described. The vir2. Cells were subsequently washed with phosphate buffered saline and then cultured in complete medium.Cells were infected when 50% confluent with the dose of adenovirus given in the appropriate table. The infection was performed in serum free DMEM media for 90 minutes at 37°C and 5% COCell viability was determined by the standard Trypan-blue exclusion test. Cells were washed with PBS and then lifted from tissue culture plates with Trypsin-EDTA solution (Invitrogen). Cells were stained with 0.2% Trypan blue solution for 5 minutes and then counted on a hemacytometer.JM performed all of the experiments outlined in this study. JAK independently replicated results for validity. JAK and MRS contributed equally to the molecular cloning of the plasmids and adenovirus propagation and purification. All authors participated in maintenance of cells in culture. The hypothesis, study design and statistical analysis were all performed by MRS. MRS drafted the entire manuscript with editing and approval by all of the authors.