Asthma is a common disease and appears to be increasing in prevalence. There is evidence linking air pollution, including that from road-traffic, with asthma. Road traffic is also on the increase. Routine surveillance of the impact of road-traffic pollution on asthma, and other diseases, would be useful in informing local and national government policy in terms of managing the environmental health risk.Several methods for exposure assessment have been used in studies examining the association between asthma and road traffic pollution. These include comparing asthma prevalence in areas designated as high and low pollution areas, using distance from main roads as a proxy for exposure to road traffic pollution, using traffic counts to estimate exposure, using vehicular miles travelled and using modelling techniques. Although there are limitations to all these methods, the modelling approach has the advantage of incorporating several variables and may be used for prospective health impact assessment.The modelling approach is already in routine use in the United Kingdom in support of the government's strategy for air quality management. Combining information from such models with routinely collected health data would form the basis of a routine public health surveillance system. Such a system would facilitate prospective health impact assessment, enabling policy decisions concerned with road-traffic to be made with knowledge of the potential implications. It would also allow systematic monitoring of the health impacts when the policy decisions and plans have been implemented. The prevalence of asthma is increasing and there is concern that the increase may in part be attributable to increasing road traffic related pollution. The concerns relate especially to childhood asthma. In this article, we set out the arguments for using modelled exposure assessment to create a surveillance system that will facilitate routine public health work, such as monitoring and health impact assessment. We first discuss the increasing prevalence of asthma and the effects of air pollution on asthma. We then set out the benefits of monitoring the link between outdoor air pollution related to road traffic and asthma and discuss methods of exposure assessment in some detail. We describe modelling air quality in the UK as an example and then address the implementation of a surveillance system.Asthma is a common disease. Figures from the International Study of Asthma and Allergies in Childhood suggest up to 25.9% of children in Oceania have ever had asthma . Althougth century, air pollution increased greatly alongside the noted increase in the prevalence of asthma. Yet the last decades of the century have seen a considerable reduction in pollutants such as sulphur dioxide (SO2) following a cut back on industrial emissions [Air pollution has been linked to morbidity and mortality of several diseases, including diverse conditions such as coronary heart disease  and Hodgmissions .2) however, still remain problematic due to the increasing number of vehicles on our roads. In fact almost 50% of NO2 is thought to be produced by vehicles and much particulate matter is produced by diesel exhaust fumes [2 exposure increases cell membrane permeability, decreases ciliary beat frequency [3 of NO2 to be associated with a relative risk of 1.175 for asthma admission [x) have also been shown to influence emergency room visits in Israel [10) and NO2 appeared to consistently increase attendances at accident and emergency units with asthma in London [Levels of pollutants such as nitrogen dioxide  to calculate the distance from an individual's residence to a road, most often a main road, carrying over a certain volume of vehicles. In certain cases the distance from a child's school to a main road has been used instead. Examples of the use of this method are studies by Livingstone et al.  and Wilk2 may be high, with levels possibly higher than outdoors if a gas stove is used in the home [2. This however, is not without difficulties, as if one is exposed to traffic there will be a combined effect from a cocktail of pollutants produced by both trucks and cars.Another popular method is considering traffic flow along the street of residence or one in close proximity, as used by English et al. . In a sithe home . Anotherthe home . It coulSome authors  have atA number of studies have used modelling to estimate pollution exposure ,36. A moEven within a model however, accounting for personal day-to-day exposures is still problematic. In order to take previous exposures into account a cohort study would be necessary . Certain2 and PM10. The model could be used to estimate exposures on all the streets within a certain radius of the home or place of work as dispersion of pollutants from these streets may also be affecting the individual. In a sophisticated model it may be possible to make adjustment for the height of an individual's residency or place of work in high rise buildings to account for the vertical dispersion of pollutants. Such a system could also be used to estimate exposures at previous residences, work places or schools of an individual so that an assessment of lifelong exposure could be made as accurately and practically as possible. However, the latter might be too complicated for a routine monitoring system.Despite limitations, a model would appear to be the most practical way of assessing traffic-related exposure where routine surveillance is concerned. Information such as vehicle density, type of vehicle, risk of traffic congestion, presence of bus stops and street crossings, distance of residences to roads, street width, type of street, building presence and type and meteorological conditions  could be collected routinely for use in a variety of models for predicting exposure to NOThe UK Government's current policy on air quality within the UK is set out in the Air Quality Strategy for England, Scotland, Wales and Northern Ireland published in January 2000 pursuant to the requirements of Part IV of the Environment Act 1995. The Strategy sets out a framework for improving air quality and for ensuring that international commitments are met. It is designed to be an evolving process that is monitored and regularly reviewed. The Strategy sets standards and objectives for ten pollutants that have an adverse effect on human health, vegetation or ecosystems and target dates for achieving them. The standards generally set concentration limits above which sensitive members of the public  might experience adverse health effects. In early 2003 an Addendum to the Strategy was published introducing standards and objectives for a new pollutant and revising those for three others.2, PM10, SO2, ozone (O3), NOx and polycyclic aromatic hydrocarbons. The predominant source of most of these pollutants is road traffic, but industrial and domestic sources are also contributors.The pollutants currently specified in the Strategy now include benzene, 1,3 butadiene, carbon monoxide, lead, NO2), there is both an annual mean guideline limit and a short-term mean guideline limit. These reflect the varying impacts on human health of exposure to some pollutants over differing time periods, .The air quality standards or guideline limits are long-term benchmarks for ambient pollutant concentrations which represent negligible or zero risk to human health, based on medical and scientific evidence reviewed by the Expert Panel on Air Quality Standards (EPAQS) and the World Health Organization (WHO). For some pollutants, . The latest available guidance is Policy Guidance LAQM.PG(03) and Technical Guidance LAQM.TG(03). Air quality modelling is key to assessing the future potential for attainment, or not, of the objectives. Part IV of the Environment Act 1995 requires a local authority to designate an Air Quality Management Area (AQMA) covering any part of its administrative area where air quality objectives are not likely to be achieved by, or at any point beyond, the relevant objective's target date at locations where the general public might reasonably be exposed. These AQMAs have been determined by modelling future scenarios. For each AQMA the local authority has a duty to draw up an Air Quality Action Plan (AQAP) setting out the measures the authority intends to introduce to deliver improvements in local air quality in pursuit of the air quality objectives. Local authorities are not statutorily obliged to meet the objectives, but they must show that they are working towards them. As of June 2004, there were 120 designated AQMAs in the UK, with 80 AQAPs produced outlining how air quality would be tackled in these areas.A routine surveillance system would be one which links modelled air quality data, such as those derived in the UK described above, with routinely collected health data. There are a number of issues regarding the technical aspects of linking spatial information on air quality with health information, typically carried out using GIS. These are discussed in detail elsewhere . The infThe majority of studies examining the link between asthma and road-traffic pollution have concentrated on the prevalence of asthma, for example studies by Ciccone et al. , JedrychA few of the studies examining the link between asthma and road-traffic pollution have used information that is readily available. For example studies by English et al.  and Lin A routine surveillance system recording spatial variation in pollutant levels would allow improved understanding of the link between road-traffic pollution and asthma, or indeed other diseases and could be used to help predict future health impact, particularly in cities and towns. The results of such assessment would allow local policy decisions concerning the routing of traffic around residential areas or schools and plans to reduce congestion to be made with knowledge of the implications of the decision on the health of the local population. It would also allow systematic monitoring of the health impacts when the policy decisions and plans have been implemented.We should point out here that to examine acute effects  using daily time series analyses, monitoring data would be required as modelling would probably be too insensitive to detect daily variation in outdoor air pollution levels. However, time series analyses are complicated research methods that are not within the realm of routine public health practice. What we have argued for here is a surveillance system that looks at spatial variation in pollution and asthma which would highlight problem areas and could monitor the effects of interventions to reduce pollution in these areas.We believe that a routine surveillance system which links modelled outdoor air pollution data to health data would provide a useful tool for facilitating routine environmental public health work. Such a system would be especially useful for monitoring the health effects of traffic related pollution and for aiding health impact assessment. Implementation of the system will require close collaboration between public health and environmental health departments, protocols for sharing data and investment in training to develop the necessary technical expertise to set up and maintain the surveillance system. Of particular importance will be the ability of high level management to interpret surveillance information within a wider policy context.RM proposed the idea for the article. ECF wrote the first draft, supervised by RM. MD contributed the section on modelling air quality in the UK. RM edited subsequent drafts.


Difficulty of reconstruction of the eyelids arises from the need to reconstruct different supporting and covering structures in a single operation. Defects in the anterior lamella of the eyelids can be readily repaired with skin grafts or flaps but posterior lamellar reconstruction needs more complex applications.We performed posterior lamellar eyelid reconstruction with posterior parts of the temporalis fascia, since their anatomical and histological features are very similar to the defects. Nine patients with skin tumors located on the periorbital region were treated with local skin flaps and deep layer of the temporalis fascia.Grafts were harvested very easily. There was no complication related with graft or donor site. Biopsy was performed in three cases and normal conjunctival elements were seen. Functional and acceptable aesthetically results were achieved in all patients.Ideal reconstructive material for replacement of the posterior lamina is still lacking. Tarsal reconstruction can be made with deep temporalis fascia with success since the thickness of the both tissues are very similar and also since the loose areolar layer of the temporalis fascia is very thin and highly vascularized, this layer can be used in reconstruction of the conjunctiva. According to our knowledge this is the first report of using of the posterior part of temporalis fascia as a composite graft in the literature. Reconstruction of eyelid defects after tumor excision should aim at obtaining full globe protection without visual disruption and restoring the area to an appearance as close to normal as possible . ReconstTarso-conjunctival grafts, buccal mucosa, hard palate mucosa and amniotic membrane have been used in the reconstruction of the conjunctival defects and all techniques were reported with individual advantages and disadvantages. Temporalis fascia has a specific and complex anatomy -6. MainlNine patients with lower eyelid defects after removal of malignant tumors were treated with local flaps combined with temporalis fascia grafting in the Department of the Plastic and Reconstructive Surgery, Adnan Menderes University Hospital. Data of the patients were summarized in Table Ages of patients were ranged from 21 to 71. Full thickness defects measured from 15 × 5 mm to 50 × 15 mm. All patients were operated under the general anesthesia. The follow up periods ranged from 6 months to 24 months. After ethical approval from our department and patient consent, biopsy performed in three cases to evaluate the technique.Tumor excision was performed with safety surgical margins in all patients. Mustardé cheek flap was prepared in four patients, forehead flap and tri-lobed flap in two patients and one bi-lobed flap were designed. "Y" skin incision was performed for harvesting of the temporalis fascia Figure  and 1c.Against possible hair damage we did not use solution containing of epinephrine for haemostasis. The scalp is incised at angle that follows the direction of the hair follicles, which is facilitated by not shaving the hair. The incision is made through the scalp to the superficial fascia. Superficial fascia was incised to the temporalis muscle in dimensions 40 × 20 mm. This layer was elevated above the loose areolar tissue as distal based flap and loose areolar layer and deep temporalis fascia was dissected and harvested  this layer is thin and will conform to the shape of underlying soft tissue or cartilage with aesthetically pleasing fidelity,(b) well vascularized, and if handled with care, will readily support split and full thickness skin graft,(c) can be harvested easily, with minimal donor-site morbidity.We used this anatomical structure for conjunctiva reconstruction. Conjunctiva is a mucous membrane that covers the posterior aspect of the eyelids  and the anterior surface of the globe (bulbar conjunctiva). Defects larger than 25% of the eyelid usually require a free graft to make for the loss of specialized tissue. Conjunctiva harvested from another lid is the ideal match and physiologically but it is thin, difficult the handle, has tendency to contract, and can only be harvested sparingly to avoid interfering to donor fornices. In addition this technique requires two operations when it is performed as a lid sharing procedure. Oral mucosa is abundant and simple to remove but tends to contract and infection. Nasal mucosa is easier to handle, and minimal contraction but this procedure has some problem of poor access and thickness. Today, most performed techniques are hard palate mucosal graft, amnion membrane, chondormucosal Septum -20. TarsOne of the most popular grafts in posterior lamella reconstruction used to come from the chondromucosal nasal septum. Major disadvantages of this technique are the difficulty of the shape and adapt to the eyelid. Palatal mucosal grafts were widely used. Such grafts meet both the mucosal and supporting requirements. The use of hard palate mucosal grafting in lower eyelid reconstruction was first described by Siegel . Hard paIn our series we did not observe irritation signs and symptoms.It is agreed among most authors that the ideal reconstruction should use identical or similar tissue to that of the original structure. Undoubtedly, reconstructions of the posterior lamella with original tissue i.e. free tarso-conjunctival grafts or flap is the best treatment modality. If the donor sites of the tarso-conjunctival grafts or flap were not suitable or enough, deep temporalis fascia grafting would be an alternative in the reconstruction of the posterior lamella.We believe that application of the deeper part of the temporalis fascia for the reconstruction of the posterior lamella has many advantages: easy to perform, single composite graft for two layers, without or minimal donor site morbidity, no infection risk, no shrinkage of the graft, easy to adapt, wide donor site, no irritation and excellent cosmetically results. There is no data about the using of the posterior part of the temporalis fascia in reconstruction of the tarsus and conjunctiva in the literature.In conclusion, loose layer of the temporalis fascia is ideal tissue for the growing of the conjunctiva and the thickness of the deep layer of the temporalis fascia is very similar to the tarsus. Both tissues can be used in reconstruction of the posterior lamellar reconstruction of the eyelids with success.None declaredEC conceived of the study, and participated in its design and coordination. NS participated in design of study. All authors read and approved the final manuscript.


Quality of care from the perspective of users is increasingly used in evaluating health care performance. Going beyond satisfaction studies, quality of care from the users' perspective is conceptualised in two dimensions: the importance users attach to aspects of care and their actual experience with these aspects. It is well established that health care systems differ in performance. The question in this article is whether there are also differences in what people in different health care systems view as important aspects of health care quality. The aim is to describe and explain international differences in the importance that health care users attach to different aspects of health care.Data were used from different studies that all used a version of the QUOTE-questionnaire that measures user views of health care quality in two dimensions: the importance that users attach to aspects of care and their actual experience. Data from 12 European countries and 5133 individuals were used. They were analysed using multi-level analysis.Although most of the variations in importance people attach to aspects of health care is located at the individual level, there are also differences between countries. The ranking of aspects shows similarities. 'My GP should always take me seriously' was in nearly all countries ranked first, while an item about waiting time in the GP's office was always ranked lowest.Differences between countries in how health care users value different aspects of care are difficult to explain. Further theorising should take into account that importance and performance ratings are positively related, that people compare their experiences with those of others, and that general and instrumental values might be related through the institutions of the health care system. Large differences between countries exist in the use, costs, quality, and accessibility of health services . Also laQUality Of care Through the patients' Eyes – that distinguishes two quality of care dimensions: performance and importance [In this study we did a secondary analysis on surveys of patient views on quality of health care. Patients' views were measured using the QUOTE-questionnaire – with the acronym QUOTE standing for portance . PerformTo select relevant quality of care aspects, a general and a disease-specific approach was followed, using focus group discussions . With thIn this article we first compare the importance dimension of QUOTE across several European countries and Israel to gain insight in the similarities and differences in people's views on quality of care. Secondly, we will look at the relationship between importance and performance ratings as part of an explorative analysis to explain differences in importance ratings between patients and/or countries. The general research question is:Do patients in different European countries think differently about the relative importance of various aspects of quality of care, and if so, how can these differences be explained?This general question is divided into the following separate questions:a. To what extent do the importance judgements of patients cluster within countries when individual characteristics of patients are taken into account?b. Does the ranking of importance judgements vary between countries?c. What is the relationship between the average performance of health care systems, as judged by patients, and the individual importance judgements?When we compare the importance judgements of patients between countries, we will take into account individual characteristics of respondents to rule out differences in the composition of the groups of respondents. With respect to the relationship between importance and performances scores it might be anticipated that in general people attach more importance to those aspects that they actually experience less often. Analogous to the economic mechanism of decreasing marginal utility, e.g. quick service without waiting time in the doctor's office might be valued as less important, if in general services are quick and people don't have to wait long. However, at the same time it can be hypothesized that it's no use aspiring to something that nobody has. If quality of care ratings, as seen through the eyes of the patient, are low on the average and if there is small variation in these performance ratings between individuals, people will probably not find these aspects important. This idea is based on a mechanism of social comparison .The First Dutch QUOTE-questionnaires  served as a starting point for our database ,12. ThesThe QUOTE-questionnaires have been translated in the context of different projects. In all but two cases backward-forward translations have been used. Answering formats of importance items were: Not important (1); Fairly important 2); Important (3); and Extremely important (4). The answering formats for the performance items were: No (1); Not really (2); On the whole, yes (3); and Yes (4). The equivalence of the answering formats in different languages has not been assessed. The wording of the importance items that were used in the QUOTE-questionnaires are presented in tables ; ImportaTable All 5133 health care users reported for each of (maximum) ten items their importance and performance ratings. The importance ratings are dependent variables in a series of statistical analyses with patients hierarchically nested in countries. In contrast to traditional forms of analysis of variance in which factors have 'fixed' effects, countries are considered to have 'random' effects. Such a variance component model is preferred over traditional analysis if the number of categories exceeds ten ,18. The The ICC is statistically defined as the variance between countries divided by the total variance. An ICC of zero therefore implies no variance between countries, indicating the absence of differences between countries in patients' importance ratings. Age and sex were included as covariates to take into account differences in the composition of responder groups in the different countries, because of their association with importance scores ,10,21-23In order to explore the relationship between importance and performance ratings Pearson correlations were calculated between the performance items, both at the individual level and aggregated to country level, and the importance rating on user level. In the introduction we have put forward two hypothetical relations between variation of performance within the countries and the importance ratings. To look at the relation between variation of performance and importance ratings, a distinction was made between countries with large variation in experienced health care quality and countries with smaller variation. Based on the mean standard deviation (SD) of all ten performance items, the countries were equally split into: Denmark, Italy, Belarus, Ireland, Ukraine and Portugal (mean SDs ranging from 1.17 to 1.40) indicating countries with high variation and Greece, Finland, Israel, Norway, Netherlands and UK (mean SDs ranging from .82 to 1.08) indicating countries with low variation. Furthermore, explore the relationship between variation in performance at country level, individual respondents performance experience and their importance ratings, we have divided the individual respondents into those who experienced high performance and those who experienced low performance. To keep these distinctions conveniently arranged in one table, we recoded the individual performance scores from the four point scale to a two point scale, with the combination of 'No' and 'No, not really', indicating poor quality, versus 'On the whole, yes' and 'Yes'.We start the presentation of the results with a description of the overall importance that respondents in all twelve countries attached to the different aspects and the clustering of their answers within countries. We then move to the differences in the ranking of aspects between countries. Finally, we will present results for the relationship between the actual experiences of respondents, both individually and aggregated to an average for each country, and the importance they attach to the different aspects.As shown in table In order to explore differences between user groups, we have computed intra-class correlation coefficients for user groups within the Netherlands only. These coefficients are on average higher than those regarding countries. We also analysed the differences between countries within the IBD patient groups. These differences are much like the figures of table The variation between the countries for each aspect is illustrated in table In order to look at the consistency of user views across the different countries, the ten importance aspects were ranked according to their mean value within each country. Table Looking at the relationship between importance and performance ratings by means of correlation coefficients, table If we take into account the variation in performance ratings within countries, the positive relationship between importance and performance ratings is somewhat stronger in the 'high variation' mode compared to the 'low variation' mode. This can be seen by comparing the difference between the first two columns of table Overall, importance scores are somewhat lower in countries with high variation in performance ratings as compared to countries with low variation in performance scores.The objective of our study was to gain insight into similarities and differences in the value users of health care in different countries attach to aspects of care. As an indicator of these values, importance scores of the QUOTE-questionnaires were used. These scores reflect what is important in evaluating health care quality according to users. Our results show that health care users in different countries to some extent think differently about the relative importance of various aspects of quality of care. Intra-class correlation coefficients were calculated to measure the difference between countries. They range from low to high. Sex-age adjusted intra-class correlation coefficients were only slightly lower. This means that demographic differences between the groups that filled in the questionnaires in different countries cannot explain the differences in average importance between the countries.Although there are differences between countries, the importance rankings of the aspects also show consensus. 'My GP should always take me seriously' is nearly always ranked highest, while the item about waiting time is always ranked as least important. Since we only analysed a small sample of countries, it is difficult to generalise this result. However, it might say something about a hierarchy of these instrumental health care values, suggesting that values concerning respectfulness are seen as more important than service aspects, such as waiting time. This is in line with the findings of Grol et al. . There aIn general, there is a positive relation between what people find important and their experiences, both on an individual level and related to the average experience in a country. The positive relation between importance and experience could probably be explained by a general tendency of cognitive consistency  or alterBy looking at the variation in the performance scores, we have tried to include social comparison mechanisms into the analysis. It can be argued that if people's individual experience indicates low performance for certain aspects in countries, they will value these aspects as more important. However, such a hypothesis has to be rejected on the basis of the results presented in this article. A contrasting hypothesis, that in countries with low variation in actual experiences, people who experience low performance themselves, will not aspire to something that is apparently (from their own and others' experience) out of reach is only partly supported by our findings. Although people in this low variation condition have significantly lower importance scores for half of the items, still the differences in the high variation condition are higher.Looking at the individual aspects, it can be argued that people don't find issues important if they are more or less guaranteed by the health care system. An example for this is the issue of prescribing pharmaceuticals that are fully covered by the health care insurance plans of patients. On the basis of the material presented in this article this hypothesis too has to be rejected. People in countries with low levels of cost sharing in this field, found this item more important than people in countries with higher levels of cost sharing. However, an alternative explanation for this finding could be that structural aspects of the health care system, e.g. on the dimension public – private, reflect general values ,29. If tIn general, we believe that further theorising about differences between health care systems in what people find important, might start from the positive relation between importance and experience, from the idea that people compare their experiences with those of others, and from the idea that general and instrumental values might be related trough the institutions of the health care system. Hofstede  has idenThe analysis presented in this article has its shortcomings. The number of countries is small. The analysis of differences between countries is based on only twelve countries, even though large numbers of respondents were involved. The number respondents increase our confidence in the averages per country, but this does not solve the problem of small numbers at the country level. Differences between user groups could not be taken into account because of the small number of groups for all countries except the Netherlands, although one might expect these differences to exist .The multilevel model now contained two levels, respondents and countries. However, as table We used existing data, collected in different studies with different aims and methods. The response rates differed across studies. The user groups in the surveys refer to are very different, ranging from GP-patients in Belarus to disabled people in the UK and IBD patients in Israel. Apart from the apparent differences between user groups, there are also differences between countries in the position and tasks of GPs. The situation in Ukraine and Belarus was transitional, even ten years after the fall of the Iron Curtain . This maThe QUOTE-questionnaires provide a general framework, and researchers have adapted them to their own aims. Against the advantage of flexible adaptation to different research aims and populations stands the disadvantage of reduced  comparability. For  comparisons more standardisation is very important. Apart from that, the ten QUOTE-aspects we used in our analyses cover different dimensions of the Quality of care concept and can be understood as being comprehensive enough for this explorative type of research.In conclusion, we believe it is important to continue to do research into health care related values because of the increasing importance of user views, both in the health policies of European countries separately and in the international debate about the performance of health care systems. There is not much ground for strong cultural relativism, saying that what is important in the eyes of health care users is so different that it is not possible to develop performance measures that can be used in a wide range of countries.There are differences between countries in the importance people attach to aspects of health care. Most of this variation is related to individual differences, but there is also significant variation between countries. The ranking of aspects shows similarities between countries. In nearly all countries, people ranked the item that their GP should take them seriously as most important, while an item about waiting time was always ranked lowest. It is difficult to explain the variation between countries. Further theorising should take into account that importance and performance ratings are positively related, that people compare their experiences with those of others, and that general and instrumental values might be related through the institutions of the health care system.The author(s) declare that they have no competing interests.PG conceived the study and wrote the first drafts of the article; JK performed the statistical analysis; HS provided data and wrote the final draft of the article; WB provided data; IvdE provided data.The pre-publication history for this paper can be accessed here:


Despite the sophisticated cross sectional image techniques currently available, a number of biliary stenosis or obstructions remain of an uncertain nature. In these pathological conditions, an "intrinsic" parietal alteration is the cause of biliary obstruction and it is very difficult to differentiate benign from malignant lesions using cross-sectional imaging procedures alone. We evaluated the efficacy of different endoluminal techniques to achieve a definitive pathological diagnosis in these situations.Eighty patients underwent brushing, and or biopsy of the biliary tree through an existing transhepatic biliary drainage route. A subcoort of 12 patients needed balloon-dilatation of the bile duct and the material covering the balloon surface was also sent for pathological examination . Pathological results were compared with surgical findings or with long-term clinical and instrumental follow-ups. Success rates, sensitivity, specificity, accuracy, confidential intervals, positive predictive value and negative predictive value of the three percutaneous techniques in differentiating benign from malignant disease were assessed.The agreement coefficient of biopsy and brushing with final diagnosis was calculated using the Cohen's "K" value.Fifty-six patients had malignant strictures confirmed by surgery, histology, and by clinical follow-ups. Success rates of brushing, balloon surface sampling, and biopsy were 90.7, 100, and 100%, respectively. The comparative efficacy of brushing, balloon-surface sampling, and biopsy resulted as follows: sensitivity of 47.8, 87.5, and 92.1%, respectively; specificity of 100% for all the techniques; accuracy of 69.2, 91.7 and 93.6%, Positive Predictive Value of 100% for all the procedures and Negative Predictive Value of 55, 80, and 75%, respectively.Percutaneous endoluminal biopsy is more accurate and sensitive than percutaneous bile duct brushing in the detection of malignant diseases (p < 0.01). Bile duct dilatation and increasing jaundice are often onset symptoms of a number of either malignant or benign pathological conditions.In most cases, common cross-sectional imaging techniques such as ultrasonography (US) or spiral-CT or cholangio-pancreatico-biliary magnetic resonance (MRCP) are highly capable of depicting the causes of obstruction by simply showing extrinsic masses in case of infiltrating tumors or benign causes of jaundice, like stones .These non invasive techniques, however, can still yield uncertain results in some particular benign or malignant pathological conditions.When stones are too small for example, or when bile duct dilatation is limited. For final confirmation or exclusion of endobiliary stones ERCP is unavoidable before the subsequent therapeutic steps.There are also some pathological conditions where an "intrinsic" parietal alteration is the cause of biliary obstruction and it is very difficult to distinguish benign from malignant lesions using cross-sectional imaging procedures alone figure  - 5.A biliary dilatation may also occur after surgical interventions involving the biliary system, like bilioenteric anastomoses for gastric or pancreatic neoplasms or cholecystectomies. It is not always possible to distinguish between a recurrent disease or a fibrotic post-surgical stenosis, especially if it occurs early and if inflammatory alterations were already present during surgery.A number of obstructions remains therefore unexplained because the aforementioned imaging modalities do not show any extrinsic compressing or infiltrating mass, nor a calcolous disease.In these cases, only the intrahepatic bile ducts dilation and the level of the obstruction can be determined, but additional diagnostic procedures should be performed for a diagnosis of the real nature of the obstruction .Patients with obstructive jaundice often undergo percutaneous biliary cholangiography and drainage (PBD) for decompression of the biliary tree.Although relatively invasive, not even PBD allows to achieve a definitive differential diagnosis between malignant and benign pathologies.Tissue sampling becomes therefore essential for the histological characterization of parietal alterations and for planning their appropriate treatment .Bile cytology, although easily feasible either through a transhepatic or an endoscopic route, does not accomplish adequate pathological diagnoses in most cases -7.In our study, three different endoluminal sampling techniques for the characterization of biliary strictures of uncertain nature have been reviewed and comparatively evaluated with the purpose of determining which one of them is the most accurate for differentiating between benign or malignant pathology.Our investigation was performed on a population of 80 patients who, from January 1992 to September 1999, underwent endoluminal brushing and/or biopsy for biliary stenosis or obstructions of uncertain nature. A retrospective evaluation of the efficacy of these techniques was carried out by comparing the pathological findings on one hand and radiological findings, clinical long-term follow ups, and/or post-surgical specimens, on the other.Patient population included 43 men and 37 women, with a mean age of 62 years (range 37–87 years). All patients presented jaundice as a common symptom, weight loss was present in 20 cases (25%), and itching in 12 cases (15%).At admission, all patients presented biliary dilation, but no masses were identifiable by imaging procedures such as US, CT, MRCP. ERCP had been performed in 23 patients with middle-low common bile duct stenosis but the nature of obstruction could not be determined nor a drainage could be placed.Obstruction were located at the biliary bifurcation in 18 cases (22.5%), the common extrahepatic bile duct in 50 (62.5%), the right (2) and left (2) hepatic duct in 4 cases (5%)Eight patients with bilioenteric anastomoses (10%) were also evaluated in order to determine the nature of the stricture. The patients came to the interventional unit needing for a biliary drainage. PBDs were performed using standard interventional techniques.The percutaneous approach was right lateral in all patients but 25 of them required also an anterior subxyphoid approach.The procedures for cytological and/or histological samples were performed in any case 3–5 days after placement of the biliary drainage in order to operate on a decompressed biliary system.One hundred and two endoluminal procedures were included in this study. Twenty-one patients underwent cytologic brushing only, 25 endoluminal biopsy only, and 22 both brushing and biopsy. A total number of 43 brushings and 47 biopsies were performed.In 12 additional cases, we obtained cytological and tissue samples from an angioplastic balloon used for the bilioplasty of strictures of uncertain nature  with techniques that will be described.Sixteen patients underwent brushing only, because the cholangioscope was not available yet and five because the diagnosis of malignancy achieved with brushing was considered sufficient, thus not requiring further diagnostic workups to planning the appropriate therapy.All the specimens were collected after PBD thus after translesional advancement of a 10 French catheter; in 12 particularly heavy stenosis a preliminary balloon dilatation was also accomplished.Brushing was performed according to the following technical steps:- the indwelling biliary drainage catheter was exchanged over a guide-wire with a 7/9-F. introducer sheath without valves at the proximal end - a flexible probe Fig.  with a c- the brush was then carefully pulled back to be removed from the patient into the sheath, to avoid malignant spreading through the transhepatic tract-samples were then placed on a glass slide, fixed with Sprayfix , immediately submitted for cytological examination and stained by the standard Papanicolau technique.Endoluminal forceps biopsy was performed under direct visualization with a 5-mm. cholangioscope  Fig. . Sometim- the biliary catheter was replaced, over a guide, by a 18-F "peel-away" introducer sheath through which the cholangioscope was advanced until it was close to the lesion;- a flexible forceps :- once deflated, the balloon  was pulled back into the sheath and removed from the patient;- the balloon was then placed in a saline solution with 10% of formalin, inflated and agitated several times to facilitate detachment of samples, which were then immediately delivered to the pathology laboratory.Pathological specimens from 14 patients were compared with open surgery findings and post-surgical pathological reports. The pathologists, hystologists and cytologists, were blinded as to corresponding results.The follow-up of the patients undergone to surgery stopped after the operation. Patients not candidate to surgery were treated with interventional procedures only such as bilioplasty, biliary drainage, interstitial radiotherapy and/or with chemotherapy or just a supportive therapy. This group of unoperated patients was followed-up on the basis of clinical and radiological data obtained by case-record reviews and by correspondence with their referring clinicians and general practitioners. The follow-up period ranged from 3 to 48 months. A minimum of 12 months of healthy negative cytological-histological diagnosis was necessary because either a clinically benign stenosing lesion or a cytohistological diagnosis negative for malignancy must be confirmed by a prolonged survival, as well as by clinical and radiological findings.Calculations of success rate, sensitivity, specificity, positive predictive value, and negative predictive value for each technique were based on the number of biopsy procedures n = 102) rather than on the number of patients (n = 80),  were determined for brushing and biopsy groups assuming a P value of .01 by using the Geigy Scientific Table . The agreement coefficient between biopsy or brushing and final diagnosis was calculated using the Cohen's "K" value using SPSS 8.0 for Windows .A final diagnosis of malignant disease was confirmed in 56 (70%) cases and a final diagnosis of benign disease in 24 (30%) cases for an overall of 80 patients.Final diagnoses in the malignant group included: cholangiocarcinoma (n = 31), adenocarcinoma (n = 6), metastatic adenocarcinoma (n = 5), pancreatic carcinoma (n = 13) and malignant endocrine tumor (n = 1). Final diagnoses in the benign group included: iatrogenic stenosis (n = 9), sclerosing cholangitis (n = 12) and primary (N = 2) and secondary biliary cirrhosis (N = 1).Thirty-nine of the 43 brushing biopsies procedures were technically adequate for the diagnostic evaluation. Four cases were considered "poorly cellular" by the pathologists, with an overall success rate of 90.7% Table . These 4Malignant cells were detected by brushing in 11 cases including: adenocarcinoma n = 2), cholangiocarcinoma (n = 6), pancreatic carcinoma (n = 2), and metastatic adenocarcinoma (n= 1)  Table .Endoluminal forceps biopsy was performed in 47 cases. Thirty-five of these were interpreted as containing malignant cells Table : cholangThree cases were erroneously interpreted as benign  but the clinical follow-up revealed 2 pancreatic carcinomas and 1 adenocarcinoma Table . With 35CIs were reported in Table As previously discussed, 12 samples were obtained in as many patients with an angioplastic balloon after dilation of biliary strictures . This technique demonstrated malignant cells in 7 58.3%) cases and benign cells in 5 41.7%). The final clinical diagnosis of the malignant group included adenocarcinoma (n = 1), cholangiocarcinoma (n = 5) and pancreatic carcinoma (n = 1) , iatrogenic stricture (n = 1) and biliary cirrhosis (n = 2) and 6 malignant diseases, such as cholangiocarcinoma (n = 3), pancreatic carcinoma (n = 2), and metastatic adenocarcinoma (n = 1) were included. All concordant diagnoses were confirmed by clinical or surgical follow-up. In the group of 6 discordant diagnoses, cholangiocarcinoma (n = 4) adenocarcinoma (n = 1) and pancreatic carcinoma (n = 1), were included. All the 6 malignancies misdiagnosed by brushing were correctly diagnosed by biopsy.Transient hemobilia (spontaneously reversed from 1 to 12 hours after the procedure), was observed in 5/47 (10.6%) patients who had undergone biopsy.In 4 of them, particular angulations of the access route was present and there were difficulties in negotiation of the stricture.No major complications directly related to the brush cytology or the endoluminal forceps biopsy procedures occurred.The evaluation of patients with biliary tract obstruction without evidence of any intrahepatic or extrahepatic growing mass has traditionally involved a variety of diagnostic imaging techniques . UltrasoThese imaging modalities, are however limited in depicting intraductal anatomy and, sometimes, in exactly determining the level and the cause of the obstruction ,9. MagneThe assessment of the level of obstruction also has been reported as highly accurate -19. HoweFine-needle percutaneous biopsy (FNPB) and fine-needle aspiration (FNA) has been reported poorly valuable in absence of a lesion clearly identifiable ,22. The Treatment protocols require pathological diagnoses for palliative or possibly curative therapy in almost all these types of conditions. In addition, the surgical technique itself can be different if a malignancy is present or not. Some Authors, when liver transplant is still indicated, suggest a large excision associated with gastrectomy, pancreatectomy and a transverse ascending colectomy .Although the efficacy of this surgical approach is still under debate, the role of a preoperative diagnosis is extremely important.Malignant cells surrounding the biliary ducts may continuously exfoliated into the bile, especially in the case of tumors which breack through the mucosa, and become available for the cytological examination when the bile is collected either percutaneously or by ERCP. Cytodiagnosis is easy to be performed, atraumatic in nature, with less potential risk and associated with relatively low charges. This technique has shown low sensitivity 15–28%) and accuracy (48–58%) rates, due to an early cellular degeneration after bile collection and to "poorly cellular" specimens. In addition, some pathological aspects can affect the efficacy of the procedure, such as in case of lesions extrinsically compressing the bile duct wall without a complete transmural infiltration or an adequately wide mucosal disruption . Endolum% and accAccording also with our cholangioscopic experience, biliary tumors may remain intramural causing an annular constriction of the biliary duct ,29 withoIn this review only patients not suitable for ERCP or coming from a failed drainage or other type of retrograde endoscopic intervention were evaluated, thus the percutaneous and endoscopic techniques can not be compared. Whenever possible especially if skilled endoscopists are available, retrograde approach could still to be considered the first step, for its potentially high sensitivity especially if repeated sampling are performed ,28Percutaneous brush cytology, if compared with bile transendoscopic cytodiagnosis, has the potential risk of a malignant spreading of cells through the transhepatic tract. To overcome this risk, in our opinion, the brush should be pulled back and removed from the patient into the introducer sheath. With this technique, in fact, no spreading along the transhepatic tract was observed in our malignant patients at imaging follow-up. Brush cytology is a easy, safe and at relatively low-cost procedure, similarly to bile collection. A single sampling however has a low possibility of detecting a malignancy. The results can improve with multiple samples. Three consecutive negative samples decrease the probability of a malignancy from more than 55% to less than 5% . The absThe differentiation degree of a tumor can affect the accuracy of the histological classification. In those cases of extrahepatic and periampullary biliary tumors, in fact, usually highly undifferentiated, the histological characterization can be difficult, although a generic diagnosis of malignancy can be made.Potential complications due to forceps biopsy, such as disruption of the ductal wall with consequent bile leak and bleeding, cholangitis and pancreatitis, are reported .Endoluminal forceps biopsy especially in patients with hemobilia or cholangitis, should therefore be performed in the remission phase of the disease, and, in any case, at least 5 days after biliary drainage, to avoid the complications related to manipulations into the biliary tree. No major complications were observed in this series, neither from percutaneous brushing nor from forceps biopsy. A transient hemobilia was observed in 5 patients who had undergone biopsy (10.6%). This series, including part of a previously analyzed smaller population  confirmsA high diagnostic value was proven by the simple examination of tissue samplings coming from balloon dilatation of biliary or bilio-enteric anastomotic strictures . Although the number of cases reported in this series is relatively small, it should be considered that it was possible to distinguish benign from malignant diseases in almost all the cases. Our suggestion, therefore is to associate a tissue collection from the balloon to bilioplasty, as a standard procedure when screening for malignant pancreatobiliary diseases is required. In this way. it is possible to save time and avoid risks related to further endobiliary procedures.Patients with obstructive jaundice, who are candidates for biliary drainage, often come to the interventional radiologist still without a definitive diagnosis of the real nature of their disease, even after high-level cross-sectional imaging procedures, such as abdominal MR, MRCP or abdominal spiral-CT. When the clinical diagnosis needs to be histologically confirmed for further therapeutic choices, the transhepatic route can be successfully used for the intraductal sampling. Forceps biopsy is highly accurate under cholangioscopic guidance. As an alternative, repeated brushings can be performed under fluoroscopy. If a balloon blioplasty, for any reason, is performed, it is advisable to collect the tissue fragments on the balloon surface and send them for pathological evaluation.None declared.MR carried out the interventional procedures, participated in the design of the study and drafted the manuscript, FMS carried out the interventional procedures, VC defined the design of the study and performed the statistical analysis, LG collected the results of the procedures, LG participated in the histological analysis, AR participated in the design of the study and drafted the manuscript,, GG participated in the imaging evaluations, EP performed the patients follow-up, VD supervised the drafting of the studyThe pre-publication history for this paper can be accessed here:


There is ample clinical evidence that inherited severe AAT deficiency predisposes to chronic obstructive pulmonary disease. Augmentation therapy for AAT deficiency has been available for many years, but to date no sufficient data exist to demonstrate its efficacy. There is increasing evidence that AAT is able to exert effects other than protease inhibition. We investigated whether Prolastin, a preparation of purified pooled human AAT used for augmentation therapy, exhibits anti-bacterial effects.Human monocytes and neutrophils were isolated from buffy coats or whole peripheral blood by the Ficoll-Hypaque procedure. Cells were stimulated with lipopolysaccharide (LPS) or zymosan, either alone or in combination with Prolastin, native AAT or polymerised AAT for 18 h, and analysed to determine the release of TNFα, IL-1β and IL-8. At 2-week intervals, seven subjects were submitted to a nasal challenge with sterile saline, LPS (25 μg) and LPS-Prolastin combination. The concentration of IL-8 was analysed in nasal lavages performed before, and 2, 6 and 24 h after the challenge.In vitro, Prolastin showed a concentration-dependent (0.5 to 16 mg/ml) inhibition of endotoxin-stimulated TNFα and IL-1β release from monocytes and IL-8 release from neutrophils. At 8 and 16 mg/ml the inhibitory effects of Prolastin appeared to be maximal for neutrophil IL-8 release  and monocyte TNFα and IL-1β release . Furthermore, Prolastin (2.5 mg per nostril) significantly inhibited nasal IL-8 release in response to pure LPS challenge.in vitro and in vivo, and provide scientific bases to explore new Prolastin-based therapies for individuals with inherited AAT deficiency, but also for other clinical conditions.Our data demonstrate for the first time that Prolastin inhibits bacterial endotoxin-induced pro-inflammatory responses  AAT is The pulmonary emphysema that is associated with inherited AAT deficiency is intimately linked with the lack of proteinase inhibitor within the lungs that is available to bind to, and inactivate, neutrophil elastase. On the basis of clinical observations involving patients with inherited AAT deficiency and various experimental studies, the elastase-AAT imbalance hypothesis became widely accepted as the explanation for lung tissue destruction in emphysema ,21. TherBased on the protease-antiprotease hypothesis, augmentation therapy of emphysema with severe AAT deficiency was introduced during the 1980s . Intravein vivo [in vitro, that both native (inhibitory) and non-inhibitory (polymerised and oxidised) forms of AAT strongly inhibit lipopolysaccharide-induced human monocyte activation [in vitro, and suppresses nasal IL-8 release in lipopolysaccharide-challenged individuals, in vivo.There is growing evidence that AAT, in addition to its anti-proteinase activity, may have other functional activities. For example, AAT has been demonstrated to stimulate fibroblast proliferation and procollagen synthesis , to up-rin vivo ,38. We rtivation . AAT app® (Lot 26N3PT2) was a gift from Bayer . This vial of Prolastin contained 1059 mg of functionally active AAT, as determined by capacity to inhibit porcine pancreatic elastase. Prolastin was dissolved in sterile water for injections provided by manufacture and stored at +4°C. Purified human AAT was obtained from the Department of Clinical Chemistry, Malmö University Hospital, Sweden. Native AAT was diluted in phosphate buffered saline (PBS), pH 7.4. To ensure the removal of endotoxins, AAT was subjected to Detoxi-Gel AffinityPak columns according to instructions from the manufacturer . Purified batches of AAT were then tested for endotoxin contamination with the Limulus amebocyte lysate endochrome kit . Endotoxin levels were less than 0.2 enzyme units/mg protein in all preparations used. The concentrations of AAT in the endotoxin-purified batches were determined according to the Lowry method [α1-antitrypsin (Human) Prolastiny method . Polymerg for 35 min, at room temperature, the cells in the interface were collected and washed 3 times in PBS-EDTA. The cell purity and amount were determined in a cell counter Autocounter AC900EO . The granulocyte fractions were less than 10%. Cells were seeded into 12-well cell culture plates  at a concentration of 4 × 106 cells/ml in RPMI 1640 medium supplemented with penicillin 100 U/ml; streptomycin 100 μg/ml; non-essential amino acids 1×; sodium pyruvate 2 mM and HEPES 20 mM . After 1 h 15 min, non-adherent cells were removed by washing 3 times with PBS supplemented with calcium and magnesium. Fresh medium was added and cells were stimulated with lipopolysaccharide  in the presence or absence of various concentrations of Prolastin (0–16 mg/ml), constant concentration of native or polymerised AAT (0.5 mg/ml) for 18 h at 37°C, 5% CO2.Monocytes were isolated from buffy coats using Ficoll-Paque PLUS . Briefly, buffy coats were diluted 1:2 in PBS with addition of 10 mM EDTA and layered on Ficoll. After centrifugation at 400 Human neutrophils were isolated from the peripheral blood of healthy volunteers using Polymorphprep TM  as recommended by the manufacture. In brief, 25 ml of anti-coagulated blood was gently layered over the 12.5 ml of Polymorphprep TM and centrifuged at 1600 rpm for 35 min. Neutrophils were harvested as a low band of the sample/medium interface, washed with PBS, and residual erythrocytes were subjected to hypotonic lysis. Purified neutrophils were washed in RPMI-1640- Glutamax-1 medium  supplemented with 0.1% bovine serum albumin (BSA) and resuspended in the same medium. The neutrophil purity was more than 75% as determined on an AutoCounter AC900EO. Cell viability was > 95% according to trypan blue staining.6 cells/ml) were plated into sterile ependorf tubes. Zymosan was boiled, washed and sonicated. Opsonized zymosan was prepared by incubating zymosan with serum (1:3) in 37°C water bath for 20 min. After, zymosan was centrifuged, washed with PBS and re-suspended at 30 mg/ml. Cells alone or activated with zymosan (0.3 mg/ml) were exposed to various concentrations of Prolastin (0–8 mg/ml), and native or polymerised AAT preparations (0.5 mg/ml) for 18 h at 37°C 5% CO2. Cell free supernatants were obtained by centrifugation at 300 g for 10 min, and stored at -80°C until analysisNeutrophils .Seven subjects  of 26–50 (median 38) years of age, non-smokers, non-allergic volunteers participated in the study. All subjects gave written informed consent before participation in the study. None of the subjects has a history of respiratory disease and none took any medication at the study time.At 2-week intervals each subject was submitted to a nasal challenge with sterile saline, LPS and LPS-Prolastin combination. All experimental sessions were done in the same room. On each provocation day, the nose was inspected and cleaned with 8 ml of isotonic NaCl. Between nasal lavages the subjects stayed in the same building and asked to keep away from known sources of nasal irritants. The night was spent in their own homes. All participants completed a symptom questionnaire. In the first session, the baseline lavage was taken after instillation to each nostril of 8 ml of sterile isotonic NaCl. In the next session, the subjects were challenged with LPS from Escherichia coli serotype 026:B6, Lot 17H4042 . The provocation solution was prepared prior to use. LPS was added to 8 ml of sterile 0.9% NaCl to obtain a final concentration of 250 μg/ml, and 100 μl of the provocation solution was sprayed into each nostril, using a needle-less syringe. In the third session, the subjects were first challenged with LPS, as described above, and after 30 min with 2.5 mg of Prolastin into each nostril. Lavage samples were taken with instillation to each nostril of 8 ml of sterile isotonic NaCl after 2, 6 and 24 h followed by assessment of symptoms by a questionnaire. All subject completed a symptom questionnaire with questions about nasal and eye irritation, and throat and airway symptoms. None of the participants reported symptoms of nasal, eye or throat irritations, and no general symptoms such as muscle pain, shivering, were mentioned.The procedure for nasal lavage was performed according to a method described by Wihl and co-workers . Each na. Tests showing p < 0.05 were considered to be significant.Statistical Package  was used for the statistical calculations. The differences in the means of cell culture experimental results were analysed for their statistical significance with the one-way ANOVA combined with a multiple-comparisons procedure (Scheffe multiple range test). The equality of means of experimental results in healthy volunteers were analysed for statistical significance with independent two sample t-test and repeated measures of ANOVA using the SPSS MANOVA procedure Various concentrations of Prolastin (0–16 mg/ml) were added to adherent-isolated human monocytes with or without LPS (10 ng/ml). Cells stimulated with LPS alone served as a positive control, while PBS stimulated monocytes served as negative controls. As illustrated in figures We recently found that simultaneous incubation of monocytes with LPS and either the inhibitory (native) or non inhibitory (polymeric) form of AAT resulted in a reduction in TNFα and IL-1β release compared to the cells stimulated with LPS alone. At 0.5 mg/ml the effects of native and polymerised AAT appeared to be maximal (41). Therefore, we selected a 0.5 mg/ml concentration of Prolastin, native and polymerised AAT, and compared their effects on LPS-stimulated cytokine release at 18 h. As shown in figures The effects of Prolastin (0–8 mg/ml) on human neutrophil IL-8 production are shown in Figure Neutrophils were stimulated with zymosan (0.3 mg/ml) or AATs (0.5 mg/ml) either alone or in combination for 18 h and IL-8 protein determined. As illustrated in figure To assess the effect of Prolastin on LPS-induced nasal provocation, IL-8 levels in nasal lavages were measured. Nasal instillation 25 μg per nostril of LPS alone or in combination with 2.5 mg/ml of Prolastin was performed in non-smoking and non-allergic volunteers . The IL-8 release in response to LPS challenge increased over time compared to baseline levels  and at least two modified (non-inhibitory i.e. polymeric and oxidised) forms of AAT can block the release of an array of chemokine and cytokines from LPS-stimulated monocytes [in vitro and in vivo.There is now, however, ample evidence that serine proteinase inhibitors (serpins), in addition to their well established anti-inflammatory capacity to regulate serine proteinases activity, may possess broader anti-inflammatory properties. Several studies have shown that the biological responses of bacterial lipopolysaccharide (endotoxin) n by LPS ,43. Our onocytes . These se.g. IL-6, IL-1β and TNFα) and the chemokines (e.g. MCP-1 and IL-8) [in vitro. The Prolastin exhibited these anti-inflammatory properties in a concentration-dependent manner. Its maximal effects were observed with 16 mg/ml in the monocyte model and with 8 mg/ml in the neutrophil model, since doubling these concentrations did not significantly modify the intensity of the effects. Indeed, Prolastin markedly prevented endotoxin-induced cell activation at 0.5–4 mg/ml concentrations, implying that these lower concentrations of Prolastin might also be sufficient to inhibit endotoxin effects. It is worth noting that in order to reduce a potential risk of transmission of infectious agents the Prolastin preparation is heat-treated in solution at 60° ± 0.5 for not less than 10 h. Data from in vitro studies show that heat-treatment results in AAT polymerization and loss of its inhibitory activity [Stimulation of human monocytes and neutrophils with bacterial endotoxin results in the release of a range of inflammatory mediators including the pro-inflammatory cytokines (nd IL-8) -46. Togeactivity ,48. Theractivity . Furtherin vivo. In particular, we were interested in concentrations of the neutrophil chemoattractant, IL-8. Endotoxin (or LPS) from gram-negative bacteria is a common air contaminant in a number of occupational conditions, especially those in which exposure to animal waste or plant matter occurs [3 air and may be associated with respiratory symptoms and nasal inflammation in exposed persons [To explore our hypothesis that AAT functions as a potent inhibitor of endotoxin-induced effects, we examined whether Prolastin also inhibits responses to LPS in the nasal airway, r occurs ,49-51. L persons . For exa persons . A numbe persons ,55. In oin vitro and in vivo experiments provide novel evidence that the Prolastin preparation is a potent inhibitor of endotoxin effects. The major concept behind augmentation therapy with pooled plasma-derived AAT has been that a rise in the level of AAT in subjects with severe inherited AAT deficiency would protect the lung tissue from continued destruction by proteinases (i.e. primarily leukocyte elastase) [Pseudomonas aeruginosa lung infection [Thus, data from both lastase) ,56,57. Rlastase) ,58. Furtnfection . Stocklenfection ,60. DataAAT, α1-antitrypsin; COPD, chronic obstructive pulmonary disease; LPS, lipopolysaccharide; ZZ, homozygous AAT-deficiency variant; MM, wild type AAT variant; PBS, phosphate buffered saline; EDTA, ethylenediaminetetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidIzabela Nita, performed cell culture experiments, made contribution to acquisition of data;Camilla Hollander, made substantial contribution to patient study design, material collection and analysis; Ulla Westin, contributed to study design and data interpretation; Sabina Janciauskiene, contributed to conception and study design, data interpretation and wrote the article


Green tea is widely consumed in Asian countries and is becoming increasingly popular in Western countries. Epidemiologically, it has been suggested that green tea consumption prevents type 2 diabetes. The present study was aimed at providing evidence of improvement in glucose metabolism in diabetic mice and healthy humans upon green tea consumption.Green tea promoted glucose metabolism in healthy human volunteers at 1.5 g/body in oral glucose tolerance tests. Green tea also lowered blood glucose levels in diabetic db+/db+ mice and streptozotocin-diabetic mice 2–6 h after administration at 300 mg/kg without affecting serum insulin level, whereas no effect was observed in control mice . The serum protein profiles of db+/db+ and +m/+m mice were analyzed for the first time by SELDI (surface-enhanced laser desorption/ionization)-TOF (time-of-flight)-MS (mass spectrometry), and then compared to investigate any effects of oral green tea administration on serum proteins. The protein profiles in db+/db+ mice showed that the spectral peak intensities at the mass/charge ratios (m/z) of 4119, 4203, 4206, 4211, 4579, 9311 and 18691 were >3 times lower, and those of 13075, 17406, 17407, 17418, 17622, 18431 and 26100 were >3 times higher than respective peak intensities in +m/+m mice. When green tea was administered to db+/db+ mice, the peak intensities were markedly decreased at m/z 11651 and 11863, and slightly decreased at m/z 4212. The peak intensities at 7495, 7595, 7808, 14983, 15614, 31204 were markedly increased after the administration.The present study provides evidence that green tea has an antidiabetic effect. Although we could not find simple reversed effect of green tea on the diabetes-induced modifications of the levels of several serum proteins, we found that the 4211 (4212) Da protein level that was decreased in the diabetic state was further decreased after green tea administration. This is the first report demonstrating that a certain serum protein may be involved in the antihyperglycemic effect of green tea. The contribution of this protein should be further studied. Camellia sinensis, Theaceae) is a popular beverage in East Asia, and also used as a herbal remedy in Europe and North America. Green tea is considered to be antiinflammatory, antioxidative, antimutagenic, and anticarcinogenic . PP is primarily expressed in the endocrine cells of the pancreas, and the plasma PP concentrations are elevated by food intake [ Da proteThe changes in serum protein profiles by green tea also demonstrate the increase in the peak intensities of hemoglobin-related multi-MS signals, suggesting the adverse side effects of green tea, although blood samples from db+/db+ mice tended to exhibit a hemolytic feature compared with those from wild-type mice (data not shown). Interestingly, the hemoglobin-related multi-MS signals shown in Fig. We observed that green tea improved oral glucose tolerance in humans. It is therefore likely that green tea is prophylactic against diabetes and ameliorates diabetic hyperglycemia. Green tea consumption at moderate doses may be associated with a reduced risk of type 2 diabetes in apparently healthy individuals by controlling postprandial hyperglycemia.The control of postprandial hyperglycemia by green tea can help reduce the risk of type 2 diabetes. In the present study, we provide evidence showing that green tea promotes glucose metabolism in healthy humans, and produces an antihyperglycemic effect in diabetic mice. In addition, we analyzed the serum protein profiles of db+/db+ and +m/+m mice for the first time using SELDI-TOF-MS, and further investigated its association with any effects of oral green tea administration on serum proteins. Among the several proteins that were significantly lowered in the serum of diabetic mice, the 4211(4212) Da protein was significantly decreased after green tea administration. This is the first report demonstrating that a certain serum protein is involved in the antihyperglycemic effect of green tea. The contribution of this protein, therefore, should be further investigated in a future study.Moreover, we speculate that the observed effects of green tea on BGL are primarily due to the promotion of insulin action in peripheral tissues, such as skeletal muscles and adipocytes. Indeed, a recent paper showed that green tea supplementation for 12 weeks ameliorates insulin resistance and increases glucose transporter IV content in a fructose-fed rat model resembling the human type 2 diabetes mellitus . Since tC57BLKS/J db+/db+ mice  and its age-matched control C57BLKS/J +m/+m mice  were purchased from SLC  and used as the type 2 diabetic mouse model. Male ddY mice  were singly injected with STZ , and then used 4–6 weeks after the injection . The age-matched normal ddY mice  were also used. The animals were housed (3–5 per cage) under a daily cycle of 12 h light and 12 h darkness, with free access to food and water. Animals were treated as approved by the Toyama Medical and Pharmaceutical University Animal Research Committee, and according to the guidelines for animal experiments established by the Japanese Pharmacological Society.Mice were deprived of food for 11–14 h. Blood samples (20 μl) were collected from mouse tail veins under ether anesthesia. In experiments for proteomic analyses and serum insulin measurements, blood samples (100 μl) were collected from the orbital venous plexus of mice under ether anesthesia. The food deprivation was continued throughout the measurement of blood glucose levels (until 6 h after the administration with green tea). In the oral glucose tolerance test as described below, blood samples (0.3 μl) were obtained from human skin microvessels using FreeStyle .Healthy human volunteers (18–24 years old) were fasted 12 h before the starting point of experiments. The participants were perorally administered with either a suspension of green tea powder or hot water at 9:20 a.m. Ten min after the administration (at 9:30 a.m.), the participants were perorally administered with 225 ml of Trelan-G75  containing 75 g glucose. BGLs were measured before and 30, 60, and 120 min after the administration with Trelan-G75 (glucose). All the subjects enrolled in this study were ethnic Japanese. Before participation, the purpose and risks of the study were carefully explained, and written informed consent was obtained from all the participants. The protocol was approved by the Toyama Medical and Pharmaceutical University Ethics Committee regulating human research.g for 1 min at 4°C, and its supernatant was immediately separated from the pellet.BGLs were measured using ANTSENSE II  in the mouse study and using FreeStyle  in the human study. Serum insulin levels were measured using an insulin-ELISA kit . To prepare the serum samples, blood samples from mice (100 μl) were kept on ice for 2 h, centrifuged at 16,000 × 2+  (Ciphergen Biosystems), were used to fractionate proteins in serum. For the proteomic analyses with Q10 ProteinChip array, samples were 10-times diluted with denaturation buffer (7 M urea/ 2 M Thiourea/ 4% CHAPS/ 1% dithiothreitol/ 2% ampholine) and incubated on ice for 10 min, and then 10-times more diluted with buffer of 50 mM Tris-HCl (pH8). When CM10 ProteinChip array was used, samples were 10-times diluted with the denaturation buffer and incubated on ice for 10 min, and then 10-times more diluted with buffer of 100 mM NaOAc (pH4) or 50 mM HEPES (pH7). When IMAC30 ProteinChip array was used, samples were 100-times diluted with phophate-buffered saline. Spots on the different arrays were equilibrated with the buffer used for sample dilution, e.g., Tris-HCl buffer for spots on Q10 array, and each sample solution (70 μl) was loaded onto two separate spots on the arrays. After incubation for 20 min with rotation spots were rinsed with water and air-dried completely. The spots were analyzed using the SELDI ProteinChip system . In each sample, data from the two spots were averaged (duplicate assay). If a signal/noise ratio was larger than 2, the peak was considered to reflect the amount of a protein. Quantitative nature of the instrument was confirmed as previously described [Quantitative serum proteomic profiles were measured with SELDI-TOF-MS . The mouse serum was prepared as described above. Several types of ProteinChip Array, i.e., Q10 , CM10  and IMAC30-Cuescribed . The m/zCamellia sinensis, Theaceae)  were prepared at the Taiwan Tea Experiment Station, picked in May at Nan-Tou County, and immediately used without drying. The leaves were steeped in hot water (95°C) for 30 min and the filtrate was condensed under reduced pressure. The dry weight of the extracts was determined, and the extracts were dissolved in saline for administration into mice. On the other hand, green tea powder was donated by Fukuju-en . The particle size (median diameter) of the green tea powder was 2.9 μm, according to the measurement using a Centrifugal Particle Size Analyzer . In the human study, the tea powder (1.5 g) was added to 150 ml of hot water (80°C) and then whipped with a bamboo whisk. In the mouse study, the tea powder was suspended in saline at room temperature using a sonicator. The contents of caffeine and catechins in these tea samples were determined by HPLC: the samples were injected into an HPLC column ; the eluent was 10 mM phosphate buffer (pH2.6)/MeCN (gradient: 5 to 15%) at a flow rate of 1.3 ml/min. The data are shown in Table Fresh raw leaves of tea  followed by the Scheffé's multiple range test. Values of P less than 0.05 were considered to be significant. Especially, to determine the significance of the time-dependent effects of green tea  and the peak intensity averages at m/z indicated . The analyzed peak is indicated by arrows in the data of mass spectral signals. **P < 0.01; significantly different from the peak in wild-type mice, by unpaired t-test. Types of ProteinChip used were described in the Fig. 6 legend.Click here for fileChanges in serum protein profiles of db+/db+ mice after green tea administration MS spectra shows typical changes in the serum protein profiles of db+/db+ mice administered with saline (left) or green tea (right). Graph shows the peak intensity averages at m/z indicated, before (open column) and after (closed column) administration with saline (n = 4) or green tea (n = 4). The analyzed peak is indicated by arrows in the MS spectra. **P < 0.01; significantly different from the peak obtained before the administration, by unpaired t-test. Types of ProteinChip used were described in the Fig. 7 legend.Click here for file


We examine results of gain-of-function experiments on retinocollicular maps in knock-in mice [Brown et al. (2000) Cell 102:77]. In wild-type mice the temporal-nasal axis of retina is mapped to the rostral-caudal axis of superior colliculus. The established map is single-valued, which implies that each point in retina maps to a unique termination zone in superior colliculus. In homozygous Isl2/EphA3 knock-in mice the map is double-valued, which means that each point on retina maps to two termination zones in superior colliculus. This is because about 50 percent of cells in retina express Isl2, and two types of projections, wild-type and Isl2/EphA3 positive, form two branches of the map. In heterozygous Isl2/EphA3 knock-ins the map is intermediate between the homozygous and wild-type: it is single-valued in temporal and double-valued in the nasal parts of retina. In this study we address possible reasons for such a bifurcation of the map.We study the map formation using stochastic model based on Markov chains. In our model the map undergoes a series of reconstructions with probabilities dependent upon a set of chemical cues. Our model suggests that the map in heterozygotes is single-valued in temporal region of retina for two reasons. First, the inhomogeneous gradient of endogenous receptor in retina makes the impact of exogenous receptor less significant in temporal retina. Second, the gradient of ephrin in the corresponding region of superior colliculus is smaller, which reduces the chemical signal-to-noise ratio. We predict that if gradient of ephrin is reduced by a genetic manipulation, the single-valued region of the map should extend to a larger portion of temporal retina, i.e. the point of transition between single-and doulble-valued maps should move to a more nasal position in Isl2-EphA3 heterozygotes.We present a theoretical model for retinocollicular map development, which can account for intriguing behaviors observed in gain-of-function experiments by Brown et al., including bifurcation in heterozygous Isl2/EphA3 knock-ins. The model is based on known chemical labels, axonal repulsion/competition, and stochasticity. Possible mapping in Isl2/EphB knock-ins is also discussed. Topographic ordering is an important feature of the visual system, which is conserved among many visual areas . Thus, tThe mechanisms responsible for topographic ordering have been lately under thorough examination. Following the original suggestion by Sperry , it was In this study we address the results of gain-of-function experiments, in which the retinocollicular maps were modified by genetic manipulations . RGCs ofIn addition to the observation of the overall map doubling in homozygous knock-ins Figure , Brown eBrown et al.  suggest absolute values of Eph/ephrin expression levels, rather than relative differences. Our model manages to reproduce all the essential features of experiments described in Brown et al. .We base our choice of parameters for the distribution of molecular markers on experimental observations in mouse retina and SC. Thus, the distribution of ephrinA2 and A5 is obtained in . The tot) Figure , etc. No2, obtained in [RA(x) = exp(-x)  = exp(-2x)  Figure .LB(y) = exp(-y) and RB(y) = exp(-y). As with the EphA/ephrinA the non-unit overall factors in these distributions are absorbed in parameter β (see below).The profiles of expression of EphB/ephrinB pair are measured in  similar Here we describe our 2D model in more detail. We consider an array of 100 by 100 RGC, which are connected to 100 by 100 different points in colliculus. Each RGC is characterized by two levels of expression for two receptors, EphAs and EphBs, described in the text. The concentration profiles are taken to be the same for EphA and EphB receptors in the wild-type species. In the homozygote and heterozygote cases the concentration of EphA is taken as shown at Figure α = β = 30.The process of development is modeled as follows. We randomly choose a pair of axons in SC separated either in RC or in ML direction. We exchange their positions with the probability given by Eq. (1) or Eq. (3) respectively. We then repeat the process until a stationary distribution of probabilities is reached in the same manner as for 1D case. Note, that this time a chosen pair of axons, say in RC direction, may not be a neighboring pair, but consist of two axons separated by any distance in SC. This procedure dramatically decreases the convergence time to the stationary distribution, which is the same as in the case when we choose the neighboring axons only. The noise level is taken to be the same for both RC and ML directions, that is 6 iterations. The temporal evolution of the map for the label in the central retina is shown at the Figure The spatial 2D distribution of the axons corresponding to labeled RGCs is shown at Figure In both 1D and 2D cases the calculations were performed on Dell PowerEdge 1600SC server. The programs, written on Matlab , are available for download in .Equations for the probability of switching of two axons (1) and (2) can yield probabilities, which are below 0 or larger than 1. Thus, in the numerical implementation of our model instead of (1) and (2) we use expressions with soft cutoff at 0 and 1, i.e.These probabilities are restricted to be between 0 and 1. In addition, when differences in ligand and receptor densities between neighboring points are not large, (4) and (5) are equivalent to (1) and (2).final probability distribution for connectivities does not depend on whether the swaps are local or global. This statement is true, for any distribution of chemical labels, in 1 or 2D. It is true however if Eq. (4) is used to calculate probabilities of transitions. In particular, we show that Eq. (4) leads to a Boltzmann distribution of the probabilities of connections, which does not depend on the locality/globality of transitions. Thus, we can present final maps for both local and global transitions interchangingly, since results pertaining to the final state of the map, such as in Figures One could use exchanges between nearest neighbors to implement map development, as described in text above and in . Alternar if Eq.  is used PA and PB. These probabilities satisfy the detailed equilibrium condition [Let us now derive the probability distribution of projections in the final map. We perform our derivation for the 1D case; in 2D it is similar. We proceed using the detailed equilibrium principle, frequently employed in statistical mechanics . Consideondition where the transition probabilities are given by equation (4). After some algebra, it is possible to show that the transition probabilities are given by a simpler than (4) formEA and EB are 'state' variables, depending on the current arrangement of axons in the targetwhere i, with L(i) being the ligand concentration and R(i) the receptor concentration. The latter, of course, depends on the arrangement of axons, corresponding to the state A. The transition probability PB → A is given by the same expression, with exchanged indexes A and B. The detailed equilibrium condition (6) leads then to Botzmann probability distribution of the states of the mapHere summation is assumed over all termination sites in SC, denoted by index not depend on the methods of reconstruction. Thus, both global and local transitions will lead to identical final arrangement of the map. This property is well-known in considering Metropolis Monte-Carlo procedures. What does depend on the methods of reconstruction is the time, which it takes to reach the final configuration. Thus, as it was mentioned above, global transitions lead to the final state much faster. With local transitions, on the other hand the map can freeze in the original state, and it may take an exponential time to reach the final configuration.Eq. (9) is instrumental in showing that the final distribution of projections in our approach does We thus conclude that our results presented in this study are universal in that they do not depend on the exact developmental mechanism, but only on the distribution of 'chemical' tags.


For more than 2,000 years, from the time of Aristotle onwards, it was thought that the complete body plan of human beings  was present in the fertilized egg. During pregnancy, a preformed miniature human being, or homunculus, grew bigger and bigger; development was simply a process of growth.Then, in the mid-18th century, Carl Friedrich Wolff described how the chick gut, basically a tube, forms from an initially flat sheet of cells, overthrowing at a stroke the preformation theory of embryology. We now know that development is a complex series of coordinated processes that transforms the amorphous ball of cells produced from the fertilized egg by cell division into an intricate body containing numerous specialized tissues and organs. And we are beginning to understand how a wide array of transcription factors—proteins that bind to regulatory sequences within genes to control their expression—guide the sequential stages involved in development. It seems that these factors form regulatory networks that control the temporal and spatial waves of gene expression that underlie and are required for organized body building.Susan Mango and her colleagues are studying the role of transcription factors in controlling organ development. The organ they are studying—the pharynx of the nematode worm—is relatively simple. This muscular tube, which passes bacteria  from the mouth to the midgut, contains fewer than 100 cells of only seven different types.To get an overall picture of the regulatory sequences within genes that are involved in the temporal control of pharyngeal development, the researchers identified 339 candidate pharyngeal genes by comparing gene expression profiles in mutant worm embryos that had excess pharyngeal cells with those in mutant embryos lacking pharyngeal cells. Then, by referring to a database that details gene expression patterns in nematode worms and embryos, the researchers classified 37 of their candidate genes as having early-onset expression and 34 as having late-onset expression.Next, the scientists carefully examined the DNA sequence of each gene for candidate regulatory regions that might contribute to its temporal regulation. Of nine candidate motifs revealed by this search, six functioned as regulatory sites in in vivo assays. The researchers estimated that these six elements, together with sites that bind PHA-4—a member of a family of transcription factors that are important in digestive tract development in many animals—account for the timing of onset of expression of about half of the nematode's pharyngeal genes. Finally, the researchers used combinations of the newly discovered temporal regulation sites and PHA-4 sites in a genome-wide search that predicted pharyngeal genes and their time of onset of expression with greater than 85% accuracy.From these results and those of previous studies, Mango and her colleagues propose a model to explain how the temporal control of pharyngeal gene expression needed for pharynx development is achieved. The earliest time for pharyngeal gene expression, they suggest, is determined by how well PHA-4 sticks to a particular gene's binding site. However, gene expression only occurs if other factors that bind to the regulatory sites are also present, and the exact combination of these factors determines which gene is active at any given time. The identity of these factors remains to be discovered. Nevertheless, at least for this simple organ, we now have a much better idea of how the complex process of organ formation is controlled at a molecular level, and it is likely that similar regulatory networks will underlie the formation of other organs as well.